Characterization of Rat Liver Proteins Adducted by Reactive

Article pubs.acs.org/crt

Characterization of Rat Liver Proteins Adducted by Reactive Metabolites of Menthofuran S. Cyrus Khojasteh,*,† Dylan P. Hartley,⊥ Kevin A. Ford,‡ Hirdesh Uppal,‡ Shimako Oishi,§ and Sidney D. Nelson∥ †

Drug Metabolism and Pharmacokinetics, Genentech, Inc., 1 DNA Way MS 412a, South San Francisco, California 94080, United States ‡ Safety Assessment, Genentech, Inc., 1 DNA Way MS 59, South San Francisco, California 94080, United States § Pharma Products Group, Abbott Japan Co., Ltd., Tokyo 108-6303, Japan ∥ Department of Medicinal Chemistry, School of Pharmacy, University of Washington, Box 357610, Seattle, Washington 98195, United States S Supporting Information *

ABSTRACT: Pulegone is the major constituent of pennyroyal oil, a folkloric abortifacient that is associated with hepatotoxicity and, in severe cases, death. Cytochrome P450-mediated oxidation of pulegone generates menthofuran, which is further oxidized to form electrophilic reactive intermediates, menthofuran epoxide and the ring-opened γ-ketoenal, both of which can form adducts to hepatocellular proteins. Modification of hepatocellular proteins by the electrophilic reactive intermediates of menthofuran has been implicated in hepatotoxicity caused by pennyroyal oil. Herein, we describe the identification of several proteins that are the likely targets of menthofuran-derived reactive metabolites. These proteins were isolated from the livers of rats treated with a hepatotoxic dose of menthofuran by two-dimensional gel electrophoresis (2D-gel) separation and detected by Western blot analysis using an antiserum developed to detect protein adducts resulting from menthofuran bioactivation. The antibody-reacting proteins were excised from the 2D-gel and subjected to tryptic digestion for analysis of peptide fragments by LC-MS/MS. Although 10 spots were detected by Western blot analysis, only 4 were amenable to characterization by LC-MS/ MS: serum albumin, mitochondrial aldehyde dehydrogenase (ALDH2), cytoplasmic malate dehydrogenase (MDH1), and mitochondrial ATP synthase subunit d. No direct adduct was detected, and, therefore, we complemented our analysis with enzyme activity determination. ALDH2 activity decreased by 88%, and ATP synthase complex V activity decreased by 34%, with no activity changes to MDH1. Although the relationship between these reactive metabolite adducted proteins and hepatotoxicity is not clear, these targeted enzymes are known to play critical roles in maintaining cellular homeostasis.

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INTRODUCTION Pulegone1 is a monoterpene present in high concentrations in pennyroyal oil, a mint oil obtained from leaves of Mentha pulegium and Hedeoma pulegoides. The oil is used as a flavoring agent in beverages and food, and it has reported folkloric use for aromatherapy and is used as an abortifacient and a remedy for colic. Unfortunately, ingestion of pennyroyal is associated with hepatotoxicity and, in some cases, death,2,3 with one of the first reported dates in 1897.4 In 1979, Sullivan reported on a post-mortem finding, in the case involving an 18-year-old girl, was massive centrilobular hepatic necrosis consistent with hepatic damages at regions of the liver with high level of cytochrome P450 enzymes (P450s).5 © 2012 American Chemical Society

Since then, many mechanistic studies have been performed to better understand the role of P450s in linking bioactivation of pulegone to hepatotoxicity.6 Early studies in rats showed that P450 induction by phenobarbital intensified hepatotoxicity, whereas the P450 inhibitor piperonyl butoxide resulted in protection against hepatotoxicity.7 Importantly, the proximate metabolite of pulegone, menthofuran, is metabolized by P450s to form γ-ketoenal-derived and epoxide-derived reactive intermediates that result in adducts with hepatocellular proteins Received: April 2, 2012 Published: October 29, 2012 2301

dx.doi.org/10.1021/tx300144d | Chem. Res. Toxicol. 2012, 25, 2301−2309

Chemical Research in Toxicology

Article

Scheme 1. Overall Scheme Depicting Oxidation of Menthofuran to Form γ-Ketoenal and Epoxide Reactive Intermediates That Covalently Modify Cellular Proteins and the Methodologies for Identifying the Modified Proteins and Determining Their Enzymatic Activities

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(Scheme 1). Covalent modification of targeted proteins has been implicated in liver injury caused by pennyroyal oil.8,9 Many xenobiotics are known to generate reactive intermediates that form covalent adducts to key proteins, thus leading to toxicity.10,11,33−35 Identification of these target proteins is crucial to understanding the mechanisms of toxicity, and a major effort is underway to collect and provide data on the target proteins of xenobiotic reactive metabolites.12 Immunochemical methods are frequently used to detect and purify adducted proteins.13 Immunogens are prepared by conjugating the chemicals of interest to proteins, such as keyhole lympet hemocyanin (KLH) and metallothionein (MT), which are not present in the target samples. Antibodies have been raised to antigens prepared by this method using several drugs and other chemicals, including acetaminophen,13−15 diclofenac,16 halothane,17 tienilic acid,18 and trichloroethylene,19 all of which form reactive metabolites. In one case, single-chain fragment variable (ScFv) antibodies were used to detect and purify several protein targets of a reactive metabolite of the furan-containing compound, teucrin A.20 Radiolabeled small molecules or targeted mass spectrometry has also been used for the detection of protein adducts.21 The use of mass spectrometry by itself to detect protein adducts tends to be very complex, though significant progress has been made in this regard. Radiolabeled probes have the advantage of not introducing bias into the detection of adducted proteins as well as producing very quantitative information, unlike antibodies, which recognize only certain epitopes. However, radioactive probes have the disadvantage of low sensitivity and a decreased limit of detection.22 These different detection methods may result in differences in identification of the targeted proteins, as has occurred in the case of tienilic acid.22,23 Advances in the separation of cellular proteins using twodimensional gel electrophoresis (2D-gel) have allowed for improved and reproducible protein separations.24 The identification of chemically modified proteins has advanced through the use of mass spectrometry of peptides generated from tryptic digests of isolated covalently alkylated proteins. Here, we applied 2D-gel separation, immunochemical detection, and mass spectrometry to fish out the likely target proteins of menthofuran reactive metabolites. No adducted peptide fragment was detected, so we complemented our data with enzyme activities of the identified peptides. Two key enzymes were shown to have decreased activities.

EXPERIMENTAL PROCEDURES

Materials. Menthofuran was purchased from Fluka (Buchs, Switzerland). Dry immobilzed pH gradient gel strips (11 cm, linear pH 3−10) and ampholines (pH 3.5−9.5) were purchased from Pharmacia-Biotech (Uppsala, Sweden). Trypsin (porcine, sequence grade modified) was purchased from Promega (Madison, WI). l-Ethyl3-[3-(dimethyl amino)-propyl]-carbodiimide hydrochloride, KLH Inject, Ellman’s reagent, and goat antirabbit alkaline phosphatase conjugate were purchased from Pierce (Rockford, IL). Cellulose dialysis tubing (molecular weight cutoff 12,000−14,000) was purchased from Spectrum Medical Industries, Inc. (Los Angeles, CA). Common chemicals used in the studies were of the highest grade available. Instruments. Enzyme-linked immunosorbent assays (ELISAs) were carried out in 96-well microtiter plates (high binding) using a THERMOmax VMax Microplate Reader with a SOFTmax data system from Molecular Devices Corp. (Menlo Park, CA). The Multiphore II electrophoresis system and EPS 3500 power supply were purchased from Pharmacia-Biotech (Uppsala, Sweden). Preparation of the Immunogen (MT-DDM). A total of 5 mg of horse kidney MT (0.735 μmol) in 20 mL of acidic aqueous solution (adjusted pH ∼5 with 1 N HCI) was added to 31.2 mg (147 μmol, 200 molar excess) of α,α′-dimethoxydihydromenthofuran (DDM) in 0.5 mL of ethanol as previously described.7 The conjugate, MT-DDM, solution was constantly stirred and allowed to incubate at 4 °C for 18 h. Aliquots (0.2 mL each) of the reaction were removed at 0, 0.5, 3, and 18 h to monitor the loss of free thiols in MT. To each aliquot, 0.8 mL of 0.1 M phosphate buffered saline (PBS; pH 7.5) was added, and the total number of free thiols remaining in MT was determined spectrophotometrically at 412 nm with Ellman’s reagent. After 18 h of stirring the reaction at 4 °C, the mixture was transferred into cellulose dialysis tubing and dialyzed against 10 mM ammonium bicarbonate buffer (pH 7.8, 2 × 3 L) maintained at 4 °C for 48 h. The dialysate was transferred to plastic vials, lyophilized, and stored at −80 °C until immunization. Immunization with MT-DDM Conjugate. All animal husbandry protocols were in accordance with the University of Washington Guide for the Care and Use of Laboratory Animals. New Zealand white rabbits (2−3 kg) were immunized subcutaneously with 300 μg of MT-DDM emulsified in saline and Freund’s complete adjuvant (1:1). At three-week intervals, rabbits were given another immunization of 300 μg of immunogen in Freund’s complete adjuvant. Production bleeds were collected one week after the final booster injection, and the serum was isolated by allowing the blood to clot at room temperature for 1 h, after which time the blood was left overnight at 4 °C. Sera were isolated by centrifugation for 30 min at 2,500g at 4 °C. Antimetallothionein-α,α′-dimethoxydihydromenthofuran (α-MT-DDM). The cross-reactivity of α-MT-DDM was assessed using competitive ELISA according to Pumford et al.’s method.16 Wells were coated with 1.5 μg of MT-DDM/100 μL in 60 mM sodium carbonate (pH 9.6) at 4 °C overnight. After washing the wells with 100 μL of 2302



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Table 1. Half Maximal Inhibitory Concentration (IC50) Used to Assess Cross-Reactivity of α-MT-DDM Antiserum in Competitive ELISA Assaysa

a

Data is expressed as the means ± SD. bStandard curves for every compound were determined in triplicate. First-Dimensional Separation: Isoelectric Focusing. Rat liver homogenates were mixed with a solution containing 5 M urea, 2 M thiourea, 2% v/v NP-40, 2% v/v 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate (CHAPS), 50 mM dithiothreitol, 2% ampholyte 3.5−10, and a trace of bromophenol blue. The incorporation of thiourea improved the resolution and solubilization of proteins.27 The mixture was stirred with a micro stirrer for 5 h. The stirrer was removed, and the mixture was centrifuged for 1 h at 100,000g. Samples were loaded during the rehydration of immobilized pH gradient gels (IPGs), which avoided the precipitation of proteins.24,27 A volume of 260 μL of supernatant was placed in a 1.0 mL pipet tube after placing the IPG strip in it. The IPG strips were rehydrated and kept frozen at −20 °C. The tubes were then placed on a shaker and left overnight. Running conditions for the firstdimensional isoelectric focusing separation were as follows: the voltage was set at 150 V for 2 h, raised to 300 V for 5 h, raised to 1000 V for 11 h, raised to 2000 V for 7 h, and finally raised to 3500 V for 20 h. Immediately after isoelectric focusing, the IPG strips were equilibrated for 2 × 15 min in Tris-HCl buffer (10 mL/strip), pH 6.8, containing 6 M urea, 2% w/v sodium dodecyl sulfate (SDS), 30% w/v glycerol, 50 mM dithiothreitol, 2% v/v β-mercaptoethanol, and a trace of bromophenol blue. Iodoacetamide (25 mM) was added to the second equilibration solution instead of dithiothreitol and βmercaptoethanol. The IPG strips that were not immediately used were frozen at −20 °C prior to equilibration. DTT (2%) was added followed by iodoacetamide (2.5%) in the second equilibration step to reduce and alkylate free thiols.

0.05% Nonidet P40 in PBS three times, excess binding sites were blocked with 200 μL/well of 2% BSA in PBS for 1 h. The wells were incubated for 2 h with 100 μL of test compound (at concentrations up to 100 μM; Table 1) plus α-MT-DDM diluted with 0.5% BSA in PBS, followed by washing 3× with 100 μL of 0.05% Nonidet P40 in PBS. Goat antirabbit alkaline phosphatase conjugated secondary antisera (100 μL; 1:5000 dilution in 0.5% BSA in PBS) were added to the wells. After incubating for 1 h, the wells were washed 3× with 100 μL of 0.05% Nonidet P40 in PBS. Plates were developed by adding 100 μL of substrate solution (p-nitrophenyl phosphate in 10 mM diethanolamine and 0.5 mM MgCl2; pH 9.5), and the product absorbance was monitored at 405 nm. Incubation of α-MT-DDM without inhibitor was used as the control to determine the maximum end-point for optical density at 405 (OD405 nm). For competitive ELISA, various concentrations of inhibitors (0.01−100 μM) in 10% ethanol were incubated with 1:100 or 1:250 dilutions of α-MT-DDM in 0.5% BSA in PBS. As a negative control, wells were incubated with buffer containing no antimetabolite sera. Inhibition curves were analyzed by a four-parameter logarithmic curve-fitting procedure obtained by the omission of zero-concentration data.26 In Vivo Hepatotoxicity and Covalent Binding Studies. Male Sprague−Dawley rats (250−280 g) from Harlan Laboratories (Indianapolis, IN) were injected intraperitoneally with 200 mg/kg (5 mL/kg) menthofuran in corn oil. After 8 h, rats were euthanized, and livers were surgically removed and perfused with saline to remove excess blood. Livers were minced and homogenized, and the protein content was determined by the bicinchoninic acid protein assay with BSA as a standard. 2303



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ALDH2 Activity Using Immunocapture. The Abcam ab115348 kit was used to determine ALDH2 in the samples. Briefly, the enzyme was captured within the wells of the microplate utilizing rat specific antibodies as an immunocapture of ALDH2, and the activity was determined by following the production of NADH in the following ALDH2 catalyzed reaction: acetaldehyde with NAD+ to acetic acid and NADH. The antibody used to isolate ALDH2 in this kit was generated by immunization with rat mitochondrial proteins. The resulting monoclonal mouse antibody isolates, by immunoprecipitation, a single ALDH2 band to purity from a number of species including rat, mouse, and human. The activity measured by this kit is only compatible with the NAD+ cofactor; there is no activity with NADP+, a feature of mitochondrial aldehyde dehydrogenase activity over other cytosolic aldehyde dehydrogenases. The generation of NADH was coupled to the 1:1 reduction of a reporter dye to yield a colored (yellow) reaction product whose concentration could be monitored by measuring the increase in absorbance at 450 nm (Dye molar extinction coefficient −37000 M−1 cm−1). The antibody in the kit immunocaptured in each well only native ALDH2 from the chosen sample; this removed all other enzymes, including unrelated aldehyde dehydrogenases. For simplicity, the activity was expressed as the change in absorbance per minute per amount of sample loaded into the well. Activity was collected using a Molecular Dynamics microplate reader. Standard curves of reference and sample data were exported to GraphPad Prism software for analysis. ATP Synthase. Other mitochondrial enzymes were measured using commercial immunoaffinity activity kits, specifically Complex I,31 Complex II,32 Complex IV,31 and ATP synthase.33 A standard curve was created, and test sample activities were derived and expressed as described above.

Second-Dimensional Separation: SDS−PAGE. The sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS−PAGE) system of Laemmli was used with no stacking gel and a 10% separation gel solution as previously described.16 Western Blots. After electrophoresis, the separated proteins were blotted to a polyvinylidene fluoride membrane as described previously.16 The membrane was blocked using 3% nonfat dry milk in tris-buffered saline (TBS) overnight at 4 °C with shaking, followed by 3 × 10 min washing with TBS. The membrane was incubated with the α-MT-DDM (diluted 1:500 in TBS) for 3 h, followed by 3 × 10 min washing with TBS. The secondary antibody used was from an ABC kit from Pierce. The membrane was incubated with the biotinylated goat antirabbit antibody from a Pierce Immunopure ABC kit for 30 min followed by 3 × 10 min washings with TBS. The membrane was then incubated with ABC solution for 30 min, followed by 3 × 10 min washings with TBS. The membrane was developed with diaminobenzidine enhanced developer. In-Gel Digestion and Mass Spectral Analysis of Tryptic Digests. An identical SDS−PAGE gel was also prepared at the same time and for image analysis, and the gel was stained with silver nitrate. Polypeptide spots from Western blot were matched to spots in the reference spots in the silver stained gel. The protein spots from the silver stained gel were carefully excised using a manual spot cutter. They were transferred to a vial and destained followed by the tryptic digest as described previously.28 The peptide fragments were analyzed by capillary LC-tandem mass spectrometry using Finnigan TSQ 7000 according to the methodology.29 Mobile phases A and B were water and acetonitrile each with 0.4% acetic acid, respectively. The running conditions started with 5% B and were gradually raised to 95% B for 30 min at a flow rate of 150 nL/min. The makeup solution was 200 nL/min methanol/water (50:50) with 0.4% acetic acid. About 1 μL of sample was injected onto the column. The collision-induced dissociation (CID) was performed on ions with intensities of at least 80,000 counts. The identity of the proteins was determined by analyzing the data generated by the CID of the peptides using SEQUEST (a registered trademark of the University of Washington and distributed by Thermo (San Jose, CA)) as previously described.30 Enzyme Activity from Liver Samples. Excised livers from rats dosed with menthofuran or corn oil were homogenized in a Dounce tissue homogenizer. Specific enzyme activities were determined based on procedures described below. In general, control activities of the proteins that had enzymatic activities were determined using serial dilutions of liver samples from rats treated with corn oil vehicle only. Test samples were measured in triplicate at concentrations within the range of the standard curves that were generated from the control samples. The relative activity was determined by interpolation from the standard curve and expressed as the relative amount of standard sample necessary to achieve the same rate as the test sample at the specified concentration. MDH1 Activity Assay. NAD+/NADH dependent cytosolic-form malate dehydrogenase (MDH1) activity was measured using the following reaction mixture: 100 mM potassium phosphate at pH 7.7, 1 mM NADH, 5 mM MgCl2, and 25 μg/mL of total cytosolic protein fraction. The reaction was initiated by the addition of 1 mM oxaloacetate, and the reaction velocity was determined by measuring the decrease in absorbance at 340 nm resulting from the oxidation of NADH. ALDH2 Preliminary Activity Assay. In the preliminary assay, the reduction of NAD+ was measured spectrophotometrically over 3 min at 340 nm. ALDH2 activity in the mitochondrial fraction was measured using the following reaction mixture: 50 mM Tris at pH 9, 1 mM NAD+, 10 μM methoxy phenazinium methylsulfate, 1 mM [2-(4iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium] (WST-1), and 25 mM propionaldehyde. The reaction was initiated by the addition of 100 μg/mL of total protein from the mitochondrial fraction. The reduction of NAD+ produced from ALDH2-mediated metabolism of propionaldehyde or acetaldehyde was coupled to the reduction of the tetrazolium dye WST-1 and measured spectrophotometrically at 450 nm after a 50 min incubation.

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RESULTS Generating and Characterizing α-MT-DDM. DDM was shown to react with semicarbazide to form 5,6,7,8-tetrahydro4,7-dimethyl-7H-cinnoline under slightly acidic conditions.7 This reaction is thought to proceed through the formation of a γ-ketoenal reactive metabolite, similar to menthofuran bioactivation by P450 enzymes.34 Hence in order to generate an antigen that mimics a reaction product of menthforuan reactive intermediate, DDM was reacted with cysteine-rich MT under acidic conditions to generate the antigen conjugate, MT-DDM (Scheme 2).7 This conjugate was then used to raise antibodies, Scheme 2. Conjugation of α,α′Dimethoxydihydromenthofuran (DMM) to Metallothionein (MT) to Form the MT-DMM Conjugate Used to Raise Antibodies in Rabbits

α-MT-DDM, in rabbits. The cross-reactivity of this antiserum was tested using competitive ELISA (Table 1). Interestingly, menthofuran and 2-(N-acety1-cysteinyl)-menthofuran were weak inhibitors of the antiserum, but mintlactone, isomintlactone, and 7a-(N-acety1-cysteinyl)-mintlactone were inhibitors with IC50 values at submicromolar concentrations. These results suggested that possibly the conjugates of DDM to MT through cysteine thiol more resemble mintlactone than menthofuran. Identifying Targeted Proteins. To identify the protein targets of menthofuran in vivo, rats were dosed with a hepatotoxic dose of menthofuran (200 mg/kg formulated in 2304



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Figure 1. (A) Western blot and (B) silver-stained gel of the two-dimensional electrophoresis of rat liver obtained from rats administered toxic doses of menthofuran (200 mg/kg). The identities of spots A to D are included in Table 2.

Figure 2. Percent activity of complexes I, II, IV, V, pyruvate dehydrogenase complex, MDH1, and ALDH2 in the liver of rats 8 h after an intraperitoneal dose of corn oil (−M) or 200 mg/kg menthofuran in corn oil (+M). N = 3. The data presented are the mean + SD.

Table 2. Rat Liver Target Proteins Identified by LC-MS/MS Followed by Tryptic Digesta spot

rat liver target proteins

gene name

Uniprot/ Swiss-Prot

number of peptide fragments matched

% amino acid sequence coverage

identified amino acid sequences

A

serum albumin

Alb

P02770

4

8

B

aldehyde dehydrogenase (ALDH2) malate dehydrogenase (MDH1) ATP synthase subunit d

Aldh2

P11884

6

12

Mdh1

O88989

4

15

DLDVAVLVGSMPR EVGVYEALKDDSWLK GEFITTVQQR FVEGLPINDFSR

Atp5h

P31399

2

13

AIGNALK ANVDKPGLVDDFK

C D

SIHTLFGDK RHPYFYAPELLYYAEK FPNAEFAEITK APQVSTPTLVEAAR LLYR LGPALATGNVVVMK VAEQTPLTALYVANLIK TFVQEDVYDEFVER ILGYIK YGLAAAVFTK

a

It includes the number of peptides, the percent amino acid sequence coverage, and the sequence identified by mass spectrometry fragmentation. The complete sequence and the mass spectrometry interpretation are included in Supporting Information, Figures S1−S20.

corn oil) or with the control vehicle (corn oil). The livers were excised and homogenized 8 h postdose. This dose strength and time point were previously shown to have resulted in the maximum liver damage from menthofuran (unpublished data). The proteins were then separated by a 2D-gel according to the isoelectric point in the pH range of 3.5−10, followed by separation in the second dimension by SDS−PAGE. Prelimi-

nary attempts were made with testing the various pH ranges for the first dimension by isoelectric point. This pH range between 3.5 and 10 provided the maximum separation of all the proteins observed following Western blotting and silver-stained gels (Figure 1). The modified proteins were visualized onto polyvinylidene fluoride membranes using α-MT-DDM antiserum for Western blotting, and 10 distinct spots were 2305



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specific monoclonal antibodies to first isolate the enzyme. This was done for Complexes I, II, IV, and V. No activity changes were observed for Complexes I, II, or IV; however, a 35 ± 15% decrease in activity of Complex V for ATP hydrolysis was observed.

observed (Figure 2A). These spots were absent in the control liver that was not exposed to menthofuran (data not shown) and happened to be located in an area with reasonably limited spots on the silver-stained gel (Figure 2B). The 10 spots were excised from the gel and digested using the tryptic digest, and the peptides were then analyzed by LC-MS/MS. Of the 10 detectable spots, only 4 provided enough quantitative and qualitative information for adequate assignment. Following product ion scans in the positive ion mode, CIDs were interpreted using SEQUEST. The assigned sequences were then compared to the sequences for rats in Swiss-Prot database. Four of the peptides using these sequencing gave adequate spectra that could be unambiguously assigned to proteins. In this study, there was no attempt to search for adducted peptides. The four proteins were serum albumin, mitochondrial ALDH2, cytoplasmic MDH1, and ATP synthase subunit d. These proteins are listed in Table 2 with their corresponding identified peptide sequences. In all, two to six peptides, each accounting for 8−15% of the total protein amino acid sequence, were identified for each protein. The complete sequence of the proteins plus the corresponding coverage and mass spectrometry and their corresponding interpretation are shown in the Supporting Information (Figures S1−S20). Changes in Enzyme Activities of Targetted Proteins. Since there was no direct detection of the adducted proteins, we complemented the studies for determining enzyme activities of the targeted proteins. The activity of mitochondria for oxidative phosphorylation and the activities of MDH1 and ALDH2 were assessed from subcellular fractions isolated from the rat livers. In the case of MDH1, no changes in hepatic MDH1 activity was detected between the control rats and the menthofuran-dosed rats, thus demonstrating that total downregulation of hepatic enzymes did not occur. In the case of ALDH2, two probe substrates were used in the mitochondria fraction. They were propionaldehyde or acetaldehyde, each at 25 mM substrate concentrations at pH 9. A 50% decrease in aldehyde dehydrogenase activity was detected in mitochondrial preparations. However, a portion of this activity may be derived from mitochondrial contamination by aldehyde dehydrogenases other than the mitochondrial isoform ALDH2. This became more apparent when aldehyde dehydrogenase activity/mg sample was analyzed for the three generated fractions: tissue homogenate, cytosol, and mitochondria. The aldehyde dehydrogenase activity using the probes described in the mitochondrial sample (ALDH2 isoform) was small (20%) compared to the cytosolic aldehyde dehydrogenase activity (up to 18 other isoforms). As a consequence, a minimal contamination of mitochondrial preparations with cytosolic isoforms would contribute significant aldehyde dehydrogenase activity unrelated to ALDH2. Concerns of cytosolic ALDH contamination of mitochondrial preparations prompted the development of a new method for the analysis of ALDH2 activity by immunoassay in tissue homogenates. Using this method, mitochondrial ALDH2 activity decreased by 88 ± 7% (Figure 2) in comparison to that of control rats. To make sure that this dramatic change in ALDH2 acitivity was not due to necrosis or downregulation of ALDH2, a Western blot analysis of this enzyme was conducted in rat liver homogenates (Supporting Information). The analysis showed decreased ALDH2 levels, but this could not account for the magnitude of the decrease in activity. For oxidative phosphorylation, the activity assays were performed on the whole tissue homogenate using highly

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DISCUSSION The toxicity of the abortifactant pennyroyal oil is in large part due to a series of reactions initiated by the bioactivation of the monoterpene pulegone, a major constituent of the oil. First, pulegone is oxidized by P450s to form menthofuran as a major metabolite. The mechanism of this transformation was studied by using deuterated analogues of pulegone to show that P450 oxidation of pulegone to form menthofuran involved radical formation and a rotation around the double bond.1 In humans, this reaction is catalyzed mainly by P450 2E1, 1A2, and 2C19.35 Second, the metabolism of menthofuran, also by P450s, produces epoxide and γ-ketoenal reactive intermediates. Studies comparing plasma alanine transaminase (ALT) levels in pulegone-dosed versus menthofuran-dosed rats showed, with matching menthofuran plasma area under the curve, that 50% of toxicity by pulegone is due to the bioactivation of menthofuran.9 The use of P450 inducers and inhibitors in in vivo assays confirmed that P450 metabolism plays a significant role in menthofuran toxicity.8 In humans, the enzymes involved in menthofuran oxidation are P450 2E1, 1A2, 2C19, and 2A6.35 Finally, these reactive intermediates form covalent adducts with cellular proteins. When mice were administered 200 mg/kg menthofuran, 7.2 nmol/mg protein of related menthofuranrelated adducts was covalently bonded in the liver and, importantly, 3.4 nmol/mg protein was covalently bonded in the microsomal fraction.8 The addition of semicarbazide, a γketoenal trapping agent, to in vitro incubations of radiolabeled menthofuran plus NADPH in human, mouse, and rat liver microsomes led to a 70−80% reduction in covalent binding to microsomal proteins. In the same incubation, the tetrahydrocinnoline derivative of menthofuran was detected, thus pointing to the formation of the γ-ketoenal reactive intermediate. It is important to note that broad spectrum inhibitors of P450, cobaltous chloride and piperonyl butoxide, dimished the toxicity−hepatotoxicity of menthofuran in rats as indicated by the ALT level in the plasma and hepatic necrosis score.1 Liver damage caused by menthofuran did not significantly decrease the hepatic GSH levels,36 which was consistent with no significant changes in ALT levels when rats were pretreated with buthionine sulfoximine. The authors argued that although glutathione conjugates may be formed to some extent it may only result in partial reduction of toxicity by menthofuran;25,37 therefore, at least some of the reactive intermediates of menthofuran metabolism are likely to form adducts on protein−SH groups. In order to raise an antibody against menthofuran-derived reactive metabolites, cysteine-rich MT was coupled with DDM under acidic conditions via the γketoenal of menthofuran, and the α-MT-DDM antiserum was then raised in rabbits.8,38 The epitope recognition of α-MTDDM using competitive ELISA showed that this antibody is selective for recognizing a mintlactone-type of structure rather than menthofuran or 2-(S-glutathionyl)-menthofuran (Table 1). This selectivity shows that either the adducted MT is more similar in structure to mintlactone than menthofuran or that the mintlactone-type structure is more immunogenic in rabbits. 2306



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The α-MT-DDM antiserum was analyzed by Western blot, and 10 distinct spots were identified. Only 4 of the 10 spots were amenable to identification by LC-MS/MS analysis: mitochondrial ALDH2, cytoplasmic MDH1, serum albumin, and the ATP synthase subunit d (Complex V). In this analysis, no adduct-bearing peptides were observed, and, for this reason, we complemented the Western blot analysis with enzyme activity assays. The largest decrease in activity, of about 88%, was seen in ALDH2, followed by the activity of Complex V, which decreased by about 34%. To ensure that these changes were not due to necrosis or downregulation of hepatic enzymes, a Western blot analysis was conducted for ALDH2. A low concentration of enzymes was present, but the magnitude of the result was not large enough to explain the 88% decrease in observed activity. Additional confirmation that a total downregulation of hepatic enzymes did not occur is that MDH1 activity did not change in the treated group compared to the untreated group. The exact relationship between menthofuran-derived adducts of the identified enzymes and toxicity is not known, but these enzymes may play a critical role in the hepatotoxicity of menthofuran in rats. Mitochondrial ALDH2 is one of the key enzymes involved in the oxidation and detoxification of aliphatic aldehydes.39,40 This enzyme, a homotetramer, is an NAD+-dependent enzyme with high affinity for the oxidation of acetaldehyde produced during ethanol metabolism.41 This enzyme is known to detoxicate many toxic reactive lipid aldehydes.42 ALDH2 has broad substrate specificity, and its active site cysteine (Cys-302) is highly conserved across species.43,44 This cysteine is thought to be the site of the initial nucleophilic attack to the carbonyl of the aldehyde substrate.45 A number of drugs and chemicals have been reported to covalently modify ALDH2. They include teucrin A,46 acetaminophen,47 bromobenezene,48 mycophenolic acid,49 and benzene.50 The exact mechanisms and the site of modification of the enzyme in many cases are not known. In the case of the nitric oxide donor, it is proposed that the inactivation of the enzyme is through direct modification of Cys-302.51 In the case of menthofuran-driven reactive metabolites, the exact site of modification is also not known, but it would not be unlikely that one of its reactive metabolites, γ-ketoenal, may serve as an aldehyde substrate that reacts with the nucleophilic cysteine to form a non-dissociable covalent adduct. Whatever the mechanism of inactivation of ALDH2, diminishing its activity could lead to an imbalance of NAD+/ NADH, which would disrupt oxidative phosphorylation in mitochondria.52 Such an effect has been observed for adducts of acetaminophen-reactive metabolites with ALDH2.53 The ATP synthase subunit d is another target of menthofuran reactive interemdiates. This enzyme is located in the mitochondrial inner membrane and is a multisubunit assembly of 16 different proteins.54 ATP synthase consists of two components: F1 and FO. F1 catalyzes the hydrolysis of ATP, and FO, which is embedded in the mitochondrial inner membrane, creates a channel that allows protons to cross the membrane. When these two components are brought together, they form F1-FO-ATPase, which consists of five proteins and synthesizes one ATP molecule with three protons that cross the membrane. We determined that reactive metabolites of menthofuran could have modified the subunit d of this complex, and it resulted in ATP synthase Complex V activity decreasing by 35 ± 15%. F1-FO-ATPase inhibition would be detrimental to cells, as has been shown with other compounds

and drugs,55 such as acetaminophen,13 naphthalene,56 mycophenolic acid,49 benezene,50 and teucrin A.46 It is interesting to note that there are several common target proteins between ATP synthase and ALDH2. Furan-mediated uncoupling of hepatic oxidative phosphorylation has been demonstrated previously in rats.57 In summary, four adducted proteins were identified in extracts of livers from menthofuran-treated rats. Diminished activities were seen in two of these proteins: ALDH2 and ATP synthase Complex V. It is important to note that the changes in activity may not be related to the direct toxicity of menthofuran observed in rats. Further studies need to be conducted to investigate whether the protein adducts of ALDH2 and the ATP synthase subunit d result in toxicity.

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ASSOCIATED CONTENT

S Supporting Information *

Sequence coverage of the proteins plus their mass spectrometry information, ALDH2 activity, and Western blot from in vivo samples. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: (650) 225-6094. E-mail: [email protected]. Present Address ⊥

Drug Metabolism and Pharmacokinetics, Array BioPharma, Inc., Boulder, Colorado 80301, United States.

Funding

This work was support by National Institute of Health Grants GM 25418 (to S.D.N.), Program Project Grant GM 32165, and the UW NIEHS sponsored Center for Ecogenetics and Environmental Health Grant P30ES07033. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge David Arnott and Peter Fan for the scientific discussions and Ronitte Libedinsky for her editorial support. DEDICATION This article is dedicated to the memory of Professor Sidney D. Nelson, who passed away on December 9, 2011. ABBREVIATIONS ALDH, aldehyde dehydrogenase; ALT, alanine transaminase; BSA, bovine serum albumin; CID, collision-induced dissociation; 2D-gel, two-dimensional gel electrophoresis; DDM, α,α′dimethoxydihydromenthofuran; ELISA, enzyme-linked immunosorbent assays; IPG, immobilized pH gradient gel; KLH, keyhole lympet hemocyanin; MDH, malate dehydrogenase; MT, metallothionein; α-MT-DDM, antimetallothionein-α,α′dimethoxydihydromenthofuran serum; P450, cytochrome P450; PBS, phosphate buffered saline; SDS−PAGE, sodium dodecyl sulfate−polyacrylamide gel electrophoresis; TBS, trisbuffered saline; WST-1, [2-(4-iodophenyl)-3-(4-nitrophenyl)5-(2,4-disulfophenyl)-2H-tetrazolium]

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Characterization of Rat Liver Proteins Adducted by Reactive

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