Xenobiotica the fate of foreign compounds in biological systems
ISSN: 0049-8254 (Print) 1366-5928 (Online) Journal homepage: http://www.tandfonline.com/loi/ixen20
Metabolism of methapyrilene by Fischer-344 rat and B6C3F1 mouse hepatocytes D. W. Kelly, C. L. Holder, W. A. Korfmacher, T. A. Getek, J. O. Lay, D. A. Casciano, J. G. Shaddock, H. M. Duhart & W. Slikker To cite this article: D. W. Kelly, C. L. Holder, W. A. Korfmacher, T. A. Getek, J. O. Lay, D. A. Casciano, J. G. Shaddock, H. M. Duhart & W. Slikker (1992) Metabolism of methapyrilene by Fischer-344 rat and B6C3F1 mouse hepatocytes, Xenobiotica, 22:12, 1367-1381, DOI: 10.3109/00498259209056688 To link to this article: http://dx.doi.org/10.3109/00498259209056688
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Date: 30 March 2016, At: 16:23
XENOBIOTICA,
1992, VOL. 22,
NO.
12, 1367-1381
Metabolism of methapyrilene by Fischer-344 rat and B,C,F, mouse hepatocytes D. W. KELLY$§, C. L. HOLDER§, W. A. KORFMACHERSg, T. A. GETEKg, J. 0. LAY§, JR, D. A. CASCIANOSS, J. G. SHADDOCK§, H. M. DUHARTS and W. SLIKKER, JRt$§ 0 National Center for Toxicological Research, Jefferson, Arkansas 72079-9502 Downloaded by [University of California, San Diego] at 16:23 30 March 2016
and $ Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA Received 19 November 1991 ;accepted 24 June 1992 1. Suspension cultures of freshly isolated F344 rat and B,C,F, mouse hepatocytes were compared for their ability to transform various concentrations of methapyrilene (MP). 2. M P metabolites were isolated and purified by h.p.l.c., and were identified by comparing their chromatographic and mass spectral properties with those of authentic standards. 3. Both rat and mouse hepatocytes transformed M P to tentatively identified 2thiophenecarboxylic acid (I), and definitively identified mono-N-desmethyl methapyrilene glucuronide (11), methapyrilene glucuronide (111), methapyrilene N-oxide (V), and monoN-desmethyl methapyrilene (VI I).
Introduction Methapyrilene (MP) is a histamine HI-receptor antagonist of the ethylenediamine chemical class, previously used for treating symptoms stemming from allergies and the common cold (Douglas 1980). M P was removed from the market place several years ago after causing hepatocarcinogenesis in rats (Lijinsky et al. 1980, Lijinsky 1984, Habs et al. 1986). Limited studies with guinea-pigs, hamsters (Lijinsky et al. 1983), and mice (Brennan and Creasia 1982) indicated that carcinogenicity of M P is species-specific, since it failed to induce cancer in these species. MP has been studied in numerous short-term toxicity assays; most indicate that M P is an epigenetic carcinogen (Probst and Neal 1980, McQueen and Williams 1981, Casciano and Schol 1984, Budroe et al. 1984, Oberly et al. 1984, Kammerer et al. 1986, Casciano et al. 1988, Steinmetz et al. 1988), although some studies indicate that M P is genotoxic (Althaus et al. 1982, Blazak et al. 1986, Lampe and Kammerer 1987, Turner et al. 1987, Glauert and Pitot 1989). Because hepatic biotransformation is a primary route by which rats eliminate M P (Kelly et al. 1990) and because M P seems to induce cancer only in rats, we used target organ cells (freshly isolated hepatocytes) from rats (a sensitive species) and mice (an apparently insensitive species) to determine whether biotransformation in part might influence the species-specific toxicity of MP. We also con.ipared these findings with previous findings from an in vivo study (Kelly et al. 1990) to see whether hepatocyte cultures were good qualitative predictors of the metabolism of M P in vivo.
t Address correspondence to: William Slikker, Jr, Ph.D., HFT-132, Chief, Pharmacodynamics Branch, Division of Reproductive and Developmental Toxicology, National Center for Toxicological Research, Jefferson, Arkansas 72079-9502, USA. 0049-8254192 $3.00 0 1992 Taylor & Francis Ltd
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Chemicals Methapyrilene (2-[(2-dimethylaminoethyl)-2-thenylamino]pyridine)as the hydrochloridi salt was purchased from Abbott Laboratories (North Chicago, IL, USA). G.1.c. (flame ionization detector) and h.p.1.c. analyses demonstrated a purity of >99%. 14C-Methapyrilene HCI (labelled at the 2-thienyl carbon) with a specific activity of 50.0mCi/mmol and a chemical purity of >98%, according to t.1.c. radiochemical analysis, was synthesized by Chemsyn Science Laboratories (Lenexa, KS, USA). T h e methods for the synthesis, isolation, and purification of the N-oxide and mono-N-desmethyl methapyrilene reference standards are described elsewhere (Cerniglia et al. 1988). Authentic 2-thiophenecarboxylic acid was purchased from Aldrich Chemical Company (Milwaukee, W I , USA). All solvents were of h.p.1.c. grade, and all reagents were of chemically pure grade. Solvents and buffers were filtered through a Rainin (Rainin Instrument Company, Inc., Woburn, MA, USA) 0.45-pm Nylon 66 filter and degassed before use. Water was purified through a Millipore Milli-Q System (Millipore Corporation, Bedford, MA, USA), fed by a deionized water source. Isolation of mouse and rat hepatocytes Adult, male B,C,F, mice ( 3 3 4 7 g) and male Fischer-344 rats (209-238 g), obtained from the National Center for Toxicological Research breeding colony, were used to derive suspension cultures of hepatocytes according to the in situ collagenase perfusion technique of Oldham et al. (1979). B,C,F, mouse and rat hepatocytes, respectively. Concentrations, prepared by dissolving M P in Williams' >92% at the beginning of all metabolism studies. Fischer-344 rat hepatocytes, obtained in similar fashion, with a single rat for each experiment, yielded cell viabilities > 88% at the start of all metabolism studies. Hepatocyte incubation and sample collection The concentrations of M P at which mouse and rat hepatocytes were incubated were chosen to determine whether the cells metabolized methapyrilene in a dose-dependent manner. Preliminary experiments indicated that final concentrations of 500 and 1 0 0 M~P ~were cytotoxic to mouse and rat hepatocytes, respectively. Concentrations, prepared by dissolving M P in Williams' Medium E (WE) (Gibco, Grand Island, NY, USA), were considered cytotoxic if cell viability was 75% for mouse and rat hepatocyte cultures containing final concentrations of 250 and 50 p~ MP, respectively. Therefore, mouse hepatocytes were incubated in a maximum concentration of 2 5 0 MP, ~ ~whereas rat hepatocyte cultures were incubated in a maximum concentration of 5 O p MP. ~ Hepatocyte viability was determined by trypan blue exclusion. Cells excluding trypan blue were considered viable. Viability was determined with a Bright Line@hernacytometer (American Optical Corporation, Buffalo, NY, USA), placed under a Wild M40 light microscope (Wild Herrbrugg, Ltd, Herrbrugg, Switzerland) equipped with a 10-X lens. Mouse hepatocytes in suspension culture were incubated in final concentrations of 250, SO, or 25 p~ MP. Rat hepatocytes in suspension culture were incubated in final concentrations of 50.10, and 5 p~ MP. Each culture contained 1OpCi of 14C-MP. Hepatocytes were incubated with M P for 2 h , and six 2-ml samples (0, 15, 30, 60, 90 and 120min) were taken from each suspension culture. T h e experiment was replicated three times for each species. A 2-h incubation was chosen based on historical information from our laboratories indicating that after 2 h , cell viability dips below 75% for hepatocytes placed in suspension culture. Viable hepatocytes (28 x lo6) suspended in cold WE medium were dispensed to each of three sterile ice-cold 50-ml Erlenmeyer flasks equipped with Teflon-lined screw caps. T h e required volume of cold WE medium was then added to each flask so that after addition of MP, each flask contained a total volume of 14ml. Substrate was added to hepatocytes from a l & n M stock solution of unlabelled MP dissolved in WE medium. The radiolabelled MP, containing 1.OpCi of 14C-MP/pI of purified water, was prepared separately from the unlabelled solution. Total radioactivity and final suspension culture volumes were equal for all concentrations of MP. Unlabelled M P was added to the suspension cultures while gently swirling the flasks. Each flask then received 1OpCi of I4C-MP. Each flask was swirled, and a 2-ml sample was removed immediately and dispensed into a sterile 15-ml conical polystyrene centrifuge tube equipped with a screw cap (Corning Glass Works, Corning, NY, USA). This sample served as the control for estimating each concentration of MP. T h e suspension cultures were then capped and placed in a gyratory water bath shaker (New Brunswick, NJ, USA) set at 37"C, operating at 160 oscillations/min. T h e 2-ml samples were immediately centrifuged at 5Og for 2min at 4°C to pellet the hepatocytes. An aliquot (20 pl) of the incubation medium was removed to estimate total radioactivity and to determine the amount of radioactivity in the hepatocytes compared with the incubation medium. At all time points, the incubation medium contained 78% or more of the estimated radioactivity present in each 2-ml sample (as determined by liquid scintillation spectrometry analysis). The remaining sample was transferred to a
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sterile 15-ml polystyrene centrifuge tube, capped, and placed on ice. This sampling procedure was used at each sampling interval. At the end of the experiment, the samples were frozen (-20°C) for later h.p.1.c. analysis. Sample preparation Before h.p.1.c. analysis, 200pl of each 2-ml sample of incubation medium was processed by adding 400p1 of 50% methanol-ethanol (v/v),vortexing the sample to precipitate protein and salts associated with the cell media, and centrifuging at 16 OOOg for 2 min in an Eppendorf model 541 5 centrifuge (Brinkmann Instruments, Inc., Westbury, NY, USA). T h e sample was transferred to a filtering unit containing a Nylon 66 filter with 0.20pm pores (Rainin Instrument Company, Inc., Woburn, MA, USA). T h e precipitate was washed with 200p1 of 50% methanol-ethanol (v/v), centrifuged, and pooled with the original sample, which was then filtered and evaporated to dryness at 37°C under a stream of nitrogen. The extraction efficiency of this procedure was > 80% for all time intervals. The dried samples were redissolved in 75pI of the initial h.p.1.c. mobile phase with which they were analysed. For samples that were not used formass spectral analyses, known amounts of authentic standards were added to the biological matrix as chromatographic markers before clean-up and h.p.1.c. analysis. Quantitation of metabolites T h e h.p.1.c. system used to isolate and quantify methapyrilene metabolites consisted of two Waters Associates (Milford, MA, USA) model 6000A pumps, a model 660 solvent programmer, and model 440 dual channel ultraviolet detector equipped with 254nm UV filters. T h e system included a Rheodyne model 7125 injector (Cotati, CA, USA), and Upchurch guard column (2.0mm internal diameter x 20mm) (Upchurch Scientific, Inc., Oak Harbor, WA, USA) packed with 40pm C,, Corasil, and a Supelco 5 pm C18reversed-phase column (4.5 mm internal diameter x 250 mm) (Technicon Corporation, Tarrytown, NY, USA). Chromatograms were recorded on an Omniscribe" D-5000 Dual Pen recorder (Houston Instruments, Austin, TX, USA). Radioactive fractions were collected on a Gilson model FC80K microfractionator (Gilson Medical Electronics, Inc., Middleton, WI, USA). Liquid scintillation
Methapyrilene N-oxide
Mono-N-desmethyl methapyrilene ethapyrileke
IV
10
20
Retention Time (mid ~ ~ Figure 1. H.p.1.c. chromatogram of the 120-min mouse hepatocyte products derived from 2 . 5 0 14C methapyrilene. H.p.1.c. was performed in the presence of 5pg of each reference standard. T h e reversed-phase system resolved the total radioactivity into seven radiolabelled products.
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spectrometry was performed on a Tracor Analytic Mark I11 6881 liquid scintillation system (Tracor Analytic, Inc., Atlanta, GA, USA). A 40-min linear gradient h.p.1.c. programme began with 10% methanol and 90% 0.01 M monobasic potassium phosphate buffer (v/v), plus 200 pl triethylamine/litre (pH7) and concluded with 100% methanol. T h e mobile phase was delivered at a rate of l.Oml/min and 1.0-ml fractions were collected. A > 98% recovery of radioactivity was observed for samples analysed with this system. Metabolites of methapyrilene were isolated and purified by repeated h.p.1.c. injections of samples containing the highest concentrations of metabolites according to I4C analysis (figure 1).
Enzymic hydrolysis Because metabolites I1 (13.2min), I11 (14.8min), and I V (20.0min) (figure 1) had h.p.1.c. retention times in the range of a previously identified glucuronide of pyrilamine (retention time 17.8min) (Kelly and Slikker 1987), they were each incubated with 8-glucuronidase. Before enzyme hydrolysis, each metabolite was eluted from a C I S Sep-Pak (Waters Associates) with methanol (100%) to remove salts associated with the h.p.1.c. solvents. T h e methanol fractions were evaporated to dryness, and the residues were incubated with 10000 units of type B-10 bovine fi-glucuronidase (Sigma, St. Louis, MO, USA) in 1.5 ml of 0.2 M sodium acetate buffer, pH 5.0. Control incubations contained metabolites 11, 111, or IV in the absence of enzyme, plus 1.5 ml of 0.2 M sodium acetate buffer pH 5.0. T h e reaction mixtures were incubated overnight at 37°C and were stopped the following day with 1 ml of 50% methanol-ethanol (v/v). The reaction mixtures were vortexed, centrifuged (9000g for 10 min), and evaporated to dryness. The samples were then redissolved in 1.5 ml of purified water, transferred to a 10-ml glass test tube equipped with a Teflon-lined screw cap, and extracted with 3 x 5 ml of ethyl acetate. T h e organic extracts were evaporated to dryness under vacuum at 37°C. reconstituted with 75 pl of the initial mobile phase, and analysed by h.p.1.c. Fractions (1 min) were collected and radioactivity monitored by scintillation spectrometry. Each radiolabelled, h.p.1.c.-resolved metabolite of M P that was definitively or tentatively identified was converted to a percentage of the total of identified metabolites, for later statistical analysis. Mass spectrometry 14C-Methapyrilene metabolites were identified with a combination of desorption chemical ionization mass spectrometry, fast atom bombardment (FAB) mass spectrometry or thermospray mass spectrometry. Instrumentation and methodologies used for antihistamine characterization are described by Korfmacher et al. (1985). Lay et al. (1986, 1989a) and Cerniglia et al. (1988). T h e metabolites were passed through C,, Sep-Paks to remove h.p.1.c. mobile phase salts from the samples before mass spectral characterization. Statistics Mouse and rat hepatocyte incubations containing equimolar concentrations of MP (50 p ~ were ) analysed with a repeated measures two-way analysis of variance including a Bonferroni correction factor to determine whether there were statistical differences among metabolite concentrations. Values were considered significant at p < 0.05.
Results Isolation and identification of methapyrilene metabolites Since rat hepatocytes were substantially more sensitive to M P and were therefore incubated with much lower concentrations, no mass spectral data were obtained for metabolites generated by these cells. T h e results reported for the metabolism of M P by rat hepatocyte incubations are based upon chromatographic comparisons with mass spectrally characterized metabolites produced in mouse hepatocyte incubations. Mouse and rat hepatocyte suspension cultures incubated with 14C-MP were serially sampled at 15, 30, 60, 90, and 120min. Known amounts of synthetic standards were then added to these samples before clean-up and chromatographic analysis. All of these samples contained seven radiolabelled products, three of which co-eluted with the reference standards methapyrilene N-oxide, methapyrilene, and mono-N-desmethyl methapyrilene (figure 1). Mouse hepatocytes were incubated in the highest concentration of M P (250 p ~ ) , and were therefore considered the best matrix from which to isolate and purify methapyrilene metabolites. Hence, all chromatographic and mass spectral data displayed are from mouse hepatocyte incubations.
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Metabolite VIZ. This had an h.p.1.c. retention time virtually identical to that of authentic mono-N-desmethyl methapyrilene (retention time 39.8 min) (figure 1). The mass spectral fragmentation pattern of metabolite VII matched that of authentic mono-N-desmethyl methapyrilene when both were subjected to ammonia desorption chemical ionization mass spectrometric analysis. Metabolite VII displayed a base peak at m/z 248 with no significant fragment ions. This is consistent with the [M + H I + ion of mono-N-desmethyl methapyrilene and agrees with published data (Korfmacher et al. 1985, Cerniglia et al. 1988). Metabolite V I . When analysed by h.p.1.c. in the presence of reference standards, metabolite VI co-eluted with authentic methapyrilene (h.p.1.c. retention time 35.3 min) (figure 1). Under ammonia chemical ionization mass spectrometry conditions, the fragmentation pattern of metabolite VI was consistent with that of authentic methapyrilene under the same mass spectral conditions. Both metabolite VI and authentic methapyrilene showed a base peak, the [M + H I + ion, at m/z 262. T h e ammonia chemical ionization mass spectral fragmentation pattern of metabolite VI was consistent with that described for methapyrilene (Korfmacher et al. 1985). Metabolite V . Metabolite V co-eluted with authentic methapyrilene N-oxide (h.p.1.c. retention time 25-3min) (figure 1). When metabolite V was analysed with ammonia chemical ionization mass spectrometry its mass spectral fragmentation pattern was virtually identical to the fragmentation pattern observed for authentic compound under the same analytical conditions. T h e mass spectral fragmentation pattern of metabolite V included the [M +HI+ ion at m/z 278, and a base peak at m/z 262, corresponding to the [M H - 01 ion. Two other significant fragment ions were observed: [M+H-(CH,O)]+ at m/z 248 and the [M+H-(HNO(CH,),)]+ at m/z 217. A minor fragment ion observed at m/z 166 was presumably the [M H -0 - (C,H,SCH)] ion. These mass spectral data agree with previously reported results (Korfmacher et al. 1985, Cerniglia et al. 1988). Four polar radiolabelled products (I-IV, with h.p.1.c. retention times < 20 min) were also isolated from mouse hepatocyte incubations (figure 1).
+
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Metabolite ZV. Definitive identification of metabolite IV was unsuccessful, because of the lack of a reference standard and interference from a matrix-associated peak (figure 1, doublet peak with h.p.1.c. retention time 19.4 min). Incubating metabolite IV with fl-glucuronidase did not change its h.p.1.c. retention time. Metabolite IZI. Was the most abundant of the polar metabolites (figure 1). H.p.1.c. and I4C analyses of B-glucuronidase-treated metabolite I I I revealed two radiolabelled peaks (figure 2). T h e principal peak had an h.p.1.c. retention time of 29.5 min and represented 76.0% of the ethyl acetate-extractable radioactivity. This metabolite was purified b y h.p.1.c. and then analysed with ammonia chemical ionization mass spectrometry. T h e metabolite displayed a base peak at m / z 278, corresponding to the protonated molecule of a ring-hydroxylated derivative of methapyrilene (figure 3). FAB mass spectrometric analysis of metabolite I11 revealed an [ M + H ] + ion at m/z 454, corresponding to the molecular weight of a protonated glucuronide of methapyrilene (mass spectrum previously published, Lay et al. 1989a). The apparent location of the glucuronic acid moiety was discerned when a significant fragment ion at m/z 409 was observed, indicating the loss of dimethylamine from the amine terminus of the conjugate, a fragmentation pattern characteristic of the protonated molecules of ethylenediamine antihistamines (Lay et al. 1989b). Several
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Figure 2. H.p.1.c.chromatograms of 120-min mouse hepatocyte radio-labelled metabolite 111 in (A) the absence of bovine p-glucuronidase and (B) after incubation with bovine 8-glucuronidase.
ions corresponding to the [ M + N a ] + , [ M + K ] + , as well as the [ M + H ] + were observed in the mass spectrum and were attributed to the h.p.1.c. mobile phases, the biological matrix, or both. Additional evidence that metabolite I11 was a glucuronic acid conjugate of methapyrilene was provided with thermospray-mass spectrometry (Lay et al. 1989a). T h e thermospray results included an [M + H I + ion at m/z 454 and a major fragment ion at m/z 278 ([M +H-176]+), corresponding to the loss of glucuronic acid. In addition, two minor fragment ions were observed in the mass spectrum. T h e ion observed at m/z 233 presumably resulted from the loss of dimethylamine from
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Hepatic metabolism of methapyrilene
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Ammonia desorption chemical ionization mass spectrum of 120-min mouse hepatocyte metabolite 111, after B-glucuronidase hydrolysis.
the base peak (the aglycone), and the ion at m/z 436, corresponded to the loss of water from the glucuronic acid moiety. Therefore, based on its chromatographic behaviour following enzymic hydrolysis and the mass spectral behaviour observed with three ionization modes of mass spectrometry, metabolite 111 was identified as the glucuronic acid conjugate of pyridyl ring-hydroxylated methapyrilene, or methapyrilene glucuronide.
Metabolite 11. This was the second most abundant polar metabolite isolated from mouse hepatocyte suspension cultures (figure 1). H.p.1.c. and 14C analyses of metabolite I I after incubation with fi-glucuronidase revealed that several compounds were liberated upon hydrolysis (figure 4). A radiolabelled peak with an h.p.1.c. retention time of 31.6 min was tentatively identified as the aglycone of the apparent conjugate, based on its similar h.p.1.c. retention time to that of the aglycone of metabolite 111. T h e 31.6min peak represented 32.0% of the ethyl acetateextractable radioactivity associated with metabolite 11. Contaminants within this fraction, however, prevented its mass spectral characterization. Another major radiolabelled peak with an h.p.1.c. retention time of about 23 min was also liberated during hydrolysis. T h e h.p.1.c. retention time of this metabolite was similar to that of methapyrilene N-oxide; mass spectral characterization of this product was unsuccessful. These results indicated that the radiolabelled fraction containing metabolite I1 contained other compounds as well. This was confirmed when FAB mass spectral analysis of metabolite I1 revealed an [M HI+ ion at m/z 440, corresponding to the molecular weight of the protonated glucuronic acid conjugate of pyridyl ring-hydroxylated mono-N-desmethyl methapyrilene, or mono-N-desmethyl methapyrilene glucuronide (Lay et al. 1989a). In
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Figure 4. H.p.1.c.chromatograms of 120-min mouse hepatocyte radiolabelled metabolite I1 in (A) the absence of bovine 8-glucuronidase and (B) after incubation with bovine B-glucuronidase.
addition, the characteristic fragment at m/z 409 was observed, corresponding to the loss of methylamine from the amine terminus of the protonated molecule. Other major ions that were assigned nominal masses were apparently unrelated to the metabolite and were therefore attributed to the matrix from which metabolite I1 was isolated. Thermospray-mass spectrometry analysis of metabolite I1 revealed an [M H]+ ion at m/z 440,corresponding to protonated mono-N-desmethyl methapyriIene glucuronide, and a fragment ion at m/z 264, presumably corresponding to the molecular weight of the aglycone produced after cleavage of glucuronic acid (Lay et al. 1989a). These thermospray mass spectral data were consistent with the FAB results concerning the presence of several additional compounds associated with metabolite 11. Given the complementary data provided with these mass spectral methods and the chromatographic behaviour which metabolite I1 displayed after
+
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hydrolysis with /?-glucuronidase, the majority of the radioactivity associated with metabolite I I was identified as the glucuronide of pyridyl ring-hydroxylated monoN-desmethyl methapyrilene.
Metabolite 1. This was the least abundant of the radiolabelled products (figure l), and was present in mouse hepatocyte incubations at all three concentrations of MP. T h e mass spectral characterization of metabolite I was unsuccessful. However, when co-chromatographed with authentic 2-thiophenecarboxylic acid, metabolite I had an h.p.1.c. retention time identical to that of the standard (retention time 42min). Furthermore, when metabolite I was methylated with diazomethane gas, the product co-eluted with the methylated reference standard, 2-thiopheneacetate, which had an h.p.1.c. retention time of 26.3 min. Therefore, based on its co-elution with authentic 2-thiophenecarboxylic acid before and after derivatization, metabolite I was tentatively identified as 2-thiophenecarboxylic acid. Kinetics of methapyrilene metabolism in mouse hepatocyte incubations Mouse hepatocytes metabolized all three concentrations of M P to three oxidative products: methapyrilene N-oxide, mono-N-desmethyl methapyrilene and 2-thiophenecarboxylic acid, plus two conjugated products, methapyrilene glucuronide and mono-N-desmethyl methapyrilene glucuronide. Although the incubations contained the same methapyrilene metabolites, they generated these products at different rates, depending on the initial concentration of MP. T h e average initial rates (0-15 min) of M P metabolism by hepatocytes in incubation medium containing 250, 50, or 2 5 p M ~ P were 765, 426, and 285ng equivalent/ml per min, respectively. T h e average final rates (90-1 20 min) of M P metabolism for these same incubations were 38, 13, and 0.1 ng equivalent/ml per min. Incubations with the lowest initial concentrations of M P (25 and 5 0 ~ ~ ) contained < 30% of the added M P after 1.5 rnin (figure 5A and B). T h e glucuronic acid conjugates of methapyrilene and mono-N-desmethyl methapyrilene were the principal metabolites isolated from these incubations. At the conclusion of the experiments, the glucuronides represented over 80% of the h.p.1.c.-resolved metabolites (figure 5A and B). T h e N-oxide and mono-N-demethylated derivatives of methapyrilene were more prominent early in these incubations ( 2 5 3 7 % of the h.p.1.c.-resolved metabolites at 15-30 min during the incubation). T h e steady elimination of monoN-desmethyl methapyrilene from these incubations presumably resulted from its conjugation with glucuronic acid. 2-Thiophenecarboxylic acid and metabolite IV were minor products in these incubations. They were produced in roughly equal amounts for 60-90min. After this time, 2-thiophenecarboxylic acid was no longer detected, indicating that it undergoes either further metabolism or interacts with cellular or incubation medium constituents. Mouse hepatocyte incubations with the highest initial concentration of M P ( 2 5 0 ~ had ~ ) nearly 10 times more methapyrilene (as a percentage of the h.p.1.c.resolved metabolites) remaining after 60 min than the other incubations (figure 5C). Although containing more methapyrilene after 60 min, the high-concentration incubations actually metabolized methapyrilene at a faster rate than did the lowconcentration incubations. This increased rate of metabolism shifted the time-
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Time (min) Figure 5.
Concentration-time protiles of ''C-labelled methapyrilene metabolites after incubation of
B,C,F, mouse and F-344 rat hepatocyte suspension cultures with '4C-methapyrilene. Mouse hepatocytes were incubated in final concentrations of 25 (A), 50 (B), and 2 5 0 MP ~ ~(C). Rat hepatocytes were incubated in final concentrations of 5 (A), 10 (B), and 5 0 p ~ MP (C). Concentrations of methapyrilene (A),N-desmethyl methapyrilene glucuronide (D), and methapyrilene glucuronide ( 0 )are expressed as ng equivalent of free base per ml of incubation medium and are derived from the molecular weight and initial specific activity of the 14C-MP. Each value is the mean SEM for three experiments.
course profiles of methapyrilene metabolites. For example, the principal products isolated from these incubations were the N-oxide and mono-N-demethylated derivatives of methapyrilene comprising 16-41% of the h.p.1.c.-resolved metabolites for the first 60min of the experiments. These products accumulated in the incubations, indicating that the pathways responsible for their production were saturated. Over the time-course of the experiments, glucuronidation became more important. But not until 120min did the glucuronides of methapyrilene and mono-Ndesmethyl methapyrilene represent the predominant products isolated from the high-concentration incubations (figure 5C).
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T h e incubations produced about equal amounts of 2-thiophenecarboxylic acid and metabolite IV during the last 60min of the experiments. Although minor metabolites, 2-thiophenecarboxylic acid was always detected, while metabolite IV was detected over the last 60min of the incubation.
Kinetics of methapyrilene metabolism in rat hepatocyte incubations H.p.1.c. and 14C analyses of the rat hepatocyte incubations revealed that they produced identical metabolism profiles at all three concentrations of MP. T h e products that were isolated had h.p.1.c. retention times corresponding to the products isolated and identified from mouse hepatocyte incubations. As with mouse hepatocytes, glucuronidation was the predominant pathway by which rat hepatocytes metabolized methapyrilene. T h e glucuronides of methapyrilene and mono-Ndesmethyl methapyrilene consistently represented over one-third of the h.p.1.c.resolved metabolites (figure 5A and B) except at 1 5 min of the 50-/AM M P incubations (figure 5C). Early in the incubations (15-30 min), N-oxidation was a major biotransformation pathway, as methapyrilene N-oxide represented about 30% of the h.p.1.c.-resolved metabolites. Over the next 30min, its rate of metabolism was fairly constant. Metabolite IV increased steadily in these incubations; and as observed in the mouse hepatocyte incubations, its concentration-time profile paralleled that of mono-N-desmethyl methapyrilene glucuronide. These two products were present in roughly equal amounts throughout the duration of the experiments. N-Dealkylation was a minor pathway by which rat hepatocytes metabolized methapyrilene, though it was more prominent in incubations containing the highest concentration of methapyrilene. T h e production of 2-thiophenecarboxylic acid, through side-chain oxidation, was a principal pathway by which rat hepatocytes metabolized methapyrilene. At the end of the experiments, tentatively identified 2-thiophenecarboxylic acid represented almost 25-30% of the h.p.1.c.-resolved metabolites. Incubations containing 50 ~ L MP M had an average peak rate of thiophenecarboxylic acid production of 45 ng equivalent/ml per min. The peak occurred between 15 and 30 min, and was nearly eight times faster than the rate of thiophenecarboxylic acid production by equivalent mouse hepatocyte incubations. Statistical analysis of the mouse and rat hepatocyte incubations containing equimolar doses of MP (50 /AM) showed significant differences in the percentages of h.p.1.c.-resolved metabolites represented by methapyrilene N-oxide, mono-Ndesmethyl methapyrilene, and 2-thiophenecarboxylic acid.
Discussion Mouse and rat hepatocytes metabolized methapyrilene through pathways that generated identical products (figure 6). Product formation depended on the initial concentration of methapyrilene and on the time at which sampling occurred. T h e apparent absence of phenolic derivatives of methapyrilene and mono-Ndesmethyl methapyrilene, and the abundance of their corresponding glucuronides, indicates that (1) ring hydroxylation is probably the rate-limiting step during glucuronidation and (2) conjugation is the predominant pathway by which rat and mouse hepatocytes is suspension culture metabolize methapyrilene. T h e accumulation of mono-N-desmethyl methapyrilene in mouse hepatocyte incubations with
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Mano.N~desrnethyl
rnemapyrilene
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Glucuronidation
'd.no*.
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lor.Uon ol "C
&-.I
Pathways of methapyrilene metabolism by B6C3F, mouse and F-344 rat hepatocytes.
increasing doses of methapyrilene indicates that phenolic glucuronidation of methapyrilene was saturated. That methapyrilene, at the concentrations tested, apparently induced dose-dependent metabolism without inducing cytotoxicity indicates that mouse hepatocytes detoxified methapyrilene. But regardless of whether detoxication occurred, the apparent saturation of the glucuronidating capacity of the hepatocytes was not deleterious, because at least 75% of the cells were alive 1 h after dosing. What is most interesting is that rat hepatocytes were more sensitive than mouse hepatocytes to equimolar doses of methapyrilene. The most dramatic difference between the hepatocytes was their production of 2-thiophenecarboxylic acid, a metabolite whose formation involves cleaving methapyrilene and presumably forming two potentially reactive intermediates: an iminum ion and an aldehydic derivative of thiophene. At 30 min, this pathway was nearly eight times faster in rat hepatocytes than in mouse hepatocytes. Conceivably, the pathways responsible for detoxifying these intermediates could have been overwhelmed in the rat hepatocytes. Metabolic activation has been proposed before to explain the carcinogenicity of methapyrilene (Ziegler et al. 1981, Singer et al. 1987). Our findings seem to support this contention indirectly, since rat hepatocytes metabolized equal concentrations of methapyrilene nearly 1.5 times faster than did mouse hepatocytes. Proving metabolic activation though, requires chemical identification of these intermediates, identifying their location within cells, and thoroughly describing their binding kinetics. Our results generally agreed with other reports describing the in vitro biotransformation of methapyrilene. Methapyrilene N-oxide and mono-N-desmethyl methapyrilene (figure 6) have been isolated from microbial preparations (Cerniglia et al. 1988) as well as liver microsomal (Ziegler et al. 1981, Kammerer and Schmitz 1987, Singer et al. 1987) and liver homogenate incubations (Kammerer and Schmitz
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1986). Except for the microbial preparations, these systems produced primarily mono-N-desmethyl methapyrilene. We found similar products during early time points in the rat hepatocyte incubations and during later time points in the mouse hepatocyte incubations. These in vitro systems produce varying amounts of the Noxide. Kammerer and Schmitz (1 987) reported levels of methapyrilene N-oxide similar to ours when they used microsomal preparations, but did not report its formation from liver homogenates (Kammerer and Schmitz 1986). Singer et al. (1987) reported no methapyrilene N-oxide production from microsomal preparations, whereas Ziegler et al. (1981) isolated small amounts of this product. In contrast, Cerniglia et al. (1988) reported methapyrilene N-oxide as the principal oxidative product isolated from microbial incubations. These differences may result from the different P450 activities and chemical reactivities associated with the various incubations. T h e other oxidative metabolite which we detected (2-thiophenecarboxylic acid) has been reported in other studies as well (Kammerer and Schmitz 1986, 1987, Kammerer et al. 1988). T h e levels previously reported were similar to levels observed in mouse hepatocyte incubations (3-5% of the h.p.1.c.-resolved metabolites at 120min). We believe this is the first study in which suspension cultures of freshly isolated rat and mouse hepatocytes have been used to characterize the metabolism of methapyrilene. These cultures seem to be good qualitative predictors of the metabolism of methapyrilene in vivo, since we have isolated similar urinary metabolites from methapyrilene-dosed rats (Kelly et al. 1990). T h e cultures can also provide a relatively efficient means of producing analytical reference standards, perhaps leading to a more complete characterization of the metabolism of methapyrilene in vivo. T o summarize, mouse and rat hepatocytes biotransformed methapyrilene via Nand side-chain oxidation, N-dealkylation, and glucuronidation. Glucuronidation predominated in both species, regardless of the initial starting concentration of methapyrilene. Rat hepatocytes were highly efficient side-chain oxidizers of methapyrilene yielding 2-thiophenecarboxylic acid; they were inherently more sensitive to methapyrilene than were mouse hepatocytes; and they metabolized equal concentrations of methapyrilene more rapidly than did mouse hepatocytes. T h e faster rate of thiophenecarboxylic acid production and hence side-chain oxidation by rat hepatocytes-at 30 min, nearly eight times that of mouse hepatocytes-may be responsible for the higher sensitivity of rat hepatocytes to MP. T h e precise reason(s) for this response, however, requires further study.
Acknowledgements We express our gratitude to Eugene B. Hansen and Dr Carl E. Cerniglia for providing the N-oxide and mono-N-desmethyl methapyrilene reference standards, and we thank Barbara Jacks and Marty J. Green for excellent secretarial assistance.
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