Comparative Toxicity and Metabolism of N-Acyl Homologues of

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Comparative Toxicity and Metabolism of N‑Acyl Homologues of Acetaminophen and Its Isomer 3′-Hydroxyacetanilide Yakov M. Koen,† Ke Liu,† Heather Shinogle,‡ Todd D. Williams,§ and Robert P. Hanzlik*,† †

Department of Medicinal Chemistry, ‡Microscopy and Analytical Imaging Laboratory, §Mass Spectrometry Laboratory, University of Kansas, Lawrence, Kansas 66045, United States S Supporting Information *

ABSTRACT: The hepatotoxicity of acetaminophen (APAP) is generally attributed to the formation of a reactive quinoneimine metabolite (NAPQI) that depletes glutathione and covalently binds to hepatocellular proteins. To explore the importance of the N-acyl group in APAP metabolism and toxicity, we synthesized 12 acyl side chain homologues of acetaminophen (APAP) and its 3′-regioisomer (AMAP), including the respective N-(4-pentynoyl) analogues PYPAP and PYMAP. Rat hepatocytes converted APAP, AMAP, PYPAP, and PYMAP extensively to O-glucuronide and O-sulfate conjugates in varying proportions, whereas glutathione or cysteine conjugates were observed only for APAP and PYPAP. PYPAP and PYMAP also underwent N-deacylation followed by O-sulfation and/or N-acetylation to a modest extent. The overall rates of metabolism in hepatocytes varied approximately 2-fold in the order APAP < AMAP ≈ PYPAP < PYMAP. Rat liver microsomes supplemented with NADPH and GSH converted APAP and PYPAP to their respective glutathione conjugates (formed via a reactive quinoneimine intermediate). With PYPAP only, a hydroxylated GSH conjugate was also observed. Thus, differences in biotransformation among these analogues were modest and mostly quantitative in nature. Cytotoxicity was evaluated in cultured hepatocytes by monitoring cell death using time-lapse photomicrography coupled with Hoechst 33342 and CellTox Green dyes to facilitate counting live cells vs dead cells, respectively. Progress curves for cell death and the areas under those curves showed that toxicity was markedly dependent on compound, concentration, and time. AMAP was essentially equipotent with APAP. Homologating the acyl side chain from C-2 to C-5 led to progressive increases in toxicity up to 80-fold in the para series. In conclusion, whereas N- or ring-substitution on APAP decrease metabolism and toxicity, homologating the N-acyl side chain increases metabolism about 2-fold, preserves the chemical reactivity of quinoneimine metabolites, and increases toxicity by up to 80-fold.

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INTRODUCTION Acetaminophen (APAP) is a widely used and generally safe analgesic, but in overdose it can cause significant, even fatal, hepatotoxicity. For these reasons, it is also the most thoroughly studied example of a small molecule hepatotoxin. As noted in recent reviews,1−3 there are many dimensions to the overall mechanism of APAP toxicity, but one obligatory early event that is universally agreed upon is that APAP must undergo bioactivation to a chemically reactive quinoneimine metabolite dubbed NAPQI (Figure 1),4,5 which expresses its reactivity intracellularly by reacting with glutathione and, as glutathione is depleted, with protein nucleophiles.6,7 An important component of the early investigations into the mechanisms of APAP hepatotoxicity involved the elucidation of structure−activity relationships (SARs) for the metabolism and toxicity of APAP analogues. The meta isomer of APAP, namely, AMAP, stood out early as being “non-toxic” in APAP-sensitive species, particularly mouse, despite its ability to covalently bind to proteins.7−10 Many other ring-substituted analogues of APAP were also eventually made and subjected to metabolism and toxicity evaluations (reviewed by Bessems and Vermeulen11). For example, disubstitution of the aromatic ring at either © 2016 American Chemical Society

Figure 1. Structures and abbreviations for APAP, AMAP, and analogues.

the 2,6-positions (adjacent to the nitrogen) or 3,5-positions (adjacent to the phenolic oxygen) greatly reduces toxicity. Collectively, the results of numerous SAR studies with ringsubstituted analogues supported the overall hypothesis that Received: August 9, 2016 Published: September 28, 2016 1857

DOI: 10.1021/acs.chemrestox.6b00270 Chem. Res. Toxicol. 2016, 29, 1857−1864

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containing 0.05% formic acid utilizing a gradient of 5−95% acetonitrile content over 2 min and then introduced to a time-of-flight mass spectrometer for HRMS (Waters Acquity UPLC with LCT Premier TOF or Agilent 1200RRLC with 6224 TOF). Accurate mass measurements for protonated molecular ions agreed with calculated values within 3 millimass units. All compounds were shown to be >97% pure, as ascertained from 1H NMR spectra (Supporting Information) and integrated HPLC peak areas detected at 214 nm. Hoechst 33342 trihydrochloride trihydrate, 10 mg/mL (16.2 mM in water, Life Technologies), and CellTox Green Cytotoxicity Assay (Promega) were purchased from Thermo Fisher Scientific (www. thermofisher.com). Hepatocyte thawing and plating medium (MATP250) and hepatocyte maintenance medium (MM250) were from Triangle Research Laboratories, LLC (www.triangleresearchlabs. net). Collagen was isolated from rat tail tendons by acidic extraction. Isolated tendons were sterilized with UV (16 h) and extracted with 0.1% acetic acid (room temperature, 48 h, with stirring). The insoluble material was then removed by centrifugation (1800g, 30 min), and the supernatant (corresponding to 3 mg tendon/mL) was kept at +4 °C in sterile culture tubes. Immediately before use, the stock solution was diluted 30-fold with sterile water. HPLC grade solvents and analytical grade inorganic salts were obtained from Fisher Scientific (www. fishersci.com). Deionized and purified water (Milli-Q, Millipore, resistivity 18 MΩ; autoclaved for cytotoxicity studies) was used for preparation of all solutions and buffers. Hepatocytes and Liver Microsomes. For biotransformation studies, freshly isolated hepatocytes were prepared from the livers of male Sprague−Dawley rats as described previously.17−19 Liver microsomes were prepared from noninduced male Sprague−Dawley rats as described previously.20 With 12 compounds to evaluate at multiple concentrations in replicate experiments on different days, it was impractical to keep isolating hepatocytes fresh as needed. For toxicity studies, we therefore used cryopreserved rat liver hepatocytes, which we obtained from Triangle Research Laboratories, LLC (catalog no. RSCP01, lot no. RSD141). Multiple small aliquots of frozen cells (10−12 × 106 cells/vial), all from a single large pool of cells derived from multiple rats, were ordered, received, and kept in liquid nitrogen (vapor phase) until used. This allowed us to use the same batch of cryopreserved cells in all cytotoxicity experiments. Biotransformation Studies. Microsomal incubations were conducted in 16 × 100 mm culture tubes. The tubes were preloaded with substrate (1000 nmol) deposited by evaporation in vacuo from 50 μL of a 20 mM methanol solution. The tubes were then charged with 1.0 mL of a suspension of rat liver microsomes (2 mg protein/mL) in 0.1 M potassium phosphate buffer (pH 7.4) containing 3 mM MgCl2 and 2 mM reduced glutathione, giving a final substrate concentration of 1.0 mM. The tubes were then capped and placed in a shaking waterbath incubator (37 °C); reactions (under an air atmosphere) were initiated by adding either 100 μL of NADPH solution (20 mM in buffer) or blank buffer as a control. After incubating for 120 min, duplicate 100 μL aliquots were removed, added to 100 μL of cold acetonitrile, and processed as described below for hepatocyte incubations. Freshly isolated rat hepatocytes were incubated in 50 mL roundbottom Corex tubes that were preloaded with substrate (400 nmol) deposited by evaporation in vacuo from 20 μL of a 20 mM methanol solution. Hepatocyte suspension (2.0 mL, 2 × 106 cells/mL) was added to each tube and swirled gently to dissolve the substrate. As a 0 time sample, a 400 μL aliquot was removed, added to 400 μL of cold acetonitrile, vortexed briefly, and centrifuged (10 000g, 5 min), and the resulting supernatant was stored at −20 °C. The tubes were then flushed with carbogen (O2/CO2 95:5), capped, and incubated with gentle shaking for 3 h at 37 °C, after which duplicate 400 μL aliquots were removed and processed like the 0 time samples. HPLC and Mass Spectrometry. Incubation supernatants were analyzed for structural identification and relative quantitation of metabolites by HPLC/MS/MS using a Waters (Medford, MA) UPLC Acquity “classic” eluting through an Acquity TUV dual-wavelength UV/vis detector in the electrospray ionization (ESI) source of a Micromass (Manchester, UK) Quattro Ultima “triple” quadrupole

metabolic activation and covalent binding was an obligatory early step in APAP hepatotoxicity. Curiously, studies of acyl side chain analogues of APAP have apparently not been reported. Since protein covalent binding is thought to be an initiating event leading to cytotoxicity, it has long been of interest to identify the cellular targets of NAPQI. Ironically, the first target identified was glutathione transferase (isoforms not specified), an enzyme involved in detoxifying NAPQI.12 Later works identified several other target proteins based on classical separations of liver proteins from mice treated with [14C]APAP (see TPDB for examples).13 A major advance in target protein identification was the introduction of the use of 2D electrophoresis to separate mouse liver proteins coupled with in-gel digestion and mass spectral analysis of tryptic peptides for protein identification.14 Today, at least 39 different liver proteins have been reported as APAP targets. This can be compared to a total of 444 nonredundant proteins targeted by reactive metabolites of 48 drugs and chemicals reported in the TPDB.13 Our laboratory has had a longstanding interest in the chemistry of reactive metabolites, their reactivity toward proteins, and the identification of cellular target proteins. As an extension of this, we became interested in trying to expand the pool of known protein targets of NAPQI. We also wanted to extend the currently meager knowledge of APAP and AMAP protein targets comparatively across different species including rat, mouse, and human and to probe deeper into differences in protein targeting by NAPQI and its analogues. To pursue these goals, it appeared attractive to employ a click-chemistry approach, whereby an APAP or AMAP analogue having an acetylenic function, such as PYPAP or PYMAP (Figure 1), could, after metabolic activation and protein covalent binding, be selectively coupled with a biotin-azide derivative, isolated using avidin-based affinity chromatography and subjected to MS-based proteomics.15,16 However, to use this approach would first require that we characterize the metabolism and toxicity of PYPAP and PYMAP to ascertain that their behavior was sufficiently similar to APAP and AMAP such that they could serve as potential surrogates for them. Since the many analogues of APAP whose metabolism and toxicity have been studied previously all involved ring modification rather than acyl group modification, we first prepared the series of higher acyl group homologues of APAP and AMAP shown in Figure 1. We then characterized the comparative metabolism of APAP, AMAP, PYPAP, and PYMAP using rat liver microsomes and isolated rat hepatocytes. Finally, we investigated the comparative cytotoxicity of all 12 APAP analogues using a unique approach that takes quantitatively into account both the time and concentration dependence of their toxic effects. In this article, we report the results of these metabolism and toxicity studies.

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MATERIALS AND METHODS

Chemicals and Media. APAP and AMAP were purchased from Sigma-Aldrich (www.sigmaaldrich.com). All other -PAP or -MAP congeners (Figure 1) were prepared by parallel synthesis in the Synthesis Core of the Center for Cancer Experimental Therapeutics at the University of Kansas using carbodiimide (EDC/NHS) coupling methodology. All compounds were characterized by 1H NMR and HPLC-MS/MS. NMR spectra were acquired on a 400 MHz Bruker AV spectrometer equipped with a X-channel broadband observe probe and a BACS-60 autosampler. For LC-MS, samples were eluted on a Waters HSS T3 2.1 × 50 mm column with acetonitrile and water 1858

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Chemical Research in Toxicology tandem mass spectrometer. Separations were achieved using a Waters BEH C18 column (2.1 × 50 mm, 1.7 μm particles) eluted with a linear gradient of 95% water (modified to pH 9.9 using aqueous ammonia) to 100% acetonitrile at a 0.6 mL/min flow rate. The mass analyzers were tuned to 0.8 u fwhh, and full scan product ion scans or selected reaction monitoring were done at a 20 V collision energy with argon. Hepatocyte Toxicity Studies. We assessed the toxicity of APAP analogues using time-lapse photomicrography. For this, we used 24well glass-bottom plates with high-performance cover glass (0.170 ± 0.005 mm; www.cellvis.com). Less than 48 h before use, wells were coated with collagen by adding 0.6 mL of a solution of rat tail collagen in water (0.1 mg/mL) to each well, allowing the plate to stand for 2 h at room temperature, and then removing the solution and allowing the plates to dry in air. Cryopreserved hepatocytes were thawed and plated in collagen-coated 24-well glass-bottom plates. Initial viability determined by trypan blue exclusion was 80−90%. Each well (1.91 cm2) was seeded with 300 000 live cells in 0.75 mL of plating medium, and the cells were allowed to attach for 3 h at 37 °C under a humidified atmosphere of 5% CO2/95% air. After attachment, the medium was removed and the wells were washed twice with 0.7 mL of maintenance medium. After washing, the wells received either fresh maintenance medium or the same medium containing certain concentrations of test compounds, along with Hoechst 33342 (160 nM) and CellTox Green dye (1000-fold dilution from the stock concentation supplied), in a final volume of 0.5 mL/well. Stock solutions of APAP and its analogues were prepared by dissolving the compounds directly in the hepatocyte maintenance medium. Because many analogues were poorly soluble in the aqueous culture media even after thorough mixing, undissolved material was removed by centrifugation (500g × 3 min) and the concentrations of the resulting stock solutions were determined spectrophotometrically using an empirically established ε250 of 8.16 mM−1 cm−1 for APAP in medium. Immediately after the addition of the test compounds and dyes to the wells, the plate was placed on the stage of an Olympus IX-81 inverted epifluorescence microscope. The stage was enclosed in an incubator box having controlled temperature (37 °C) and a humidified atmosphere of 5% CO2 in air. The microscope was equipped with an automated stage (Prior Scientific), a Flash 4.0 CMOS digital camera (Hamamatsu), and a Xenon excitation source (Sutter Instrument Company) with corresponding excitation and emission wheels, with a 20× 0.5 NA objective (Olympus). The following filter sets were used to acquire data for the Hoechst and CellTox Green channels: excitation, 387/11 nm; emission, 440/20 nm (Hoechst); and excitation, 485/10 nm; emission, 525/15 nm (CellTox Green). Three well-separated zones per well were selected for monitoring via computerized time-lapse photography. Each observation zone (0.653 × 0.653 mm) initially contained 200−300 live cells. Images from the Hoechst, CellTox Green, and brightfield channels were collected once every 60 min for 24 h using acquisition and processing software SlideBook 6 (Intelligent Imaging Innovations). The exposure time for each channel was calculated to maximize the full 16-bit intensity range of the camera (on average, 200−400 ms for Hoechst, 5 ms for CellTox Green, and 10−50 ms for brightfield). The number of live and dead cells (containing Hoechst- or CellTox Green-stained nuclei, respectively) at certain time points was determined using ImageJ software (NIH, https://imagej.nih.gov/ij), and select results were verified by visual observation of false-colored images.

glutathione conjugate APAP-SG (Figure S2), formed via the reactive intermediate NAPQI, showed the M + H+ precursor ion at m/z 457 and a number of product ions representing diagnostic fragmentations of the glutathione moiety, as shown in Figure S3. PYPAP gave rise to a somewhat larger amount of GSH conjugate (Figure S4). We presume that glutathione addition occurs at C-3 (meta to nitrogen), as it does with NAPQI, because this position is activated for Michael addition by the inductive effect of both the quinoneimine nitrogen and acyl oxygen, whereas the alternative C-2 position is activated for Michael addition only by the quinoid oxygen. The PYPAP− GSH conjugate showed the expected molecular ion at m/z 511 and an analogous series of confirmatory daughter ions (Figure S5). LC-MS/MS analysis of the PYPAP incubation also showed one additional metabolite peak not seen with APAP, namely, PYPAP + O + GSH, presumably formed via 3-hydroxylation, further oxidation of the catechol to a quinone and conjugation with glutathione, as shown in Figures S4 and S6. All of the above-mentioned metabolites are consistent with a vast body of literature on the oxidative metabolites of APAP itself.7,21,22 These results suggest that despite its longer acyl side chain, PYPAP, like APAP, undergoes oxidative metabolism to a reactive quinoneimine intermediate that is trapped in part by reaction with glutathione. As these experiments were intended to give an initial look at the oxidative bioactivation of a longerchain analogue, and no significant differences from APAP were observed, we did not investigate the metabolism of the other analogues using microsomes. To gain a deeper understanding of possible differences in their metabolism, APAP, AMAP, PYPAP, and PYMAP (0.2 mM) were separately incubated with freshly isolated rat hepatocytes (4 × 106 cells/2.0 mL) and the supernatants were subjected to LC-MS/MS analysis (Table 1). Because of Table 1. Metabolite Profiles of APAP, AMAP, PYPAP, and PYMAP in Rat Liver Hepatocytes APAP consumption (%/3 h) 45 Metabolites Formed (% of total)a O-glucuronide 13 O-sulfate 38 GSH conjugate 4 Cys conjugate 1 aminophenol nd -sulfate nd N-acetylaminophenol mp -sulfate mp

AMAP

PYPAP

PYMAP

74

80

100

42 30 nd nd nd nd mp mp

42 1 4 nd nd 5 4 19

60 1 nd nd nd 4 2 7

a

Metabolite quantitation is approximate based on integrated peak areas in UV chromatograms. Abbreviations: nd, not detected; mp, masked by unmetabolized parent compound or its sulfate conjugate (see text).

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RESULTS AND DISCUSSION Metabolism of APAP and Analogues. Incubation of APAP or PYPAP (1.0 mM) with rat liver microsomes supplemented with glutathione (2 mM) and NADPH (2 mM) led to the formation of their respective glutathione conjugates with approximately equal efficiency (∼10% conversion), as shown by LC-MS/MS analysis of incubation supernatants. The conjugates were characterized by their mass and fragmentation patterns, and approximate quantitation was based on peak areas in SRM chromatograms (Figure S1). The

the absence of authentic standards, the quantitation of metabolites is approximate, based on relative peak areas in SRM ion chromatograms (Table 1). APAP was converted to its glucuronide conjugate (m/z 328 → 152, ∼13%) and its sulfate conjugate (m/z 232 → 152, ∼38%), both identified based on their precursor (M + H+) mass and neutral loss fragmentation patterns. Clear evidence for oxidative metabolism to NAPQI was also observed in the form of its glutathione conjugate APAP-SG (m/z 457 → 328, ∼4%). 1859

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using microscopy, which revealed very significant differences in the rates of cell killing among the various APAP analogues. This lead us to evaluate the use of time-lapse photomicrography, coupled with differential staining for counting live and dead cells vs time, for quantitatively assessing the relative toxicity of our compounds. To facilitate cell counting, we used a mixture of Hoechst 33342 dye, which stains the nuclei of all cells, and CellTox Green, which stains only cells whose membrane integrity has been compromised. The transition of individual cells from live cells that exclude CellTox Green to dead cells having a brightly stained nucleus was strikingly sharp in time (see examples of time-lapse Movies S1, S2, S3, and S4 in the Supporting Information). An important consideration in this using this approach was the potential for phototoxicity caused by the presence of Hoechst 33342 in the nucleus and the bright light used to excite the dye for photography. Hoechst 33342 has been reported to be phototoxic to Chinese hamster V79 cells24 and Myc-transfected rat embryonic fibroblast cells.25 However, in the latter work, it was shown that one could avoid phototoxicity by reducing the concentration of Hoechst 33342 and the exposure parameters. We thus experimented with the concentration of dye used to find the minimum that would stain cell nuclei enough to allow us to count cells easily while avoiding detectable toxicity, and we found that 160 nM was sufficient (data not shown). Then, we reduced the number of photos taken across the 24 h observation period to just one every hour. This still gave us good resolution for measuring the time dependence of cell death at a given concentration of test compound. As observed microscopically, once isolated hepatocytes are dispensed into wells they begin to attach to the glass bottom and they start spreading out to contact their neighbors. After the 3 h attachment period, the medium was replaced by medium containing Hoechst 33342, CellTox Green dye, and a test compound (APAP or congener) at a predetermined concentration. Untreated cells (no APAP analogue) continue spreading out during the succeeding 24 h and show little or no cell death during this time, but when observed in time-lapse movies, considerable internal motion of organelles can be seen (see representative Movies S1, S2, S3, and S4 in the Supporting Information). As the concentration or potency of a given APAP analogue increases, individual cells start to contract and round up. This characteristic behavior is followed by blebbing and then by a very sudden uptake of CellTox Green dye, by which time all cellular motion has ceased. Dose (or concentration) and time are independent variables in toxicological responses.26,27 Toxicologists often fix the time variable and examine the concentration variable to determine LD50, ED50, or EC50 values from dose−response curves. The converse experiment of fixing dose or concentration and assessing the time variable is much less commonly done; thus, useful or important information may be missed. For a given APAP analogue at a given concentration, counting the number of dead cells as a function of time generates a progress curve for cell death. An example of this is shown in Figure 2 for three concentrations of APAP and AMAP (5, 10, and 15 mM). From these results, it is readily apparent that the relative toxicity of these two analogues to rat hepatocytes is identical at the high dose and very nearly the same at the lower doses, with APAP appearing slightly more toxic than AMAP at intermediate times. In addition to the obvious dependence of toxicity on concentration, Figure 2 also shows a significant difference in the

Under identical conditions, PYPAP was more extensively metabolized than APAP (∼80%/3 h vs ∼45%/3 h, respectively). Like APAP, PYPAP was also converted to its glucuronide (m/z 366 → 190, ∼42%), sulfate (m/z 270 → 190 → 110, ∼1%), and glutathione (m/z 496 → 366, ∼4%) conjugates (Table 1). In addition, we observed that PYPAP was hydrolyzed to release p-aminophenol, which was not observed as such but was detected in the form of its sulfate conjugate (m/z 190 → 110, ∼5%), its N-acetylation product (i.e., APAP, 152 → 110, 4%), and the sulfate conjugate of APAP (m/z 232 → 152, 19%). These latter metabolites accounted for about 28% of the original amount of PYPAP present in the incubation. AMAP was also metabolized more extensively than its isomer APAP in rat hepatocytes (∼74%/3 h vs ∼45%/3 h), but we detected only its glucuronide and sulfate conjugates as metabolites; no glutathione, cysteine, or mercapturic acid conjugates were observed. Finally, hepatocytes metabolized PYMAP completely in 3 h. The major metabolite was its glucuronide conjugate, formed along with small amount of its sulfate conjugate. As with AMAP, no glutathione-derived metabolites of PYMAP were observed. Interestingly, with PYMAP, a moderate amount of hydrolysis occurred to release m-aminophenol, which was then conjugated with sulfate or acetylated to form AMAP, which was then further sulfated. This hydrolysis and reacetylation phenomenon could not have been observed for APAP and AMAP without using isotopic tracers, but futile cycling of deuterated APAP to p-aminophenol with reacetylation back to nondeuterated APAP in vivo has previously been reported.21 Likewise, liver and kidney S9 (microsomes plus soluble fraction) of rats and dogs catalyzed both the N-acetylation and deacetylation of S-allyl-Nacetylcysteine.23 Thus, the overall conclusion is that differences in metabolism and metabolic activation between APAP and PYPAP and between AMAP and PYMAP are relatively modest and quantitative in nature. The longer chain analogues are less efficiently sulfated, but they are more efficiently glucuronidated and more extensively metabolized overall. On the other hand, differences between para isomers and meta isomers are more pronounced and relate chiefly to the formation of glutathione conjugates with the para-substituted compounds but not with the meta isomers. It is interesting to note that we saw no evidence of metabolism directed toward the acyl side chains, saturated or unsaturated, other than simple hydrolysis of the amide bond. Cytotoxicity of APAP Analogues. Initially, we used freshly isolated rat hepatocytes for assessing the relative toxicity of our compounds. While mice and hamsters are the species most sensitive to APAP hepatotoxicity, and mice are most commonly used in mechanistic studies of APAP hepatotoxicity, rats are not insensitive to APAP, and we already had experience working with rat hepatocytes and materials for this were readily available. Cells were dispensed into wells of a 24-well plate and allowed to attach for 3−4 h before changing the medium to one containing test compound, and toxicity was assessed after 24 h by measuring LDH released into the medium. We were concerned about the arbitrary nature of the end point time selected and by the fact that with some of the more toxic analogues the total LDH releasable by detergent treatment appeared to be decreased compared to that observed with the less toxic analogues or untreated cells. We then began observing the cells throughout the 24 h exposure period 1860

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Figure 4. Relative cytotoxicity of saturated analogues of APAP (A) vs AMAP (B), as indicated by 24 h AUCs of progress curves for cell death. Data points show the mean and SEM for 6−23 experiments. In some cases, SEM error bars are within the size of the symbol and hence are not plotted. Figure 2. Comparative toxicity of APAP (5, 10, or 15 mM) vs AMAP (5, 10, or 15 mM) to rat hepatocytes. Data points show the mean and SEM for 6−9 experiments. In some cases, SEM error bars are within the size of the symbol and hence are not plotted.

increases. It is remarkable, in view of the modest changes in chemical structure, that the relative toxicity of these compounds (i.e., the relative concentration required to produce a given level of AUC response decreases) increases by approximately 80-fold across this series. The major chemical property change that occurs with stepwise chain lengthening is an increase in the lipophilicity of each successive homologue, as reflected in cLogP, the calculated logarithm of the octanol−water partitioning coefficient of the homologue. Upon going from APAP to C5PAP, cLogP increases from 0.494 to 2.08, corresponding to a 40-fold increase in lipophilicity. Among congeners in a series, both rates of cellular uptake (by passive diffusion) and rates of oxidative metabolism by P450 enzymes are generally known to increase with lipophilicity. While relatively little is known about the effect of lipophilicity on the intracellular distribution of reactive metabolites once they leave the active site in which they are formed, it is not unreasonable to suspect that, in addition to outright chemical reactivity, the partitioning behavior of a reactive metabolite could also be important in determining its overall disposition between detoxication and protein covalent binding. Another factor in the distribution of a reactive metabolite from its site of formation to its target is its diffusivity, which, according to Graham’s law, decreases with the inverse square root of molecular mass. The fact that the C5, PE, and PY analogues have almost the same molecular masses could help rationalize their similar toxicity despite their differing cLogP values. In the meta series of saturated analogues, the differences in toxicity among individual members are much less pronounced than in the para series, but three of the four correlation lines show essentially the same slopes as their para isomers. The exception is C3MAP, which for unknown reasons is much less soluble than any of the other congeners. This made it physically impossible to test it at concentrations higher than those shown in Figure 4B. The meta−para differences in toxicity behavior are unrelated to cLogP, however, because cLogP is the same for each meta/para pair of isomers (see below). Evaluating the toxicity of the unsaturated analogues PYPAP and PYMAP was important in relation to our interest in using these acetylenic analogues to help isolate and identify a broader range of APAP and AMAP metabolite target proteins. In the approach envisioned, the acetylenic side chain of a protein adduct of PYPAP or PYMAP would be coupled to a biotin azide derivative via click chemistry and isolated, either as such or as tryptic peptides, using an affinity medium like neutravidin.15,16 To determine whether the chemical change of introducing the alkyne function would distort the biological

time dependence or rate of cell killing as a function of analogue concentration, with higher concentrations also giving higher rates of cell killing commencing sooner after the start of exposure. The time required for a given concentration of test compound to kill 50% of the cells (i.e., the ET50) can be found by interpolation of the curves in Figure 2, and the results can be presented as shown in Figure 3. Here, again one sees that at a given exposure concentration the differences between APAP and AMAP are small and essentially negligible.

Figure 3. ET50 values for cell killing by APAP vs AMAP. Bars show the mean and SEM for 6−9 experiments.

To capture the effect of both concentration and time in a single index that could be used to evaluate the relative toxicity of a series of congeners, we computed the area under the curve (AUC) for plots of cell death vs time (e.g., Figure 2) determined using different concentrations of test compound. In this format, the maximum possible AUC for cell death is 100% × 24 h or 2400%·h. The integrated AUC results for various concentrations of the saturated homologues of APAP and AMAP are shown in Figure 4, panels A and B, respectively. The maximum observed AUCs are considerably less than the theoretical maximum of 2400%·h because of the variable and sometimes substantial lag time before cell death commences, during which time no AUC is accrued. For the para series of homologues (Figure 4A), the data generate a set of nearly parallel lines that shift to the left in a regular progression as the acyl chain length of the compound 1861

DOI: 10.1021/acs.chemrestox.6b00270 Chem. Res. Toxicol. 2016, 29, 1857−1864

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moments than most organic chemists would predict for hydrocarbon moieties lacking electronegative substituents.29 Finally, since click labeling of protein adducts could also be based on coupling an azido analogue of APAP with an acetylenic-biotin derivative, we also examined the cytotoxicity of N3PAP and N3MAP, the azidopropionyl analogues of APAP and AMAP, respectively. AUC analysis of their toxicity at several concentrations showed that adding the azide functionality to C3PAP and C3MAP leads to a modest but significant increase in cytotoxicity compared to the parent compounds (Figure S7). We did not evaluate the metabolism of the azido analogues as we did for the acetylenic analogues, but since the latter were hydrolyzed to a significant extent, we evaluated the toxicity of the acids that would be released (i.e., azidopropionic acid, 4-pentenoic acid, and 4-pentynoic acid). At concentrations that equaled or exceeded those that would have resulted from complete hydrolysis of their corresponding APAP analogues, neither 4-pentenoic acid nor 4-pentynoic acid showed any cytotoxicity, whereas 3-azidopropionic acid was as toxic or somewhat more toxic than N3PAP or N3MAP (Figure S7).

response, we evaluated the toxicity of the acetylenes PYPAP and PYMAP and their olefinic analogues PEPAP and PEMAP. Compared to the aliphatic analogue C5PAP, the removal of two or four hydrogens to create PEPAP or PYPAP, respectively, has relatively little impact on their toxicity (Figure 5A). The same is

Figure 5. Relative cytotoxicity of APAP analogues (A) and AMAP analogues (B) having five-carbon saturated, olefinic, or acetylenic side chains, as indicated by 24 h AUCs of progress curves for cell death. Data points show the mean and SEM for 6−15 experiments. In some cases, SEM error bars are within the size of the symbol and hence are not plotted.

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SUMMARY AND CONCLUSIONS The metabolism and toxicity of APAP and AMAP analogues having varied acyl side chains have been investigated for the first time. Rat hepatocytes converted APAP, AMAP, PYPAP, and PYMAP primarily to O-glucuronide and O-sulfate conjugates in varying proportions. Glutathione or cysteine conjugates were observed only for APAP and PYPAP. Interestingly, the only metabolic transformation involving the unsaturated side chains of PYPAP and PYMAP was hydrolysis followed by N-acetylation or O-sulfation of the corresponding aminophenol. The overall rates of metabolism varied approximately 2-fold in the order APAP < AMAP ≈ PYPAP < PYMAP. In microsomes supplemented with NADPH and glutathione, APAP and PYPAP both gave rise to their respective glutathione conjugates (formed via a reactive quinoneimine intermediate); a hydroxylated GSH conjugate (presumably formed via ring hydroxylation, oxidation to an oquinone, and addition of glutathione) was observed only with PYPAP. Thus, differences in biotransformation among these analogues were modest and mostly quantitative in nature. The cytotoxicity of our APAP and AMAP analogues to rat hepatocytes was evaluated using time-lapse photomicrography in combination with dual staining with Hoechst 33342 and CellTox Green to observe increases in cell death as a function of both exposure time and analogue concentration. As the rate of cell killing was considerably faster with compounds having longer acyl chains, we calculated AUC values for each treatment from cell death progress curves. APAP and AMAP were essentially identical in cytotoxicity, as reflected in their progress curves for cell death at three different concentrations. Homologating the acyl side chain from C-2 to C-5 led to progressive increases in toxicity in both the para (APAP) and meta (AMAP) series of congeners, with the overall increase being about 80-fold in the para series. This correlated well with the progressive increase in cLogP across the series of saturated analogues, but unsaturated C-5 acyl analogues (i.e., 4-pentenoyl and 4-pentynoyl) were similar in toxicity to their saturated pentanoyl analogue and thus did not conform to the cLogP correlation. Nearly all ring substitutions on the APAP molecule reduce toxicity by slowing oxidative metabolism and/or blocking sites of nucleophilic attack on the reactive quinoneimine intermediate, thus decreasing both glutathione

true for the corresponding meta series of analogues: C5MAP, PEMAP, and PYMAP (Figure 5B). However, in the meta case, the whole series is less toxic (i.e., the curves are shifted to the right and compressed toward the curve for AMAP) compared to their para counterparts. Thus, compared to the effect of moving the hydroxyl group from para to meta (as in APAP vs AMAP), removing two or four hydrogens from the acyl side chain has little effect on toxicity in either series. On the other hand, removing hydrogens has a relatively large effect on cLogP, as shown in Figure 6. While this latter effect might seem surprising, or even

Figure 6. Variation of cLogP with side chain in APAP analogues. The values and plot for the AMAP analogues are identical, as cLogP is insensitive to positional isomerism.

counterintuitive, it is quite consistent across a number of sets of molecules.28 Lipophilicity (or hydrophobicity) is a simple empirical measurement that integrates the energetics of a number of physicochemical interactions in the solution phase, including hydrogen bonding, dipole−dipole interactions, and enthalpic and entropic effects on water structure and solvation. The reason for the strong decrease in lipophilicity with increasing unsaturation is unclear, but it is worth noting that unsymmetrical olefins and acetylenes have much larger dipole 1862

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Chemical Research in Toxicology depletion and protein covalent binding.11 In contrast, extending the N-acyl chain on APAP or AMAP increases cytotoxicity, possibly by increasing the rate of metabolic activation and/or the partitioning of the reactive metabolite between covalent binding vs detoxication, while preserving the fundamental reactivity of the APAP quinoneimine or AMAP amidoquinone metabolites.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemrestox.6b00270. Summary of m/z values; HPLC profiles; product ion spectra; cytotoxicity data; and 1H NMR spectra and tabulated HRMS data for synthesized APAP and AMAP analogues (PDF) Time-lapse movie S1, untreated control cells (MPG) Time-lapse movie S2, cells exposed to 5 mM APAP (MPG) Time-lapse movie S3, cells exposed to 10 mM APAP (MPG) Time-lapse movie S4, cells exposed to 15 mM APAP (MPG)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: 785-864-3750. Funding

Funding for this work was provided by a grant from the University of Kansas. Support for the NMR instrumentation was provided by NIH grant P50 GM069663. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS APAP, acetaminophen; AUC, area under the curve; cLogP, calculated logarithm of the octanol/water partition coefficient; GSH, glutathione; HRMS, high-resolution mass spectrometry; NAPQI, N-acetyl-p-benzoquinoneimine; TPDB, Reactive Metabolite Target Protein Database

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REFERENCES

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