Identification of Potential Genomic Biomarkers of Hepatotoxicity

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Chem. Res. Toxicol. 2006, 19, 1270-1283

Identification of Potential Genomic Biomarkers of Hepatotoxicity Caused by Reactive Metabolites of N-Methylformamide: Application of Stable Isotope Labeled Compounds in Toxicogenomic Studies Abdul Mutlib,*,†,⊥ Ping Jiang,‡ Jim Atherton,† Leslie Obert,§ Seva Kostrubsky,§ Steven Madore,‡ and Sidney Nelson| Departments of Pharmacokinetics, Dynamics and Metabolism, DiscoVery Biomarkers Group and Safety Sciences, Pfizer Global Research and DeVelopment, Michigan Laboratories, 2800 Plymouth Road, Ann Arbor, Michigan 48105 Department of Medicinal Chemistry, School of Pharmacy, UniVersity of Washington, H364C Health Sciences Center, P.O. Box 357631, Seattle, Washington 98195-7631 ReceiVed May 3, 2006

The inability to predict if a metabolically bioactivated compound will cause toxicity in later stages of drug development or post-marketing is of serious concern. One approach for improving the predictive success of compound toxicity has been to compare the gene expression profile in preclinical models dosed with novel compounds to a gene expression database generated from compounds with known toxicity. While this guilt-by-association approach can be useful, it is often difficult to elucidate gene expression changes that may be related to the generation of reactive metabolites. In an effort to address this issue, we compared the gene expression profiles obtained from animals treated with a soft-electrophileproducing hepatotoxic compound against corresponding deuterium labeled analogues resistant to metabolic processing. Our aim was to identify a subset of potential biomarker genes for hepatotoxicity caused by soft-electrophile-producing compounds. The current study utilized a known hepatotoxic compound N-methylformamide (NMF) and its two analogues labeled with deuterium at different positions to block metabolic oxidation at the formyl (d1) and methyl (d3) moieties. Groups of mice were dosed with each compound, and their livers were harvested at different time intervals. RNA was prepared and analyzed on Affymetrix GeneChip arrays. RNA transcripts showing statistically significant changes were identified, and selected changes were confirmed using TaqMan RT-PCR. Serum clinical chemistry and histopathologic evaluations were performed on selected samples as well. The data set generated from the different groups of animals enabled us to determine which gene expression changes were attributed to the bioactivating pathway. We were able to selectively modulate the metabolism of NMF by labeling various positions of the molecule with a stable isotope, allowing us to monitor gene changes specifically due to a particular metabolic pathway. Two groups of genes were identified, which were associated with the metabolism of a certain part of the NMF molecule. The metabolic pathway leading to the production of reactive methyl isocyanate resulted in distinct expression patterns that correlated with histopathologic findings. There was a clear correlation between the expression of certain genes involved in the cell cycle/apoptosis and inflammatory pathways and the presence of reactive metabolite. These genes may serve as potential genomic biomarkers of hepatotoxicity induced by soft-electrophile-producing compounds. However, the robustness of these potential genomic biomarkers will need to be validated using other hepatotoxicants (both soft- and hard-electrophile-producing agents) and compounds known to cause idiosyncratic liver toxicity before being adopted into the drug discovery screening process. Introduction Adverse drug reactions (ADRs) are one of the most common causes for pharmaceutical product recalls as well the sixth leading cause of death in the United States (1). It has been shown that some drugs are capable of eliciting ADRs without any * Corresponding author. Tel: (484) 865-7525. E-mail: mutliba@ wyeth.com. † Department of Pharmacokinetics, Dynamics and Metabolism, Pfizer Global Research and Development. ‡ Discovery Biomarkers Group, Pfizer Global Research and Development. § Department of Safety Sciences, Pfizer Global Research and Development. | University of Washington. ⊥ Present Address: Wyeth Pharmaceuticals, Inc., S3324, P.O. Box 8299, Philadelphia, PA 19101.

metabolic conversion. However, most drugs that produce ADRs are also metabolized to reactive metabolic intermediates. The link between the bioactivation of these drugs and the ADRs has been only circumstantial in most instances. Nonetheless, there are numerous examples of drugs that cause ADRs and are also known to be bioactivated (2-6). Among many different types of ADRs, drug-induced liver disease (DILD) is one of the common causes for pharmaceutical product recalls and the cessation of late stage clinical programs. Most drug-induced hepatotoxicities are unpredictable and poorly understood. Because the liver is the principal site of metabolism, it is often the target of toxicity (7). Most drugs that elicit an ADR form reactive metabolites capable of interacting with macromolecules such as proteins (8-10). There are no simple rules to predict the target macromolecules for a particular reactive metabolite

10.1021/tx060093j CCC: $33.50 © 2006 American Chemical Society Published on Web 09/08/2006

Genomic Biomarkers of Hepatotoxicity

Chem. Res. Toxicol., Vol. 19, No. 10, 2006 1271

Figure 1. Structures of N-methylformamide (NMF) and its deuterated analogues, d1-NMF and d3-NMF, used in this study.

or the biological consequences of a particular modification. In order to better understand the potential role of reactive metabolites in causing toxicities, a number of studies involving toxicogenomic (gene expression changes) and toxicoproteomic (protein expression changes) approaches have recently been undertaken (11-20). However, one of the major challenges has been to pinpoint gene/protein expression changes solely attributable to the reactive metabolites. Hence, there is a need for a method that will differentiate gene/protein responses due to the generation of the reactive metabolites from off-target effects. The ability to modulate reactive metabolite formation by selectively replacing hydrogen with deuterium offers the opportunity to focus the search for predictive biomarker genes of hepatotoxicity. Carbon-deuterium bonds are more difficult to break than carbon-hydrogen bonds and lead to diminished metabolism at that site (21-23). It has been well documented that a stable isotope-labeled compound and its nonlabeled counterpart are similar in terms of target interaction. However, because of the diminished metabolism of the deuterated analogues, the onset and severity of various target organ toxicities attributed to reactive metabolite formation can be modulated effectively (24). Previously, deuterium analogues of compounds have been used to effectively demonstrate the involvement of reactive intermediates in causing organ toxicities (25-27). Thus, the decreased toxicities demonstrated by deuterium-labeled analogues should also show concurrent reduction in gene or protein changes compared to those from treatment with nonlabeled compounds. These differences in toxicity produced by the stable isotope and nonlabeled analogues can be used to better understand specific gene or protein changes elicited by reactive metabolites. The current study utilized a known hepatotoxic compound N-methylformamide (NMF1) and its two analogues labeled with deuterium at different positions to block metabolic oxidation at the formyl (d1) and methyl (d3) moieties (Figure 1). NMF was considered as an ideal molecule to study because of its small size (MW ) 59 amu) and the limited number of metabolites that could be formed from this compound. The metabolism of NMF and related N-alkylformamides has been adequately described in the literature (2830). NMF has been shown to be metabolized primarily by two metabolic pathways: (a) hydroxylation of the N-methyl group, leading to the formation of a fairly stable carbinolamide, N-(hydroxymethyl)formamide (pathway a, Figure 2) and (b) 1 Abbreviations: ALKP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; NMF, N-methylformamide; MIC, methyl isocyanate; ESI, electrospray ionization; LC-ESI/MS, liquid chromatography electrospray ionization mass spectrometry; MS/MS, mass spectrometry/mass spectrometry.

Figure 2. Metabolic pathways of NMF leading to the formation of N-(hydroxymethyl) formamide (pathway a) and methyl isocyanate (MIC) (pathway b). The deuterium on the formyl position is lost during the bioactivation process leading to MIC. The formation of formaldehyde and formamide from the carbinolamide, N-(hydroxymethyl) formamide, is postulated to take place via pathway a.

formation of S-linked conjugates via a reactive metabolic intermediate, methyl isocyanate (MIC) (pathway b, Figure 2). The formation of N-(hydroxymethyl)formamide has been considered as a detoxification pathway and has been shown to be a minor urinary metabolite in mice (30). However the overall contribution of this pathway to the disposition of NMF has not been fully explored. The pathway leading to MIC has been studied more extensively and has been implicated in NMFinduced liver toxicity (28, 29). Studies conducted with [14C]formyl-labeled NMF showed that at least 39% of the dose was eliminated via the lungs as 14CO2 (30). The release of CO2 from NMF could be attributed to the MIC reactive intermediate, which can either form a GSH adduct or react with water to form an unstable carbamic acid. Subsequently, the carbamic acid decomposes by releasing CO2 and methylamine, a major urinary metabolite in mice dosed with NMF (30). Alternatively, it has been postulated that the facile base-catalyzed chemical hydrolysis of GSH-related adducts could also be responsible for the formation of methylamine and CO2 (28). Direct hydrolysis of NMF to CO2 and methylamine was subsequently ruled out by observing a significant deuterium isotope effect in the formation of methylamine (28). MIC is categorized as being a reactive soft electrophile capable of reacting with various cellular components. The reactivity of the carbinolamide is not as well defined; however, it is quite possible that it could be further metabolized to formamide and formaldehyde, which is considered to be a harder electrophile, mostly affecting DNA bases (31). Hence, we hypothesized that these two metabolic pathways would differentially affect various genes and proteins. It has been demonstrated that introduction of deuterium at the formyl position (N-methylform-d1-amide or d1-NMF) leads to a significant kinetic deuterium isotope effect (28) in reducing the formation of MIC (trapped as N-acetylcysteine or glutathione adducts). Not surprisingly, studies conducted in mice showed that the administration of stable-isotope-labeled d1-NMF led to a reduction in the degree of hepatotoxicity (28). Studies conducted previously by other investigators have shown that the centrilobular hepatic toxicity caused by NMF is most likely due to the formation of MIC (28, 32). Hence, modulating the formation of MIC by introducing a deuterium atom in the molecule gives us an opportunity to decrease hepatotoxicity as well as study the gene changes brought about by the altered

1272 Chem. Res. Toxicol., Vol. 19, No. 10, 2006

drug metabolism. It is postulated that a deuterium at the formyl position would decrease MIC formation and/or shift the metabolism of NMF to hydroxylation of the N-methyl group. Hence, a comparison of gene changes in the livers of mice dosed with d0- and d1-NMF could aid us in identifying potential genomic biomarkers responsive to a reactive metabolite such as MIC. To further assist us in this process, the d3-NMF analogue was also included in this study. The d3 analogue (Figure 1) was postulated to lead to a decrease in the formation of N(hydroxymethyl)formamide as a result of a deuterium isotope effect. It was expected that d3-NMF would produce greater quantities of reactive MIC than the nonlabeled NMF because a greater fraction of the dose would be shunted through oxidation to MIC. However, the consequences of incorporating deuterium into the methyl group have not been previously examined. Therefore, in Vitro and in ViVo studies were conducted to evaluate the effect of deuterium substitution either in the methyl group or in the formyl position on the bioactivation of NMF to reactive MIC. A comparison of the gene expression changes observed in the livers of mice dosed with stable isotope-labeled and nonlabeled NMF analogues would reveal distinct expression patterns that could be attributed to differences in metabolism of these compounds. A number of steps were taken to identify gene changes as a result of incorporating deuterium atoms into the NMF molecule. The first step involved an overall assessment of gene changes induced by NMF, d1-NMF, and d3-NMF compared to those by the saline control. The signal intensities of each gene were obtained for NMF-treated and deuterated NMF-treated animals and were compared to the same saline control at each time point. A comparison of d1-NMF dosed and d0-NMF dosed animals (e.g., at 6 h) was made, and all of the genes showing a 2-fold change between the two groups were identified. Any gene showing an overall change in signal intensity in the order d0NMF > d1-NMF > saline control was considered to be upregulated, whereas the inverse (i.e., d0-NMF < d1-NMF < saline control), implied down-regulation as a result of MIC formation. Furthermore, any significant changes to gene expression in d1NMF, compared to that in d0-NMF, could be attributed to either the effect of decreased metabolism to MIC (pathway b, Figure 2) or due to metabolic switching, leading to an increased oxidation of the methyl group (pathway a, Figure 2). Next, the gene changes produced by d3-NMF were compared to those of d0-NMF, d1-NMF, and saline control groups. Preliminary data (in Vitro and in ViVo) suggested that d3-NMF did lead to a greater production of MIC than d0-NMF. If a particular gene showed changes in signal intensity in the order d3-NMF > d0-NMF > d1-NMF > saline control (up-regulated) or d3-NMF < d0-NMF < d1-NMF < saline control (down-regulated), it was more likely to be associated with pathway b (MIC formation). However, if the order was d1-NMF > d0-NMF > d3-NMF > saline control (up-regulated) or d1-NMF < d0-NMF < d3-NMF < saline control (down-regulated), that gene’s regulation was more likely to be associated with metabolic pathway a. Some of the specific gene changes observed were confirmed by real-time polymerase chain reaction (RT-PCR). Furthermore, mice were dosed with a synthetic standard of methyl isocyanate in order to confirm gene changes postulated to be due to reactive MIC formation from NMF. In addition to deciphering the liver genomic changes produced by administering NMF and its stable isotope-labeled analogues, we also monitored changes in serum enzymes indicative of liver injury (i.e., ALT and AST). In addition, a time-matched

Mutlib et al.

histopathologic evaluation of the liver samples was conducted. The intention was to demonstrate that the gene expression changes were being monitored before the onset of full-blown hepatotoxicity. In order to reduce and simplify our findings; only the 6 h time point will be discussed. The effect of the differential labeling of NMF with deuterium at various positions on metabolic pathways also was investigated both in Vitro and in ViVo. Because some of the gene changes were directly attributed to the formation of reactive intermediates, a deuterium isotope effect leading to significant metabolic switching had to be demonstrated. For example, it was postulated that having three deuteriums on the methyl group of NMF would slow the formation of the carbinolamide metabolite favoring the alternate metabolic pathway of MIC formation (Figure 2). Hence, some of the gene changes in the livers of d3-NMF-dosed animals compared to those in d0-NMF-dosed animals could be attributed directly to MIC formation. Finally, we demonstrated corroboration between gene changes, serum biomarker enzyme levels, and histopathology data. At the 6 h time point, there were minimal histopathologic findings, whereas significant alterations in the expression of potential biomarker genes was evident. However, before these genes can be utilized as potential biomarkers of liver injury, further studies will need to demonstrate that these genes are similarly affected by other compounds that form reactive electrophilic metabolites.

Materials and Methods Chemicals and Supplies. Control mice liver microsomes were obtained from Xenotech (Kansas City, KS). NMF, NADPH, MgCl2, and phosphate buffer (pH 7.4) were purchased from Sigma-Aldrich (St. Louis, MO). Methyl isocyanate was obtained from Supelco (Bellefonte, PA). The isotopically labeled d1-NMF and d3-NMF were obtained from CDN Isotopes (Pointe-Claire, Quebec, Canada). HPLC-grade water, methanol and acetonitrile were purchased from Mallinckrodt Chemicals (Phillipsburg, NJ). All general solvents and reagents were of the highest grade commercially available. Expression Profiling. Total RNA was isolated from mouse livers (3 animals per time point) using RNeasy Mini Kits from Qiagen. Expression profiling experiments were performed using an Affymetrix Standard Labeling Procedure. Briefly, 5 µg of RNA was primed with an oligo dT24 primer and converted into double stranded cDNA using the Invitrogen Superscript III cDNA synthesis kit. Affymetrix Gene Chip Expression 3′ Amplification Reagents were used for the in Vitro transcription of cDNA into biotin-labeled cRNA. The Affymetrix clean up module was used for the purification of cDNA and labeled cRNA. The quality and quantity of the labeled cRNA was assessed, and 15 µg of cRNA was fragmented by a metal-induced hydrolysis at 94 °C. The fragmented, labeled cRNA was mixed with a cocktail containing hybridization controls and hybridized to Affymetrix Mouse MOE430 2.0 arrays for 16 h at 45 °C, rotating at 60 rpm. Following hybridization, the arrays were placed into the Affymetrix Gene Chip Fluidics Station 450, where they were washed and stained with streptavidin phycoerythrin and an anti-streptavidin biotinlylated antibody. Arrays were then scanned on the Affymetrix Gene Chip Scanner 3000 and tested for quality control parameters using Affymetrix GeneChip Operating Software (GCOS 1.2). Statistical Data Analysis. Raw data files, including signal intensity values and present/absent calls, were generated using the Affymetrix GeneChip Operating Software (GCOS 1.2) statistical algorithm with default settings. A target intensity of 600 was used for global scaling each array. Analysis of variance (ANOVA) was used to measure the similarity for those groups having three or more animal replicates. Outliers were identified and excluded from further analysis. A correlation coefficient was used to measure the similarity between duplicates. Genes were identified as changing significantly according to the following criteria: (a) t-test p value e 0.05; (b) fold changes g2

Genomic Biomarkers of Hepatotoxicity and e2; (c) mean signal intensity g100 (arbitrary value) for either control or treated group; and (d) at least 50% of the samples in either control or treated group called Present by the Affymetrix algorithm. Quantitative PCR. TaqMan Assay-On-Demand Gene Expression reagents for the detection of ccl3, ccl4, cd83, pdk2, pdk4, sgk, dusp10, tnfaip6, kif2c, ndr1, hspb1, fosl1, tgm1, ly96, and rgs1 mRNAs were obtained from Applied BioSystems. cDNA was synthesized from 500 ng of RNA using the High Capacity cDNA Archive Kit (Applied BioSystems) and real-time PCR reactions were run using the ABI Prism 7900HT Sequence Detection System. All samples were run in triplicate in 10 µL reaction volumes using the TaqMan Universal PCR Master Mix without AmpErase UNG (Applied BioSystems), and the level of GAPDH mRNA in each sample was used as the control for normalization. The ∆∆Ct method (see Applied Biosystem technical manual) was used to calculate fold changes. Studies in ViWo. Male mice (BALB/c) weighing between 1520 g were obtained from Charles River. All animal husbandry procedures were in accordance with the Guide for the Care and Use of Laboratory Animals (NIH Publication, Volume 25, 1996, http://grants1.nih.gov/grants/guide/notice-files/not96-208.html), and the Institutional Animal Care and Use Committees (IACUC) of the facilities in which the studies were conducted approved all experimental procedures. All animals were housed in suspended, stainless steel, and wire-mesh cages equipped with an automatic watering system. The study room was environmentally controlled for temperature (72 ( 4 °F), relative humidity (40-70%), and light (a 12 h light/dark cycle). Mice had free access to water and were given a specific amount of certified Purina rodent chow each day. The dosing solution was prepared in 0.9% saline at 30 mg/mL for each compound. The dosing volume used in this study was 10 mL/ kg. Groups of mice (4 animals/group) were dosed ip with deuterated (at d1 or d3 positions) and nondeuterated N-methyl formamide at 300 mg/kg. Groups of mice (n ) 4) were also dosed with 0.9% saline and sacrificed at various time points to obtain control serum and liver samples. Urine samples were collected from mice (24 h groups) housed in metabolism cages. Animals dosed with either saline, d0-NMF, d1-NMF, or d3-NMF were sacrificed at 1.5, 6, and 24 h postdosing. Blood samples, collected by cardiac puncture at the time of sacrifice, were transferred to serum separator microtubes (Sarstedt, Germany) and centrifuged at 10 000g for 5 min. The serum samples were stored frozen at -4 °C until analyzed. After excision, livers were examined for gross pathologic changes and representative samples fixed in 10% neutral buffered formalin. Four to six sections from each animal were examined microscopically, and histopathologic changes were recorded. The remaining liver samples were snap frozen with liquid nitrogen and stored at -80 °C for genomic analysis. In another study, male BALB/c mice weighing between 15-20 g were dosed with either a 1:1 (w/w) mixture of d0/d3-NMF or d1/d3-NMF at 300 mg/kg (ip) and housed in groups of 5 in metabolism cages. Urine (0-24 h) from three separate groups of mice (n ) 5) per treatment group were collected over ice. The 1:1 composition of the substrate mixtures was confirmed by LC/MS analysis prior to dosing the animals. Aliquots (10 µL) of urine, pooled from 5 animals per group, were injected onto LC/MS to determine the ratio of N-acetylcysteine (NAC) conjugates derived from non-deuterated and deuterated NMFs. The average of ratios from three groups of mice per treatment was obtained. To study the gene changes produced by methyl isocyanate, three groups of male BALB/c mice (3 animals/group) weighing between 15-20 g were dosed with either saline or MIC at 5 mg/kg or 10 mg/kg. Appropriate safety precautions were taken to minimize human exposure to MIC during the dose preparation and administration. The animals were sacrificed at 6 h and the livers removed and analyzed for gene changes as described above. Histopathology. Liver histopathology was conducted at t ) 0, 1.5, 6, and 24 h only on surviving animals. Formalin-fixed samples were embedded in paraffin wax, and 3 µm sections were cut and stained with hematoxylin and eosin (H&E). Routine periodic acid

Chem. Res. Toxicol., Vol. 19, No. 10, 2006 1273 Schiff (PAS) special staining was performed to label hepatocellular glycogen content. In addition, adipophilin immunohistochemistry was performed to characterize potential hepatocellular vacuolation as lipid accumulation. Immunohistochemical staining was executed on a Discovery automated stainer (Ventana Medical Systems, Tucson, AZ) using CC1 Antigen Retrieval, a heat protocol, the endogenous biotin blocking kit, and the basic DAB detection kit. The adipophilin primary antibody (Progen, Immuno-Diagnostika, Heidelberg, Germany) was applied at a dilution of 1:1000 for 30 min followed by an anti-guinea pig biotinylated secondary antibody (Vector Labs-Burlingame, CA) at a dilution of 1:1000 for 30 min. Serum Chemistry. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALKP) levels were determined by Johnson and Johnson Vitros 250 or 950 Analyzers according to standard protocols. Studies in Vitro to Assess the Deuterium Isotope Effect on Metabolism of NMF. Incubations were conducted with 1:1 (w/w) mixtures of either d0-NMF/d3-NMF or d1-NMF/d3-NMF (1 mM) with naı¨ve mice liver microsomes (1 mg) fortified with NADPH (2 mM), MgCl2 (3 mM) and N-acetylcysteine (2 mM) in a total incubation volume of 1 mL made up with 0.1 M phosphate buffer (pH 7.4). The 1:1 composition of the substrate mixtures was confirmed by LC/MS prior to the microsomal incubations. The incubations were performed at 37 °C in a water bath for 30 min. At the end of the reaction, 2 mL of cold acetonitrile was added to precipitate the protein. The samples were vortexed and centrifuged at 3000g for 5 min before transferring the supernatants to clean borosilicate glass tubes. The organic phase was removed under a stream of nitrogen at 30 °C, and an aliquot (30 µL) of the remaining aqueous phase was injected onto the LC/MS. Synthesis of S-(N-Methylcarbamoyl)-N-acetylcysteine Conjugate. The synthesis of S-(N-methylcarbamoyl)-N-acetylcysteine was performed using a previously described procedure (33). Briefly, N-acetylcysteine (0.72 g, 3 mmol) was dissolved in methanol (10 mL), and methyl isocyanate (0.50 g, 8.8 mmol) was added slowly with stirring. The mixture was stirred at ambient temperature overnight. Following the removal of the solvent under a stream of nitrogen, the crude product was purified on a 10 g/60 cm3 C18 solid-phase extraction cartridge (Varian Sample Products, Freemont, CA). The crude mixture was dissolved in 0.1 M HCl and loaded on the C18 cartridge. After allowing the sample to adsorb on the C18 under gravity, the cartridge was washed with 30 mL of 0.1 M HCl. Subsequently, the metabolite was eluted from the cartridge using methanol/0.1 M HCl mixture (5:95 v/v). The eluent was dried under vacuum, and approximately 5 mg of the sample was submitted for NMR and LC/MS analysis. LC-ESI/MS showed [M + H]+ at m/z 221. 1H NMR: δ 1.87 (3H, s, CH3CO), 2.50 (3H, s, NHCH3), 2.75 (1H, dd, CH-CH2), 3.07 (1H, dd, CH-CH2), and 4.32 (1H, dd, CH-CH2). The coupling between the cysteine protons were confirmed by a 2D-COSY experiment. Liquid Chromatography Mass Spectrometry (LC/MS) Analysis of NMF Metabolites. Injections of urine samples or reconstituted microsomal incubation extracts were made directly onto an HPLC column (Synergi 4µ Hydro-RP, 150 × 2.0 mm, Phenomenex) coupled to a LCQ ion trap mass spectrometer (ThermoFinnigan) equipped with a Z-spray ion source. The capillary temperature was held at 350 °C. The electrospray needle was maintained at 4600 V. Ultrapure nitrogen was used as the sheath and auxiliary gas with arbitrary settings of 65 and 15, respectively. The capillary, tube lens offset, entrance lens, and trap DC voltages were set at 26, 26, -29, and -10 V, respectively. The mass spectrometer was operated in the positive ion LC/MS/MS mode. The mass transitions for the metabolites were 221 f 164 (N-acetylcysteine (NAC) conjugate from either d0-NMF or d1-NMF) and 224 f 164 (NAC from d3-NMF). The peak areas from each of these transitions were obtained, and the ratio of the NAC conjugates produced by each NMF analogue was calculated. The metabolites were separated on the HPLC column using a gradient solvent system consisting of acetonitrile and 10 mM ammonium acetate (pH 3.5) with the flow rate set at 0.23 mL/min. The initial conditions consisted of a mixture of acetonitrile and ammonium acetate (2:98 v/v). The percentage


Mutlib et al.

Table 1. Ratios of NAC Conjugates Produced in Witro and in WiWo by 1:1 Mixtures (mol/mol) of d0-NMF/d3-NMF and d1-NMF/d3-NMF peak area ratios of NAC conjugates Vitroa

in in ViVob

estimation of kH/kD

d0/d3

d1/d3

d0/d3:d1/d3

0.91 ( 0.05 0.48 ( 0.09

0.19 ( 0.02 0.10 ( 0.05

4.79 4.80

a Microsomal incubations conducted in triplicate as described in Materials and Methods. b Urine from three separate groups of mice (n ) 5) per treatment group were analyzed by LC/MS, and average ratios were obtained.

of acetonitrile was maintained at 2% for the first 4 min and, subsequently, increased linearly to 80% over the next 6 min. After an additional 5 min at 80% acetonitrile, the column was reequilibrated with the initial mobile phase for 10 min before the next injection.

Results Studies in Vitro. The metabolism in Vitro of NMF and its deuterated analogues were conducted to confirm an isotope effect on reactive metabolite formation. The metabolism of NMF was assessed by LC/MS analysis of MIC trapped as its N-acetylcysteine (NAC) conjugate. The LC/MS peak area ratios of the NAC conjugates produced by 1:1 mixtures of d0-NMF/ d3-NMF and d1-NMF/d3-NMF are shown in Table 1. A significant isotope effect was observed for the formation of MIC when the formyl proton was replaced with deuterium. Because d0-NMF and d1-NMF produce the same NAC conjugate with the same molecular weight (loss of deuterium during metabolism), it was impossible to calculate the kH/kD by conducting studies with a 1:1 mixture of d0-NMF/d1-NMF. Therefore, an apparent kinetic isotope effect (kH/kD) associated with the metabolic process leading to the NAC conjugate of MIC was indirectly estimated from the ratios of the peak areas shown in Table 1. The kinetic isotope effect, upon incorporating deuterium at the formyl position, was estimated to be kH/kD ) 4.8 from the in Vitro studies. The overall effect of placing deuterium at the formyl position on metabolic pathway a (carbinolamide formation) could not be assessed because there were no analytical methods to reliably quantitate the carbinolamide or its breakdown products. It was observed that d3-NMF produces a slightly greater amount of the NAC conjugate than the nonlabeled NMF (Table 1), indicating a relatively small isotope effect in the formation of MIC from d3-NMF. Studies in ViWo. Primary Kinetic Isotope Effect and Metabolic Switching. Mice dosed with a 1:1 mixture of d1/ d3-NMF showed an isotope effect similar to that obtained from in Vitro studies. The substitution of the formyl proton with deuterium appears to reduce bioactivation pathway b (MIC formation) of NMF quite significantly in ViVo. A considerable isotope effect in forming NAC from d1-NMF was observed (Table 1). Because metabolic pathway b is a significant route of metabolism for NMF in ViVo (28, 30), it is assumed that a significant metabolic switching to carbinolamide formation could occur with d1-NMF. An analysis of urine samples from mice dosed with d0/d3NMF showed higher levels of d3-NAC than d0-NAC (Figure 3). As shown in Table 1, the NAC produced from the nonlabeled NMF was less (approximately 50%) than that produced by d3NMF. The results from the in ViVo studies are consistent with the expected higher levels of MIC produced from d3-NMF as a result of metabolic shunting. As stated earlier, it was impossible to obtain kH/kD values by administering the animals a 1:1 mixture of d0/d1-NMF. Instead, an indirect approach was used, whereby the ratios of NAC conjugates produced by d0/d3-NMF and d1/

Figure 3. Total ion currents (TIC) of the NAC conjugates present in the urine of mice dosed with (A) d0/d3-NMF and (B) d1/d3-NMF at 300 mg/kg (ip). The peak areas (listed on the chromatograms adjacent to the peaks) of the N-acetylcysteine conjugates produced from d0-, d1-, and d3-NMF were used to calculate the ratios, d0/d3 and d1/d3 (listed in Table 1) for assessing the in ViVo deuterium isotope effect. Table 2. AST, ALT, and ALKP Serum Activities in U/L after Administering Mice with Saline, d0-NMF (300 mg/kg), d1-NMF (300 mg/kg), or d3-NMF (300 mg/kg)a control (saline)

NMF

d1-NMF

d3-NMF

1.5 h 6h 24 h

84 ( 8 120 ( 25 99 ( 15

AST 86 ( 27 393 ( 165b 13589 ( 17668b

74 ( 6 169 ( 45 193 ( 44b

109 ( 20 432 ( 319b 148 ( 72

1.5 h 6h 24 h

48 ( 4 46 ( 6 56 ( 1

ALT 74 ( 37 202 ( 63b 14112 ( 17470b

49 ( 4 84 ( 16c 215 ( 96b

61 ( 8c 199 ( 136b 130 ( 100

1.5 h 6h 24 h

180 ( 12 212 ( 16 206 ( 23

ALKP 221 ( 11b 216 ( 19 284 ( 120

197 ( 3 217 ( 26 183 ( 23

206 ( 19b 242 ( 16 185 ( 16

a For each group, the values are expressed as the mean (n ) 4) ( standard deviation. b The mean value is significantly different from the saline control mean at the 1% level by pairwise comparison within one-factor analysis of variance. c The mean value is significantly different from the saline control mean at the 5% level by pairwise comparison within one-factor analysis of variance.

Table 3. Principal Liver Histopathology Findings in BALB/c Mice Dosed Intraperitoneally with d0-NMF, d1-NMF, and d3-NMF. time postdose compound necrosis, hepatocellular single cell necrosis, hepatocyte vacuolation (lipid), hepatocellular depletion, glycogen congestion sinusoidal a

6h 6h 6h 24 h 24 h 24 h d0- NMF d1-NMF d3-NMF d0- NMF d1-NMF d3-NMF 1/4+a

0/4

0/4

4/4+++

0/4

2/4+

3/4+

0/4

2/4+

4/4+++

4/4++

2/4+

0/4

4/4+

0/4

4/4++

4/4+

1/4+

4/4+++

4/4+

4/4+++

4/4++

4/4++

4/4++

1/4+

0/4

0/4

3/4++

0/4

1/4+

Averaged severity score: +, minimal; ++, mild; +++, moderate.

d3-NMF were used to calculate this value (approximately 4.8) (see Table 1). This value obtained for the kinetic isotope effect due to incorporation of deuterium at the formyl position is consistent with previous findings by other investigators (28).



Figure 4. (A) Twenty-four hour saline control, normal liver, H&E stain, 10× magnification; (B) 6-h NMF, central lobular hepatocellular necrosis (arrows) with mixed cell infiltrates, hemorrhage, H&E stain, 10× magnification; (C) 6-h d3-NMF, rare central lobular hepatocyte single cell necrosis (arrows), H&E stain, 20× magnification; (D) 24-h NMF, central lobular hepatocellular glycogen depletion (arrows) indicated by a lack of PAS dark pink staining, 10× magnification; (E) 24-h NMF, central lobular hepatocellular lipid accumulation (arrows) indicated by increased adipophilin immunohistochemical staining (dark brown), 10× magnification; (F) 24 h-NMF, acute central lobular to mid-zonal hepatocellular necrosis (arrow) with sinusoidal congestion, H&E stain, 10× magnification; (G) 24-h d1-NMF, rare central lobular hepatocyte single cell necrosis (arrows), H&E stain, 20× magnification; (H) 24-h d3-NMF, central lobular hepatocellular necrosis with mixed cell infiltrates (arrows), H&E stain, 10× magnification.

The peak area ratios of the NAC conjugate produced from d0/d3- and d1/d3-NMF dosed mice were lower than those produced from the in Vitro studies (Table 1). Studies in Mice to Evaluate Clinical Chemistry, Histology, and Genomic Changes. 1. Clinical Chemistry and Histopathology. The changes in the serum enzyme activities after dosing mice with saline, NMF, d1-NMF, and d3-NMF are shown in Table 2. The principal liver histopathology findings are summarized in Table 3. At 1.5 h postdosing, there were no significant clinical chemistry or histopathology findings. Overall,

the treatment of mice with d1-NMF produced less significant changes in liver enzyme levels and in liver histopathology than nonlabeled NMF. 6 h Postdosing. NMF- and d3-NMF-treated mice had similar liver enzyme level changes and histopathology findings, whereas d1-NMF treated mice exhibited far less toxic clinical pathology and histopathology responses. None of the 6 h d1-NMF treated mice had evidence of necrosis. However, one mouse dosed with d0-NMF had minimal acute hepatocellular necrosis (Figure 4B), and the other three d0-NMF-treated mice and two d3-NMF


Mutlib et al.

Table 4. Summary of the Number of Genes That Showed Significant Up- or Down-Regulation in the Presence of d0-NMF, d1-NMF, and d3-NMF comparisons

up-regulated

down-regulated

total

d0-NMF vs control d1-NMF vs control d3-NMF vs control

659 1062 482

529 732 665

1188 1794 1147

treated mice exhibited minimal single cell necrosis (Figure 4C). Mild to moderate centrilobular depletion of cytoplasmic glycogen stores was evident as the loss of dark pink PAS staining within hepatocytes of all mice dosed with NMF and d3-NMF (Figure 4D). In contrast, only minimal glycogen depletion was evident in mice dosed with d1-NMF. However, hepatocellular vacuolation of central lobular to mid-zonal hepatocytes characterized as intracellular lipid accumulation via adipophilin immunohistochemistry was only present in mice dosed with d1NMF (Figure 4E). 24 h Postdosing. Nonlabeled NMF produced more drastic changes in AST and ALT levels than d1-NMF. Histologically, all mice dosed with NMF had mild-to-severe acute hepatocellular necrosis of central lobular to mid-zonal hepatocytes, single cell necrosis in the non-necrotic central lobular regions, and sinusoidal congestion (blood pooling) in the necrotic areas, often paralleling severity (Figure 4F). However, only minimal to mild single cell necrosis was present in d1-NMF-treated mice (Figure 4G). Toxic changes in the d3-NMF-treated mice were intermediate to those of the other compounds. Only two d3-NMF-treated mice exhibited minimal hepatocellular necrosis, minimal-to-mild single cell necrosis, and minimal liver enzyme elevations. (Figure 4H). In addition, clinical chemistry and histopathology data suggested a delayed toxic response for d1-NMF treated mice such that liver enzyme elevations (AST and ALT) were greater at the 24 h time point compared to the 6 h values. Minimalto-mild single cell necrosis was present in the centrilobular regions in all d1-treated mice at the 24 h time point (Figure 4G), whereas none of the 6 h d1-treated mice had evidence of necrosis. Minimal-to-mild and mild-to-moderate centrilobular depletions of cytoplasmic glycogen stores were evident in NMFand d3-NMF-treated mice, respectively, whereas, mild glycogen depletion was present in mice dosed with d1-NMF. Hepatocellular vacuolation (lipid accumulation) was observed minimally to moderately in all mice dosed with NMF and d1-NMF and only minimally in one mouse dosed with d3-NMF. 2. Genomic Changes. The genomic changes produced by NMF and its deuterated analogues were assessed using the GeneChip Mouse Genome 430A 2.0 Array that contained over 22 600 probe sets to analyze over 14 000 well-characterized mouse genes. Identification of differentially expressed genes in response to d0-NMF, d1-NMF, and d3-NMF compared to control (saline) was performed. NMF and its deuterated analogues induced changes to a significant number of genes. A summary of the number of genes that were up- or downregulated by NMF and its deuterated analogues are shown in Table 4. Because of the large number of genes affected by these compounds, we limited our further studies to genes that were affected more than 2-fold at the 6 h time point, a time at which the clinical chemistry (Table 2) and histology (Table 3) data showed evidence of on-set of hepatotoxicity. Table 5 lists those genes whose signal intensities changed the greatest after treatment with d3-NMF and significantly less after d1-NMF compared to saline controls. Changes caused by d0-NMF were generally intermediate, though for several genes, the changes were similar to those caused by d3-NMF. Therefore, Table 6 lists genes whose signal intensities changed the most

Table 5. Group 1 Genes Showing the Order d3-NMF > d0-NMF > d1-NMF of Fold Change in Signal Intensities Compared to That of the Saline Controla gene symbol

d1-NMF d0-NMF d3-NMF

Down-Regulated Genes thyroid hormone responsive Thrsp -3.9 SPOT14 homologue nuclear distribution Nde1 -3.7 gene E homologue 1 sterol regulatory element Srebf1 -3.6 binding factor 1 lipase, endothelial Lipg -3.6 annexin A6 Anxa6 -3.3 epidermal growth factor receptor Egfr -2.8 zinc finger protein 467 Zfp467 -2.8 transmembrane protein 7 Tmem7 -2.7 adenosine monophosphate Ampd2 -2.6 deaminase 2 (isoform L) BetaGlcNAc beta 1,3B3galt3 -2.6 galactosyltransferase, polypeptide 3 potassium channel, Kcnk5 -2.5 subfamily K, member 5 interleukin 1 receptor Il1rap -2.4 accessory protein serum deprivation response Sdpr -2.4 ankyrin repeat domain 10 Ankrd10 -2.4 fibronectin 1 Fn1 -2.3 cyclin D1 Ccnd1 -2.2 inositol polyphosphateInpp1 -2.2 1-phosphatase dual specificity phosphatase 7 Dusp7 -2.1 baculoviral IAP Birc2 -2.1 repeat-containing 2 solute carrier family 25 Slc25a19 -2.0 (mitochondrial deoxynucleotide carrier) Up-Regulated Genes hyaluronic acid Habp2 binding protein 2 UDP-glucose Ugp2 pyrophosphorylase 2 fibroblast growth Fin15 factor inducible 15 ubiquitin specific protease 9, Usp9x X chromosome solute carrier family 39 Slc39a8 (metal ion transporter), member 8 UDP-glucose Ugp2 pyrophosphorylase 2 GABA(A) receptor-associated Gabarapl1 protein-like 1 GrpE-like 2, mitochondrial Grpel2 angiopoietin-like 4 Angptl4 UDP-glucose pyrophosphorylase 2 Ugp2 nuclear receptor subfamily 1, Nr1d2 group D, member 2 serum/glucocorticoid Sgk2 regulated kinase 2 dual specificity phosphatase 6 Dusp6 DNA segment, Chr 17, D17Ertd8 ERATO Doi 808, expressed chemokine (C-C motif)ligand 3 Ccl3 serum glucocoticoid regulated kinase Sgk

-8.7

-13.3

-3.9

-4.0

-5.6

-5.8

-6.2 -4.7 -3.5 -3.1 -4.4 -8.1

-10.4 -7.4 -5.4 -3.3 -6.1 -10.9

-5.5

-7.1

-4.8

-10.1

-4.0

-4.8

-3.1 -4.7 -4.2 -3.8 -2.5

-4.6 -7.0 -6.1 -5.1 -4.3

-2.6 -2.9

-3.3 -4.0

-2.8

-3.1

2.0

2.3

2.9

2.0

2.5

4.4

2.1

2.6

3.2

2.1

2.6

3.3

2.1

2.4

3.1

2.2

2.6

4.5

2.3

2.9

3.2

2.3 2.4 2.5 2.8

3.3 3.9 3.1 3.1

3.9 4.4 4.5 3.4

3.1

3.6

3.7

4.6 5.1

6.6 9.7

9.0 10.2

3.4**b 1.5**b

9.7**b 9.3

12.1**b 13.8

aNegative numbers indicate down-regulated genes and positive numbers, up-regulated genes. Pathway b (MIC formation) for the metabolism of NMF most likely produced these gene changes. b The P values for each comparison were less than 0.05 unless otherwise stated. * 0.05 < p < 0.1; ** p > 0.1.

after treatment with d0-NMF and least by d1-NMF. A graphic representation of some of the genes from Table 5 is shown in Figure 5. On the basis of the pattern of gene signal intensities (d3-NMF > d0-NMF > d1-NMF), it appears that metabolic pathway b (Figure 2) most likely effected these gene changes. Genes showing the following order of fold change in signal intensities compared to saline control, d1-NMF > d0-NMF > d3-NMF, are listed in Table 7. A graphic representation of some of the genes from Table 7 is shown in Figure 6. On the basis of



Table 6. Group 1 Genes Showing the Order d0-NMF > d3-NMF > d1-NMF of Fold Change in Signal Intensities Compared to that of the Saline Controla gene symbol

d1-NMF d0-NMF d3-NMF

Down-Regulated CCR4 carbon catabolite Ccrn4l -5.0 repression 4-like (S. cereVisiae) cytokine inducible Cish -1.2**b SH2-containing protein F-box and leucine-rich Fbxl10 -2.9 repeat protein 10 histocompatibility 2, class II, H2-DMb2 -2.4 locus Mb2 transducer of ErbB-2.1 Tob1 -2.4 Interleukin 15 receptor, Il15ra -2.2 alpha chain heterogeneous nuclear Hnrpa2b1 -1.1 ribonucleoprotein A cyclin D2 Ccnd2 -1.1**b growth arrest specific 2 Gas2 -2.0 cell division cycle 45 Calsyntenin 2 DnaJ (Hsp40) homologue, subfamily A, member 1 aldehyde oxidase 1 calcyclin binding protein sirtuin 1 heat shock protein 1 (chaperonin) zinc finger protein X-linked DnaJ (Hsp40) homologue, subfamily A, member 1 serine/arginine rich protein specific kinase topoisomerase 1 binding c-fos induced growth factor Connective tissue growth factor lipin 2 solute carrier family 38, member 2 growth differentiation factor 15 high mobility group box transcription factor 1 growth differentiation factor 9 heat shock protein 8 bromodomain containing 2 heat shock protein 105 polo-like kinase 3 FBJ osteosarcoma oncogene DnaJ (Hsp40) homologue, subfamily B, member 1 fibroblast growth factor 21

Up-Regulated Cdc45 Clstn2 Dnaja1

-12.1

-11.3

-7.7

-5.9

-4.2

-3.7

-3.5

-3.3

-4.1 -3.9

-2.6 -2.9

-2.7

-1.7

-2.7 -2.5

-2.0 -2.3

1.5**b 1.2**b 2.2

3.2 3.4 3.2

2.9 2.2*b 2.4

Aox1 Cacybp Sirt1 Hspd1

2.2 2.2 2.2 2.3

3.8 2.6 3.6 3.5

3.1 2.4 2.8 2.6

Zfx Dnaja1

2.3 2.4

3.7 3.2

2.6 2.6

Srpk2

1.2**b

2.5

1.5**b

Topors Figf

1.6*b 2.7

3.8 4.1

2.3 3.4

Ctgf

1.2**b

4.3

2.6

Lpin2 Slc38a2

2.7 2.1*b

3.9 4.5

3.7 2.7

Gdf15

3.3

6.7

6.2

Hbp1

4.4

5.8

5.8

Gdf9 Hspa8 Brd2 Hsp105 Plk3 Fos Dnajb1

1.8**b 4.6 7.3 8.8 3.1*b 15.8 22.9

9.0 10.6 14.1 16.2 29.5 55.4 44.6

2.0 10.2 8.1 10.4 18.2 18.0 24.7

Fgf21

20.5*b

92.5

25.7

a

Negative numbers indicate down-regulated genes and positive numbers, up-regulated genes. Pathway b (MIC formation) for the metabolism of NMF most likely produced these gene changes. b The P values for each comparison were less than 0.05 unless otherwise stated. * 0.05 < p < 0.1; ** p > 0.1.

the pattern of gene signal intensities (d1-NMF > d0-NMF > d3-NMF), it appears that pathway a (Figure 2), involved in the metabolism of NMF, most likely produced these gene changes. Some of the genes that showed significant changes in the d0-NMF- versus d1-NMF-dosed groups were classified into various categories and are shown in Table 8. A significant number of genes involved in metabolism, transcription, immune response, and cell cycle apoptosis were identified by this comparison (Table 8). To further confirm the findings, RT-PCR was conducted to evaluate the relative levels of some of the genes affected in the

NMF-, d1-NMF-, and d3-NMF-dosed animals. The RT-PCR Taqman quantitative results for selected genes are shown in Figures 7 and 8. The Taqman PCR expression of Group 2 genes (associated with pathway a (Figure 2)) in various treatment groups at 6 h are shown in Figure 7. These results confirmed the microarray findings that at 6 h, the following Group 2 genes displayed a relative order of gene signal intensities: d1 > d0 > d3: tgm1, dusp 10, fosl 1, hspb1, ly96, ndr1, pdk4, and tnfaip6 (Figure 7). Similarly, the Group 1 genes, more associated with the formation of MIC, from the RT-PCR studies are shown in Figure 8. The genes that showed relative intensities in the order d3 > d0 > d1, included ccl3, ccl4, kif2c, rgs1, sgk, and cd83 (Figure 8). To confirm that the Group 1 genes were indeed a consequence of MIC formation (via pathway b, Figure 2), mice were dosed with MIC, and RT-PCR analysis was performed on harvested liver samples. The Taqman PCR results shown in Figure 9 clearly demonstrate a link between MIC formation and the Group 1 genes. However, an analysis of the Group 2 genes (including dusp10, hspb1, ly96, pdk4, and tgm1) in the same experiment showed that these were not affected at all by MIC (data not shown).

Discussion Metabolism studies in Vitro and in ViVo with d0-NMF and its deuterated analogues confirmed that the incorporation of deuterium had a significant effect on the metabolic pathways shown in Figure 2. The formation of MIC, trapped and detected as the NAC conjugate, was specifically studied to assess the effect of incorporating deuteriums in the NMF molecule. A significant isotope effect was observed when the formyl proton was replaced with deuterium (Table 1). This substitution of a single hydrogen with a deuterium led to a considerable decrease in the formation of MIC as evidenced by the reduced levels of NAC produced both in Vitro and in ViVo (Table 1). The apparent kinetic isotope effect (kH/kD ∼ 4.5-5.0) associated with the metabolic process leading to the NAC conjugate is consistent with that reported before in the literature (28). There was no significant difference in the levels of NAC produced by d0- and d3-NMF in the presence of mice liver microsomes. However, in ViVo studies showed that d3-NMF produced higher levels of MIC (trapped as the NAC conjugate) than d0-NMF (Table 1). Similarly, lower peak area ratios were obtained from in ViVo studies conducted with d1/d3-NMF compared to the in Vitro results suggesting a more pronounced isotope effect in ViVo. This apparent discrepancy (in the deuterium isotope effect) between in Vitro and in ViVo results could be due to a number of factors (34). One likely reason is that the in Vitro experiments were not run at concentrations of NMF relevant to the in ViVo situation (34). It is very likely that the NMF concentrations achieved in Vitro were saturating for the P450 enzyme(s) responsible for its metabolism, whereas the same may not have been true for the in ViVo studies. Nonetheless, the ratios obtained from the in ViVo study were more relevant to interpreting the data obtained from gene analysis conducted on the liver samples of mice dosed with NMF and its deuterated analogues. It has been previously shown that metabolic pathway a, leading to the formation of carbinolamide, is an important route of disposition for NMF in mice (28, 30). Hence, incorporating deuteriums on the methyl group could significantly affect the in ViVo formation of metabolites via the two metabolic pathways (Figure 2). However, it should be emphasized that the relative contributions of metabolic pathways a and b in metabolizing NMF have not been fully elucidated at the dose (300 mg/kg)


Mutlib et al.

Figure 5. Selected genes showing the following order of fold change in signal intensities compared to those of the saline control: d3-NMF > d0-NMF > d1-NMF. Pathway b for the metabolism of NMF most likely produced these gene changes. Genes shown displayed p < 0.05 when comparisons were made with saline treated samples. Two sample t-test assuming equal variance was used to calculate the p value.

used in this study. However, on the basis of previous results from studies conducted with 14C-NMF (dosed at 400 mg/kg) (30), it is expected that the pathway leading to MIC (pathway b) is still the major route of metabolism for NMF administered at 300 mg/kg. These significant deuterium isotope effects on the metabolism of NMF was used as a basis to understand some of the gene changes triggered by the formation of metabolites via the two metabolic pathways shown in Figure 2. Male mice dosed with d0-NMF showed more extensive liver histopathologic changes than those dosed with deuterated NMF indicating that the deuteration of NMF resulted in an attenuation of NMF-induced hepatotoxicity. In addition, mice dosed with d3-NMF exhibited more liver histopathologic changes than those dosed with d1-NMF, suggesting that the location of deuterium on a molecule can modulate the degree of attenuation of NMFinduced hepatotoxicity. Also, animals dosed with deuterated NMF and sacrificed at the 24-h time point often had more severe liver histopathologic findings than those sacrificed at the 6-h time point. The delay in the onset of toxicity with d1-NMF was very obvious, where the animals dosed with d1-NMF exhibited greater degree of liver toxicity at 24 h than those sacrificed at 6 h. A comparison of the serum enzyme levels, especially AST and ALT, at 6 and 24 h (Table 2) corroborated the histopathologic findings. The results from the d1-NMF dosed group supports the notion that deuterium at the formyl position slowed the onset and decreased the severity of liver toxicity. The clinical chemistry data corroborated with the histopathologic findings for each sample. The purpose of assessing the clinical chemistry and histopathology in parallel with genomic analysis was to ensure that we picked an appropriate time point to evaluate gene changes in the absence of gross toxicity. The 6 h time point was found to be suitable because we found evidence of minimal hepatocellular injury by d0-NMF and d3-NMF. d1-NMF, however, showed much reduced toxicity at this time point (Tables 2 and 3). This was ideal because we wanted to assess gene changes prior to the onset of gross hepatocellular toxicity. We

focused on gene changes at 6 h because the onset of overt hepatic toxicity caused by d0-NMF was delayed until 24 h. Apparently, the dose chosen was suitable enough that we could discern the deuterium isotope effect on the liver toxicity. In this study, we focused on gene changes at 6 h, the time point at which the onset of hepatotoxicity was demonstrated, especially by serum enzyme levels (Table 2). An earlier time point (1.5 h) showed very little change in gene expressions, and this correlated very well with negative findings in the liver histopathologic evaluation and in serum clinical chemistry (Table 2). The later time point (24 h) showed that toxicity had progressed too far (especially with d0-NMF) and was not considered useful in trying to understand some early genomic changes in response to a toxic insult. The ability to monitor the expression of thousands of genes using microarray technology is well established (35, 36). This tool can offer unique insights about mechanisms of toxicity (37, 38), and is currently being evaluated for its potential in predictive toxicology (39). Attempts have been made recently to apply microarray technology to generate unique gene expression profiles (also called fingerprints) for various types of toxicants including hepatotoxicants (11, 12, 16-18). A number of hepatotoxic compounds have been categorized on the basis of their similarity in signature patterns of gene expression changes (11). The general approach has been to dose intact animals or hepatocytes with various hepatotoxic compounds (different doses) and harvest samples at different time points. The gene expression changes are subsequently monitored against control samples. However, because of the large sets of data produced in these studies, various bioinformatics tools need to be used to create fingerprints or signatures of compounds (11). Subsequently, compounds have been categorized on the basis of similarities in their fingerprints. Although this approach may have its use in categorizing potential therapeutic agents with unknown mechanisms of toxicity in early screening studies, there are certain drawbacks that need to be highlighted. One major drawback of this



Table 7. Genes Showing the Order d1-NMF > d0-NMF > d3-NMF of Fold Change in Signal Intensities Compared to that of the Saline Controla gene symbol d1- NMF d0-NMF d3 -NMF Down syndrome critical region homologue 1 3-hydroxy-3-methylglutarylCoA reductase A disintegrin-like and metalloprotease musculoaponeurotic fibrosarcoma oncogene ZW10 interactor UDP-glucose dehydrogenase phosphogluconate dehydrogenase ADP-ribosylation factor-like 4 GTP binding protein 2 casein kinase, delat Coxsackievirus and adenovirus receptor inhibitor of kappaB kinase gamma proline rich nuclear receptor coactivator 1 RNA binding protein gene with multiple splicing general transcription factor II repeat domain heat shock protein 1 DNA methyltransferase 3A ectonucleoside triphosphate diphosphohydrolase5 ubiquitin-associated protein 1 lectin, galactose binding, soluble 7 induced in fatty liver dystrophy glutathionetransferase alpha 2 crystalline, alpha C neoplastic progression 3 Thioredoxin reductase 1 glutamate-cysteine ligase sequestosome 1 core promoter element binding protein heat shock protein 1B heme oxygenase (decycling 1) heat shock protein 1 tumor necrosis factor alpha induced protein 6 lymphocyte antigen 96 N-myc downstream regulated-like dual specificity phosphatase 10

7.5

3.7

-4.5

-4.6

-3.7

-2.9

Adamsts7 -4.2

-2.6

-2.4

6.5

5.8

2.0

3.9 4.4 3.6 14.0 5.6 3.2 7.3

2.6 2.9 2.3 8.9 4.0 2.6 6.1

2.0 2.0 2.0 2.1 2.1 2.1 2.2

Ikbkg

3.6

3.1

2.6

Pnrc1

6.4

4.2

2.6

Rbpms

18.6

10.4

2.7

Gtf2ird1

5.5

4.5

2.8

Hspb1 Dnmt3a Entpd5

97.5 4.2 4.9

14.9 3.7 3.6

2.9 2.9 3.0

Ubap 1 Lgals7 Ifld2 Gsta2 Cryac Npn3 Txnrd1 Gclc Sqstm1 Copeb

6.0 7.2 20.0 16.4 9.1 37.0 6.1 7.9 10.5 57.8

5.3 5.3 14.5 8.9 8.1 14.4 4.9 6.1 8.8 34.6

3.0 3.0 3.3 3.5 3.6 3.6 3.6 4.1 4.5 5.5

Dscr1 Hmgcr

Mafk Zwint Ugdh Pgd Arl4 Gtpbp2 Csnk1d Cxadr

Hspa1b Hmox1 Hspb1 Tnfaip6 Ly96 Ndr1 Dusp 10

50.7 39.1 50.3 38.5 798 112.3 5.3**b 3.4 19.9 16.9 15.5

11.2 4.8 4.1

9.6 13.7 17.5 1.1**b 1.4 2.1 1.5**b

aPathway a for the metabolism of NMF most likely produced these gene changes. Negative and positive numbers imply down-regulation and upregulation of genes, respectively. b The P values for each comparison were less than 0.05 unless otherwise stated. * 0.05 < p < 0.1; ** p > 0.1.

experimental design is that there is no way to discriminate between genes affected as a result of pharmacological response to a drug versus genes that are directly or indirectly influenced by the bioactivating metabolic pathway(s). One of the aims of the current study was to use a stable isotope-labeled compound (NMF) and relate some of the gene changes to particular metabolic pathways. It is well established that most nonidiosyncratic hepatotoxicities are due to the formation of reactive intermediates. Hence, there is a need for a method that will differentiate gene responses due to the generation of the reactive metabolites from off-target effects. Our ability to modulate reactive metabolite formation by selectively replacing hydrogen with deuterium offers us the opportunity to focus the search for predictive biomarker genes for organ toxicities caused by some compounds. By understanding the bioactivating pathway of a compound, one can perform certain structural modifications to a compound that will mitigate reactive metabolite formation.

One way of performing this structural modification has been to introduce deuterium as the stable isotope at the bioactivated site. Carbon-deuterium bonds are more difficult to break than carbon-hydrogen bonds (21-23) and, hence, can lead to diminished metabolism at that site. This ultimately leads to a reduction in the amount of reactive metabolite being formed. Thus, in some cases, attempts can be made to modulate toxicities in preclinical species by using this approach to aid in demonstrating the involvement of reactive metabolites in causing organ toxicities (25-27, 34). In this study, we used stable isotopelabeled compounds along with nonlabeled compounds to identify gene changes selectively induced by two metabolic pathways. By comparing the data sets generated from the various treatment groups, we were able to map gene expression changes, which were attributed to differences in the metabolic pathways. One of the aims of this study was to use this alternate approach to help understand some of the genomic changes produced by the known hepatotoxicant, NMF (28, 29, 40). Because of its small size and limited number of possible metabolic pathways, we attempted to modulate the two routes of metabolism (Figure 2) by employing NMF analogues that were selectively labeled with deuterium at metabolic soft spots on the molecule, d1-NMF and d3-NMF (Figure 1). It has been reported that the incorporation of deuterium at the formyl position leads to a significant isotope effect, resulting in a reduction in the formation of a reactive MIC metabolite, as well as amelioration of hepatic toxicity compared to that if nonlabeled NMF (28). However, having three deuteriums on the methyl group did not significantly decrease the toxicity because the formation of MIC was not decreased in d3-NMF (Table 1). The inference from all these studies has been that MIC is primarily responsible for the centrilobular hepatotoxicity caused by NMF (28). Therefore, in this study, the deuterium kinetic isotope effect was used to shift metabolic pathways of NMF (Figure 2) and measure and compare gene changes with those produced by d0-NMF. Two approaches were taken to simplify the analysis of gene expression changes. Comparisons of the treatment groups (d0-, d1-, and d3-NMF) with saline controls at the 6 h time point were obtained to calculate absolute fold changes in gene expressions. The fold changes induced by nonlabeled NMF were then used as baseline values to which other groups (d1NMF and d3-NMF) were compared. This allowed us to further evaluate whether the expression of a particular gene was increased or decreased in the d1- and d3-NMF groups relative to that in the d0-NMF group. If a change in signal intensity followed the relative order d1 > d0 > d3, this suggested that the changes to the gene were linked to metabolism through pathway a (Figure 2). If the order was d3 > d0 > d1 or d0 > d3 > d1, then metabolism through pathway b was implicated. It must be emphasized that more than 14 000 genes were evaluated in this study, and a significant proportion of the genes were not affected by treatment with NMF and its analogues. The overall gene changes as a result of administering NMF, d1-NMF, and d3-NMF are shown in Table 4. 1. Group 1 Genes (Signal Intensity in the Order d3-NMF > d0-NMF > d1-NMF). Genes affected by metabolic pathway b (Figure 2) were collectively classified as Group 1. Table 5 is a list of these genes that showed (fold) changes in signal intensity in the order d3-NMF > d0-NMF > d1-NMF. The basis for using this criterion was that d3-NMF produced the greatest amount of MIC, followed by d0-NMF and d1-NMF (Table 1). Overall, a greater proportion of the genes affected by this pathway were down-regulated.


Mutlib et al.

Figure 6. Selected genes showing the following order of fold change in signal intensities compared to that of the saline control: d1-NMF > d0-NMF > d3-NMF. Pathway a for the metabolism of NMF is most likely implicated in these gene changes. Genes shown displayed p < 0.05 when comparisons were made with saline treated samples. Two sample t-test assuming equal variance was used to calculate the p value. Table 8. Classification of Genes That Were Significantly Altered in the d0-NMF Versus d1-NMF Comparison categories

number of genes

cell cycle apoptosis metabolism immune transcription GSH-antioxidant mitochondrial genes

38 47 15 83 34 11

Figure 8. RT-PCR of selected Group 1 genes associated with pathway b. * p < 0.05 and ** p < 0.01. Two sample t-test assuming equal variance was used to calculate the p-value.

Figure 7. RT-PCR of selected Group 2 genes, associated with pathway a. * p < 0.05 and ** p < 0.01. Two sample t-test assuming equal variance was used to calculate the p-value.

Down-Regulated Genes. Some of the genes that were downregulated as a result of MIC formation include those involved in lipid metabolism/catabolism (e.g., Lipg), protein dephosphorylation (Dusp7), prostaglandin synthesis regulation (Anxa6), cell proliferation (Egfr), calcium ion transport (Anxa6, Egfr), purine metabolism (Ampd2), protein glycosylation (B3galt3), potassium ion transport (kcnk5), regulation of transcription (Ankrd10), cell division regulation (Ccnd1), cellular morphogenesis, and adhesion and wound healing (Fn 1). The Thrsp,

Lipg, Ampd2, Kcnk5 genes showed the greatest fold change from the saline control in the presence of d3-NMF. Up-Regulated Genes. The changes in the expression levels of the up-regulated genes as a consequence of deuterium incorporation were not as dramatic as those of the downregulated genes (Table 5). Some of the genes that were upregulated as a result of MIC formation include those involved in glycogen metabolism (Ugp2), calcium signaling, protein dephosphorylation (Dusp6), and negative regulation of apoptosis (Angptl4). The up-regulation of Ugp2 appears to be in response to glycogen depletion, which was evident from the histopathologic evaluation of the liver samples. As shown in Figure 4D and E, the glycogen depletion in the centrilobular regions was accompanied by a corresponding increase in lipid deposits. This suggested that the cells were probably depending on gluconeogenesis for glucose supply. UDP-glucose pyrophosphorylase (Ugp2) was probably up-regulated in response to the cells’ need to replenish glycogen stores. Ugp2 mediates the synthesis of UDP-glucose from glucose-1-phosphate and uridine triphosphate. UDP-glucose is the activated form of glucose utilized in the biosynthesis of glycogen. Angiopoietin like 4 (Angptl4) gene


Figure 9. RT-PCR of genes affected by methyl isocyanate administered to mice at 5 mg/kg and 10 mg/kg. * p < 0.05 and ** p < 0.01. Two sample t-test assuming equal variance was used to calculate the p-value.

was particularly interesting because it was up-regulated, and its changes are usually in response to starvation and hypoxia. It is also involved in the positive regulation of angiogenesis and lipid metabolism. Ccl3 (chemokine (C-C) ligand 3), which plays a role in chemotaxis, was up-regulated perhaps in response to the inflammation caused by MIC. Sgk (serum glucocorticoid regulated kinase), which participates in apoptosis and protein amino acid phosphorylation was also found to be up-regulated as a result of MIC formation. The pattern of up-regulation for these genes, d3-NMF > d0-NMF > d1-NMF, is consistent with the hypothesis that d3- and d0-NMF were expected to produce more MIC than d1-NMF. 2. Group 1 Genes (Signal Intensity in the Order d0-NMF > d3-NMF > d1-NMF). The list of genes showing an overall pattern of d0-NMF > d3-NMF > d1-NMF was also classified as belonging to Group 1 (Table 6). This list was compiled because the effect of d3-NMF on MIC formation was not as dramatic as that produced by d1-NMF (Table 1). Furthermore, the toxicity profile (clinical chemistry and histopathology data) was fairly comparable between the two groups at the 6 h time point, even though d0-NMF showed greater overall toxicity than d3-NMF at 24 h. Hence, it is postulated that d3-NMF and d0NMF may have had similar effects on those genes affected by the metabolic pathway leading to MIC. For example, a number of genes, including Ccrn41, Cish, Ccnd2, Cdc45, Aox1, Gdf15, Hbp1, and Hspa8, were affected similarly by d0-NMF and d3NMF yet were very significantly affected by the presence of deuterium on the formyl position of d1-NMF. Down-Regulated Genes. Some notable gene changes included the cytokine-inducible SH2-containing protein (Cish) and cyclin D2 (Ccnd2). Cish is involved in the regulation of cell growth, protein kinase C activation, and the negative regulation of signal transduction. Ccnd2, a nuclear component, is involved in cell division and the regulation of progression through the cell cycle. Up-Regulated Genes. Some of the genes identified using the criteria d0-NMF > d3-NMF > d1-NMF were found to be involved in the regulation of cell growth and inflammatory response. Those involved in the regulation of cell cycle and adhesion included Cdc45 (cell division cycle 45 homologue), Clstn2 (calsyntenin), Ctgf (connective tissue growth factor), and Gdf15 (growth differentiation factor 15). Heat shock proteins, Hsp40, Hspa8 and Hsp110, were also up-regulated as a result of MIC formation 3. Group 2 Genes (Signal Intensity in the Order d1-NMF > d0-NMF > d3-NMF). Genes affected by metabolic pathway


a (Figure 2) were collectively classified as Group 2. Table 7 is a list of these genes that showed (fold) changes in signal intensity in the order d1-NMF > d0-NMF > d3-NMF. Most of the genes showing significant changes appeared to have been up-regulated as a result of this metabolic pathway. Furthermore, many more genes in this category showed dramatic fold change differences between the various treatment groups. For example, the gene encoding for heat shock protein 1 (Hspb1) showed a tremendous increase in expression in the presence of d1-NMF (798-fold higher than that of the saline control) compared to that of d0- and d3-NMF groups (112- and 18-fold higher than that of saline, respectively). Some of the genes that were affected include those involved in lipid metabolism (Hmgcr), intracellular protein transport (Arl4), and protein biosynthesis (GtPbp2). Other genes that showed significant changes in their expressions between the various treatment groups include Rbpms, Ifld2, Cryac, Npn3, Txnrd1, Sqstm1, Copeb, Hmox1, Ly96, Ndr1, and Dusp10 (Table 7). The expression changes observed for some of these genes were further confirmed by RT-PCR studies. Overall, there was a fairly good correlation between the two methods in evaluating the pattern of overall fold changes in gene expressions relative to those of the control (see Table 7 and Figure 7). It appears that the introduction of deuterium on the formyl position led to significant changes in the expression of genes, such as Hspb1, Hspa1b, Ifld2, Gsta2, Rbpms, Copeb, Npn3, and Hmox1. Some of these genes are indicative of oxidative stress (heat shock proteins, Hmox1). It is postulated that these gene changes are produced as a consequence of (metabolic switching leading to) the greater production of carbinolamide via pathway a (Figure 2). These genes are minimally affected by the d3-NMF analogue as shown in Table 7 and Figures 6 and 7. It is also interesting to note that although d3-NMF produces more MIC (Table 1), it appears to be less toxic than d0-NMF (this study and ref 28). Hence, one possible explanation for the greater degree of toxicity observed with d0-NMF compared to that with d3-NMF (especially at the later time point) is that although liver toxicity is triggered by MIC, other factors including the presence of metabolites such as formaldehyde (generated from N-(hydroxymethyl)formamide) could be important contributors toward the overall onset and progression of toxicity. Although metabolic pathway a could lead to reactive compounds such as formaldehyde, it does not appear that this bioactivation pathway can trigger hepatotoxicity on its own as evidenced by the significant decrease in toxicity by d1-NMF (which also produces significant gene changes attributed to pathway a). In this study, we made an attempt to associate gene changes with two metabolic pathways of NMF. The formation of methyl isocyanate and its potential implication in causing hepatic toxicity has been discussed in the literature (28, 29, 32, 33). The introduction of a deuterium at the formyl position apparently decreased the extent of liver injury (compared to d0-NMF), as demonstrated by clinical chemistry and histopathologic data. At the 6 h time point, d0-NMF and d3-NMF showed greater degrees of toxicity than d1-NMF. The gene chip analysis of livers from mice dosed with the various analogues of NMF demonstrated characteristic quantitative differences in the expressions of a number of genes. Some of these genes, which showed the order of signal intensities d1-NMF > d0-NMF > d3-NMF, were categorized as Group 2 genes and were associated with metabolic pathway a (Figure 2). Similarly, those genes that produced signal intensities in the order d3-NMF > d0-NMF > d1-NMF (or d0-NMF > d3-NMF > d1-NMF) were categorized as Group 1 genes and were associated with the formation of


MIC via metabolic pathway b. Real-time PCR was conducted to confirm some of the gene chip data. A good correlation was obtained between the two methods. Furthermore, mice were dosed with MIC and RT-PCR conducted to confirm that the selected Group 1 genes were indeed linked to metabolic pathway b of NMF.

Mutlib et al.

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This study clearly showed that the degree and onset of liver toxicity could be dramatically influenced by the incorporation of deuterium at a metabolic soft spot on NMF. This minimal structural alteration in a molecule, which leads to significant differences in the toxicity profile between nonlabeled and labeled analogues, can be used as a tool to help understand genomic changes produced by reactive metabolites. NMF was used as a model compound to understand some of the genomic changes in response to the formation of reactive intermediates via two metabolic pathways. Methyl isocyanate (MIC), which has been previously described as the reactive intermediate responsible for liver injury by NMF, was found to produce changes in the expression of genes (Group 1) involved in the cell cycle, apoptosis, and inflammation pathways. However, metabolites formed as a result of oxidation on the methyl group of NMF produced changes to a different set of genes. Some of these genes (Group 2) are implicitly involved in the cell’s response to oxidative stress (such as the heat shock proteins). These significant alterations in Group 2-gene expression appear to play a role in liver injury believed to be caused by MIC (formed through pathway b, Figure 2). The higher degree of the metabolic conversion of d3-NMF to MIC (Table 1) should have led to greater liver damage than that produced by d0-NMF. Instead, d0-NMF exhibited an overall greater degree of liver toxicity than d3-NMF, implying that MIC alone is not responsible for liver damage. It is possible that a combination of reactive intermediates (produced via metabolic pathways a and b) are important in contributing to NMF-induced liver injury. This is consistent with recent findings that suggest a combination of factors, including the presence of reactive intermediates and additional factors such as oxidative stress, can lead to liver damage (20, 41). Further studies are warranted using other softand hard-electrophile-producing compounds to evaluate and compare gene changes with those reported in this study. Investigations on gene changes in rats and humans need to be conducted in a similar manner to demonstrate that we can extrapolate our findings to other species. The findings from this study may eventually be used to better understand some of the gene changes produced by other compounds classified as idiosyncratic hepatotoxicants, and perhaps potential biomarker genes can be identified. Acknowledgment. We acknowledge Theresa Cody and Zachary Stewart for performing the necropsies and processing and staining the tissue sections. We also thank Greg Walker for providing the NMR data on the synthetic N-acetylcysteine conjugate of MIC.

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Identification of Potential Genomic Biomarkers of Hepatotoxicity

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