Chem. Res. Toxicol. 1990, 3, 204-211
204
Mechanistic Studies on the Metabolic Activation of Acetaminophen in Vivo Kurt-Jiirgen Hoffmann,’ Donald B. Axworthy,2 and Thomas A. Baillie* Department of Medicinal Chemistry, School of Pharmacy, BG-20, University of Washington, Seat t 1e, Washington 98195 Received December 1, 1989
Three analogues of acetaminophen (APAP), labeled at specific positions with either oxygen-18 or deuterium, were administered by ip injection to male BALB/c mice a t the moderately hepatotoxic dose of 200 mg kg-’ in order t o probe the mechanism by which APAP undergoes metabolic activation in vivo. The thioether conjugates of APAP present in bile, urine, and feces, which are believed to derive from the electrophilic intermediate N-acetyl-p-benzoquinone imine (NAPQI), were isolated following aqueous-phase derivatization, separated by HPLC, and converted to a common volatile derivative for analysis by GC-MS. T h e observed labeling patterns of these conjugates indicated that APAP undergoes metabolism t o NAPQI by a process that does not involve the generation of a free oxygenated intermediate, but which more likely entails the sequential removal of two electrons from the substrate. On the basis of these findings, an integrated metabolic scheme is proposed which invokes initial cytochrome P-450 mediated generation of a caged oxygen-centered APAP radical species. Subsequent reactions of this intermediate may account for the formation of all known oxidative metabolites of APAP.
Introduction Metabolic activation of the widely used analgesic drug acetaminophen (APAP;3 Figure 1)to the corresponding quinone imine derivative N-acetyl-p-benzoquinone imine (NAPQI; Figure 1) has been the focus of considerable interest in recent years since the latter species is believed to act as the primary mediator of APAP-induced hepatic necrosis in both laboratory animals and humans (1-8). In support of this contention, several studies have demonstrated that NAPQI is highly cytotoxic in vitro ( 5 , 7, 9 ) , that it is a powerful chemical oxidant and a protein cross-linking agent ( 1 0 , 1 1 ) ,and that it is a reactive electrophilic species which undergoes addition reactions with both glutathione (GSH) and protein sulfhydryl groups to yield covalently bound thioether adducts of APAP ( 6 , 12-1 7). Although details of the biochemical processes by which NAPQI causes cell death remain poorly understood, evidence from work with both synthetic NAPQI (18) and structural analogues thereof ( 1I , 1 S 2 2 ) suggests that both the oxidative and arylating properties of this quinone imine may be important determinants of its toxic effects. However, it should be noted that NAPQI appears not to act as a classical chemical oxidant when generated in vivo, in that administration of hepatotoxic doses of APAP to mice fails to produce an increase in the efflux of GSSG in bile (23,241. On the basis of these and other considerations (25),it has been speculated that NAPQI may be “trapped” at its initial site of formation as an ipso adduct (Meisenheimer complex) with a cellular nucleophile such as GSH
’
On leave from the Department of Pharmacokinetics and Drug Metabolism, AB Hiissle, 5-431 83 Molindal, Sweden. * Present address: NeoRx Corp., 410 W. Harrison St., Seattle, WA
98119.
Abbreviations: APAP, acetaminophen; 3-OH-APAP, 3-hydroxyacetaminophen; APAP-SG, 3-(glutathion-S-yl)acetaminophen;APAPCyS, 3-(~-cystein-S-yl)acetaminophen; APAP-NACyS, 3-(N-acetyl-~cystein-S-y1)acetaminophen;NAPQI, N-acetyl-p-benzoquinone imine: BZQ, p-benzoquinone; TBA+HS04-, tetrabutylammonium hydrogen sulfate; MBSTFA, N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide: MBTFA, N-methyl-N-(trifluoroacety1)trifluoroacetamide;DMF, N,N-dimethylformamide; GSH, glutathione. 0893-228x/90/2703-0204$02.50/0
( 1 7, 23). Such an ipso conjugate might then serve as a vehicle for the transport of NAPQI to distant sites where arylation of cellular macromolecules could occur, either by direct reaction of the adduct with nucleophilic centers or following spontaneous regeneration of NAPQI at these loci (17,23). Oxidation of protein free thiols presumably would depend on the latter mechanism. With respect to the metabolic generation of NAPQI, the microsomal cytochrome P-450 mixed-function oxidase system has been shown to catalyze the two-electron oxidation of APAP, and NAPQI has been detected directly as a product of APAP metabolism in purified and reconstituted preparations of cytochrome P-450 ( 6 , 2 6 ) . From the evidence currently available, it appears that the oxidation of APAP to NAPQI is highly isozyme-dependent, in that marked differences have been noted in the rates of APAP metabolism by different members of the cytochrome P-450 family (26-31). However, the precise nature of this oxidation reaction remains controversial, and mechanisms have been proposed involving Nhydroxylation of APAP followed by dehydration of the resulting hydroxamic acid (pathway A, Figure 1) (32), formation of an intermediate APAP 3,4-arene oxide, rearrangement to the gem-diol, and subsequent loss of water (pathway B) ( 3 , 3 3 ) ,and “direct” oxidation (as opposed to oxygenation) in which two electrons are removed sequentially from APAP by cytochrome P-450 to yield the quinone imine (pathway C) (8,26, 34-37). With respect to the latter mechanism, both the nitrogen atom and the phenolic OH moiety of APAP have been proposed as sites for initial electron abstraction, and both free and hemebound radical and radical cation intermediates have been invoked in the process leading to NAPQI (36). Kinetic studies with synthetic N-hydroxy-APAP, together with carrier pool trapping experiments in vitro, have indicated that pathway A is unlikely to contribute significantly to the metabolic activation of APAP in liver tissue (37, 38), while the failure of epoxide hydrolase (but not of ascorbate) to inhibit the covalent binding of APAP to liver microsomal proteins in vitro has been taken as 0 1990 American Chemical Society
Chem. Res. Toxicol., Vol. 3, No. 3, 1990 205
Metabolic Activation of Acetaminophen
6 'CH,
OH
6cH3
/
0 OH
I
OH
QSG + GSH
OH
APAP-SG
methylsily1)-N-methyltrifluoroacetamide(MBSTFA), CHJ, and AgzSOI. Methanol, glacial acetic acid, and water (HPLC grade) were supplied by the J. T. Baker Chemical Co. (Phillipsburg, NJ), and CHzClz was from Mallinckrodt (St. Louis, MO). Ethyl chloroformate was obtained from Sigma Chemical Co. (St. Louis, MO), and N,N-dimethylformamide (DMF) was from Pierce Chemical Co. (Rockford, IL). N-Methyl-N-(trifluoroacety1)trifluoroacetamide (MBTFA) was purchased from Macherey-Nagel (Duren, FRG). [4-'80]APAP (48.1 atom % excess), [3-2H]APAP (consisting of 16.3% 2Ho,80.3% 2Hl, and 3.4% 2Hzspecies), and and 74.0% [3,5-2Hz]APAP(consisting of 2.2% 2Ho,23.8% 2Hl, 2Hz species) were obtained as gifts from Dr. S. D. Nelson (University of Washington). T h e preparation of these labeled analogues of APAP has been described elsewhere ( 4 4 , 4 5 ) . Authentic samples of 3-(glutathion-S-yl)-APAP (APAP-SG), 3 4 ~ cystein-S-y1)-APAP (APAP-CyS), and 3-(N-acetyl-~-cystein-Sy1)APAP (APAP-NACyS) were obtained by synthesis (43). Instrumentation. Equipment for gas-liquid chromatography (GC), combined gas chromatography-electron impact mass spectrometry (GC-MS), and analytical high-performance liquid chromatography (HPLC) was identical with that described in an earlier paper (43). HPLC separations, which were carried out isocratically under reversed-phase conditions, employed mobile phases consisting of methanol/glacial acetic acid/water [45:1:54 v/v (mobile phase A) or 40:1:59 v/v (mobile phase B)]. In each case, the flow rate WBS 1.0 mL min-', and compounds eluting from the column were detected by monitoring UV absorbance a t 254 nm. Biological Experiments. Adult male BALB/c mice (18-30 g), purchased from Simonsen (Gilroy, CA), were housed in individual metabolic cages which permitted the separate collection of urine and feces. The mice, which had free access to food and water throughout the study, were pretreated for 3 days prior t o use with sodium phenobarbital (75 mg kg-' day-', administered by ip injection in 0.9% saline solution). Groups of three animals each were then dosed with one of the stable-isotope-labeled analogues of APAP (200 mg kg-', administered ip as a 20 mg mL-' solution in 0.9% saline), and pooled urine and fecal samples were collected for 24 h. Following a recovery period of a further 24 h, each mouse then received a second ip dose of the same labeled analogue of APAP (again a t 200 mg kg-' ip) and 4 h later, the animals were sacrificed by decapitation. T h e livers of the mice were removed promptly, and as much bile as possible was collected from each gall bladder by means of a syringe. T h e bile from individual animals in each treatment group was then pooled and taken for analysis of thioether metabolites. Analytical P r o c e d u r e s . Urine samples were processed for the analysis of parent drug (measured as "total" APAP present after enzyme-mediated hydrolysis of glucuronide and sulfate conjugates) and also for the analysis of the thioether adducts APAP-CyS and APAP-NACyS. In the former case, specimens of urine were adjusted to pH 4.5 by the addition of 0.1 M sodium acetate buffer, treated with arylsulfatase/b-glucuronidase,and incubated overnight a t 37 "C. T h e hydrolyzed samples were extracted with 10 volumes of ethyl acetate, and the organic extracts were taken to dryness and derivatized with MBTFA/pyridine (1:l v/v) a t 120 "C for 30 min. Aliquots of these final solutions were analyzed directly by GC-MS using a 60-m DB-5 fused silica capillary column; following splitless injection and "cold trapping" on the column (40 "C for 30 s), components of the derivatized extracts were separated by temperature programming (rapid increase to 120 "C, followed by linear ramp at 10 "C min-' to 250 "C). Under these conditions, both reference samples of APAP and extracts of mouse urine exhibited a single major GC peak whose relative retention time corresponded to a methylene unit value (43) of 13.1. This compound was identified by mass spectrometry as N,O-bis(trifluoroacety1)-p-hydroxyaniline,a derivative formed by both trifluoroacetylation of the phenolic OH group of APAP and net replacement of the N-acetyl function by an N-(trifluoroacetyl) moiety. (Similar treatment of an authentic sample of p-aminophenol yielded an identical product.) T h e isotopic content of the "total" APAP recovered from urine, and of the original ['%I- and [*H]APAP species administered to mice, was determined by selected ion monitoring GC-MS analysis of the relative abundances of the M" ions (at m/z 301 in the unlabeled derivative). Measurements of isotopic excess included
NAPQI
F i g u r e 1. Alternative pathways for the biotransformation of APAP to NAPQI: (A) N-hydroxylation followed by loss of water; (B) formation of a 3,4-arene oxide, tautomerization to the gem-diol, and elimination of the elements of water; and (C) "direct" oxidation, entailing the net removal of two electrons from APAP without the liberation of a free oxygenated intermediate. T h e NAPQI thus formed undergoes conjugation with GSH to yield APAP-GS.
evidence against the involvement of a reactive arene oxide in the bioactivation process (pathway B) (39). At this juncture, therefore, the weight of experimental evidence favors the "direct" oxidation route to NAPQI (pathway C), which is consistent with current views of the mechanism of cytochrome P-450 mediated reactions which include net two-electron oxidations which proceed without the release of free oxygenated intermediates (40-42). The present study was carried out in an attempt to obtain mechanistic information on the bioactivation of APAP in vivo and employed the BALB/c mouse as an animal model in view of the relative importance in this species of the metabolic pathway of interest which leads, via GSH conjugation, to a series of thioether conjugates of APAP which are excreted into bile, urine, and feces. Analogues of APAP labeled at specific sites with stable isotopes (oxygen-18 or deuterium) served as metabolic probes, and advantage was taken of novel methodology for the analysis of thioether metabolites of APAP in biological fluids (43) to establish the labeling patterns of these adducts and, indirectly, those of NAPQI itself.
Experimental Procedures Materials. T h e following materials were purchased from Aldrich Chemical Co. (Milwaukee, WI): APAP, tetrabutylammonium hydrogen sulfate (TBA+HS04-), N-(tert-butyldi-
206 Chem. Res. Toxicol., Vol. 3, No. 3, 1990 corrections for both background ion current and natural isotopic abundance in the ions of interest and were based on the results of at least three replicate analyses. Thioether conjugates of APAP present in mouse urine were analyzed according to the following procedure. Urine samples (50 r L ) were treated with sodium carbonate buffer (pH 9; 3 mL) and ethyl chloroformate (50 WL).After a period of 30 min at r c " temperature, the mixtures were washed with CH2C12(5 mL), and after removal of the organic phases, the samples were treated with additional carbonate buffer (pH 9; 1 mL) and TBA+HS04- solution (0.1 M; 100 pL). The resulting mixtures were then shaken a t room temperature for 1 h with a 10% solution of CHJ in CHzClz(10mL), following which the organic phases were removed and washed with a saturated aqueous solution of Ag2S0,. After evaporation of the solvent, the residue in each case was dissolved in mobile phase A (100 p L ) and aliquots were injected into the HPLC system. Column fractions were collected at retention times corresponding to the N,O-bis(ethoxycarbony1) methyl ester derivative of APAP-CyS and the 0-(ethoxycarbonyl) methyl ester derivative of APAP-NACyS (9.8 and 5.5 min, respectively). Following evaporation of these samples under reduced pressure, the derivatized APAP thioether metabolites were converted to a common volatile benzo[ 1,3]thioxolane by reaction with MBSTFA in DMF (1:l v/v) a t 120 "C for 30 min (43). Aliquots of these final solutions were injected directly into the GC-MS system, and the isotopic enrichments of the labeled conjugates were determined by selected ion monitoring of the respective [M - CqHg]+ ions (at m/z 426 in the spectrum of the unlabeled derivative). For the analysis of APAP thioether conjugates present in feces, the entire 0-24-h fecal collection from each animal was suspended in sodium carbonate buffer (pH 9 10 mL) and ethyl chloroformate (100 pL) was added under vigorous stirring. After reaction for 30 min a t room temperature, samples were centrifuged and the clear supernatants were removed, adjusted to pH 2 with H C l ( l 2 M), and extracted into ethyl acetate (3 X 10 mL). The combined organic extracts were evaporated to dryness, and the residues were reconstituted in mobile phase B (100 p L ) for analysis by HPLC. The peaks eluting at 6.1 and 11.1min, which corresponded to the ethoxycarbonyl derivatives of APAP-NACyS and APAP-Cys, respectively, were collected and converted to the volatile silyl derivative for GC-MS analysis. Specimens of bile (IO p L ) were treated with ethyl chloroformate, as described above for the fecal metabolites, and the resulting N-(ethoxycarbonyl) derivative of APAP-GS was isolated by extraction into ethyl acetate. Purification by HPLC (mobile phase B)gave a single major peak (retention time 7.7 min), which was collected and silylated, as before, for analysis by GC-MS. T h e above analytical protocols resulted in the conversion of the urinary thioether adducts (APAP-CyS and APAP-NACyS) to their respective ethoxycarbonyl methyl ester derivatives. In the case of extracts of both feces (which contained APAP-CyS and APAP-NACyS) and bile (which contained only APAP-GS), the methylation step was omitted since esterification of the conjugates of interest frequently proved to be incomplete. However, reaction of all of the isolated APAP thioether derivatives (whether in methyl ester or free acid form) with MBSTFA in DMF resulted in the formation of a single volatile product (the benzo[ 1,3]thioxolane species referred to above) which was analyzed by selected ion monitoring GC-MS (43).
Results Pilot experiments with the synthetic APAP conjugates indicated that extractive methylation of the ethoxycarbonyl derivatives of APAP-CyS and APAP-NACyS formed in urine represented an effective procedure for the recovery of these adducts into organic solvents. However, less satisfactory results were obtained with the ethoxycarbonyl derivative of APAP-GS formed in bile, which typically afforded a mixture of products upon extractive alkylation (apparently due to incomplete methylation of the carboxylic acid groups). For this reason, the ethoxycarbonyl derivative of APAP-GS was extracted directly from acidified bile specimens and purified as the free acid
Hoffmann et al. URINE
I
I I
A=O.I&A=I,O
I REFERENCE
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12
Figure 2. HPLC separation (mobile phase A) of derivatized thioether conjugates of APAP. Peak 1 corresponds to the 0(ethoxycarbonyl) methyl ester of APAP-NACyS and peak 2 t o the N,O-bis(ethoxycarbony1) methyl ester of APAP-CyS. T h e lower chromatogram illustrates the separation obtained from analysis of a mixture of the reference, synthetic materials, while the upper chromatogram was recorded from an extract of urine from a mouse dosed with APAP (200 mg kg-' ip). In the latter case, the attenuation of the W recorder was changed (to a 10-fold less sensitive setting) just prior to elution of the derivatized cysteine conjugate.
by HPLC. A similar procedure was followed for metabolites present in fecal homogenates. It should be noted, however, that both free acid and methyl ester forms of the derivatized conjugates of APAP undergo conversion to the same volatile silyl species, identified tentatively as a benzo[ 1,3]thioxolane, when treated with MBSTFA in DMF (43). When extracts of derivatized 24-h urine collections from mice given APAP were analyzed by HPLC, both APAPCyS and APAP-NACyS were found to be present in appreciable quantities. A representative chromatogram illustrating the separation of these derivatives is reproduced in Figure 2, which also depicts the separation of the corresponding reference thioether conjugates of APAP under identical conditions. Although no attempt was made in the present study to quantify the formation of thioether adducts of APAP, the detector responses in Figure 2 (obtained a t different sensitivity settings) suggest that APAP-CyS was present in urine at appreciably higher levels than the corresponding mercapturic acid. This observation is consistent with the results of a previous study on the metabolism of APAP (200 mg kg-l ip) in the mouse, where the cysteine conjugate was found to account for 25% of the metabolites present in 24-h urine collections whereas the mercapturate accounted for only 2% (46). Both APAP-CyS and APAP-NACyS again were detected in derivatized extracts of mouse feces, although in this case it appeared that the two conjugates were present in comparable amounts (Figure 3). Extracts of mouse bile,
Chem. Res. Toxicol., Vol. 3, No. 3, 1990 207
Metabolic Activation of Acetaminophen
"1
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Figure 3. HPLC separation (mobile phase B) of derivatized thioether conjugates of APAP. Numbered peaks correspond to the following compounds (free acids): 1, 0-(ethoxycarbony1)and 3, APAP-NACyS; 2, N,O-bis(ethoxycarbony1)-APAP-GS; N,O-bis(ethoxycarbony1)APAP-CyS.The center chromatogram illustrates the separation obtained during analysis of a mixture of the reference, synthetic materials, while the upper and lower chromatograms were recorded from extracts of feces and bile, respectively, from mice dosed with APAP (200 mg kg-' ip).
on the other hand, did not contain measurable quantities of either of these adducts but instead afforded chromatograms dominated by a single peak with a retention time (7.6 min) intermediate between those of the derivatized mercapturate (5.5 min) and cysteine conjugate (9.8 min) (Figure 3). This peak corresponded to the expected glutathione adduct, APAP-GS, in the form of its N,O-bis(ethoxycarbonyl) derivative. Treatment of each of the derivatized thioether metabolities of APAP with MBSTFA/DMF led to the formation of the common silyl derivative whose proposed structure is depicted in Figure 4. Although the cysteinyl moiety and its substituents are eliminated during the conversion of the three different thioether adducts of APAP to this silyl derivative, the latter retains both the phenolic oxygen and the aromatic hydrogen atoms of the parent conjugates. Thus, measurements of isotopic enrichment of this silyl derivative will reflect the labeling
l , l , , ,
350
mlr
Figure 4. Electron impact mass spectrum, recorded under GCMS conditions, of the silyl derivative obtained by treating (unlabeled) ethoxycarbonyl derivatives of APAP thioether conjugates with MBSTFA/DMF (43).
patterns of the respective thioether precursors and, therefore, of NAPQI itself. Upon GC-MS analysis of derivatized extracts of urine, [2H1]-, feces, and bile from mice which had received [180]-, or [2H2]APAP,full mass spectra were recorded (of which selected examples are reproduced in Figure 5) in order to confirm the identity of the gas chromatographic peaks attributed to the above silyl derivative. Accurate measurements of isotopic enrichment were then made under selected ion monitoring GC-MS conditions, and the results of these andlyses are summarized in Table I. In the case of the S-linked conjugates derived from [180]APAP, an average of 96.8 f 2.8% of the oxygen-18 label present in the parent drug was retained in the adducts recovered in bile, urine, and feces. The APAP recovered in enzymehydrolyzed urine had an l80content that did not differ significantly from that of the administered compound. When [2H2]APAPwas given to animals, the parent drug liberated by hydrolysis of urine specimens again was found not to have lost any of the isotopic labels. However, the thioether metabolites of t2H2]APAP were, as expected, monodeuterated. On the basis of the premise that all molecules of [2H2]APAPundergoing biotransformation to C-3 thioether adducts would lose one atom of deuterium in the process, the isotopic distribution of the products would be predicted, on statistical grounds, to be 14.1% unlabeled, 85.9% 2H1, and 0% 2H2. The actual values (expressed as the means of all conjugates) were found to be 14.1% unlabeled, 84.2% 2H1,and 1.7% 2H2,which are in excellent agreement with the above predictions if one assumes that the [2H2]APAPcontained some 1-2% of dideuterated molecules which were labeled at sites other than C-3 or C-5. Following administration of [2H,]APAP to mice, the acetaminophen excreted into urine exhibited a labeling pattern almost identical with that of the dosing compound. The thioether conjugates, on the other hand, exhibited partial loss of label and, if one excludes the somewhat anomalous values recorded for APAP-CyS recovered from feces, the average distribution of molecular species was found to be 61.8% unlabeled, 37.8% 2H1,and 0.4% 2H2. Assuming that conjugation of NAPQI with GSH occurs with equal probability at the (equivalent) C-3 and C-5 positions, leading to loss of exactly half of the deuterium present at C-3, then the predicted deuterium distribution of the thioether adducts would be 56.5% unlabeled and 43.5% 2H1. Thus, deuterium is lost from [2H1]APAP
208 Chem. Res. Toxicol., Vol. 3, No. 3, 1990
H o f f m a n n et al.
Table I. Labeling Patterns of Metabolites of APAP Present in the Urine, Bile, and Feces of Mice Dosed with [IsO]APAP, [2HIIAPAP,or r2H21APAP(200 mg kg-I ip) isotopic content, atom % excessa metabolite
compound administered [lsO]APAP (50.1% unlabeled, 48.1% lBO)
[2H1]APAP(16.3% unlabeled, 80.3% 'HI, 3.4% 'Hz)
['HZIAPAP (2.2% 'H,, 23.8% 'HI, 74.0% 'Hz)
analyzed APAP-CyS APAP-NACVS APAP-GS APAP-CyS APAP-NACyS APAPb APAP-CyS APAP-NACyS APAP-GS APAP-CyS APAP-NACyS APAP* APAP-CyS APAP-NACyS APAP-GS APAP-CyS APAP-NACyS APAPb
source urine urine bile feces feces urine urine urine bile feces feces urine urine urine bile feces feces urine
unlabeled 51.6 f 1.3 51.3 f 0.7 50.6 f 0.6 52.5 f 0.7 51.9 f 0.6 50.6 f 0.1 57.0 f 0.7 63.5 f 2.2 58.5 f 1.4 69.5' 68.3 f 5.2 18.2c 14.3 f 0.3 14.3 f 0.3 14.1 f 0.4 13.4 f 0.6 14.2 f 0.4 2.3 f 0.2
'80
'HI
'H2
45.9 f 0.8 47.9 f 0.9 44.6 f 0.5 47.0 f 0.5 47.5 f 0.3 48.7 f 0.2 43.0 f 0.7 35.8 f 3.5 41.1 f 1.1 26.2' 31.3 f 4.7 78.OC 84.0 f 1.1 83.3 f 0.9 84.5 f 0.9 85.6 f 1.2 83.4 f 2.9 24.4 f 1.7
0
0.7 i 0.7 0.5 f 0.5 4.3c 0.4 i 0.4 3.8c 1.7 f 0.8 2.1 f 0.3 1.4 f 0.6 1.0 f 1.0 2.3 f 2.3 73.3 f 2.0
Values represent means f SD ( N = 3). bAPAP in urine was isolated following enzymatic hydrolysis of glucuronide and sulfate conjugates and therefore represents "total" drug. Means of two values.
during biotransformation to NAPQI and subsequent conjugation with GSH to a degree which is only slightly greater than that predicted by the above model.
Discussion The present investigation has shown that when APAP labeled in the phenolic OH group with l80is administered to mice, no loss of label occurs during metabolic conversion of this compound to thioether conjugates excreted in bile, feces, or urine (Table I). This finding is in agreement with the results of an earlier study by Hinson et al. (44),who administered [180]APAPto hamsters and determined the l80 content of APAP-NACyS in urine by reductive cleavage of the adduct with Raney Ni and analysis of the free APAP released. These authors also performed a similar experiment in vitro with hamster liver microsomes fortified with GSH, from which the APAP-GS adduct was isolated and reduced chemically to APAP. In each case, it was shown that metabolic activation of APAP was accompanied by negligible loss of l80from the labeled site (44). Although applicable to the I80-enriched conjugates, the above Raney Ni reduction technique cannot be employed in work with deuterated compounds labeled in activated aromatic rings owing to the complication of proton-deuterium exchange which occurs on the surface of the catalyst (47). The new analytical methodology adopted for use in the present study circumvented this difficulty and permitted the use of [2H1]-and [2H2]APAP as additional metabolic probes. From the labeling patterns of the deuterated conjugates (Table I), it may be concluded that (i) a deuterium atom from either the C-3 or (equivalent) C-5 position is lost quantitatively during the formation of sulfur conjugates of APAP, (ii) this metabolic pathway is not accompanied by any intramolecular migration of deuterium (NIH shift) from C-3 (C-5) to other sites in the APAP nucleus, and (iii) neither the metabolic oxidation of APAP to NAPQI nor the subsequent conjugation reaction is subject to a discernible isotope effect (which would have produced an excess of monodeuterated thioether conjugates from [2H,]APAP relative to the population of labeled molecules predicted solely on the grounds of statistical probability). It may also be noted that the isotopic content of the free APAP excreted in urine was essentially identical with that of the [180]-, [2H1]-,or [2H2]APAPadministered to the mice, notwith-
standing the fact that some fraction of this "unchanged" drug likely would have undergone redox cycling to NAPQI in vivo prior to excretion in urine (6). Additionally, the observation that the labeling patterns of the GSH conjugates present in bile were closely similar in all cases to the respective cysteine and N-acetylcysteine adducts excreted in urine and feces is consistent with the premise that the latter pair are indeed products of further metabolism of APAP-GS (30). With respect to the mechanism by which APAP undergoes cytochrome P-450-mediated bioactivation in vivo, the results of the present study with [180]APAPserve to eliminate pathway B (Figure 1)as a significant contributor to NAPQI formation, since dehydration of the putative free gem-diol intermediate would be expected to proceed with loss of half of the oxygen label present orginally at the 4-position of [180]APAP,a phenomenon that was not observed in practice. Moreover, the fact that each of the metabolites of [180]APAPexhibited complete retention of this label indicates that NAPQI does not exist in biological media as the (potentially more stable) hydrate formed by addition of the elements of water to C-4. In contrast, the results of this investigation are consistent with the "direct" oxidation pathway of APAP metabolism (pathway C, Figure 11,which may be viewed as proceeding by way of either oxygen- or nitrogen-centered radical intermediates (reviewed in ref 36). In this context, it appears that the mechanism depicted in Figure 6 has not been discussed previously, and it is forwarded here as a viable alternative to published schemes that have invoked sequential one-electron transfers in the metabolic oxidation of A P A P (8, 26, 34-37). According to this proposal, the first electron is abstracted by cytochrome P-450 from the phenolic oxygen of the substrate to yield the phenoxy radical (Ia). Recombination of this caged radical species with the heme iron bound oxygen radical generates the labile ferry1 hydroperoxide intermediate (11) which, in turn, collapses to NAPQI. Alternatively, delocalization of the unpaired electron on l a to the aromatic nucleus affords either the C-3-centeredcaged radical (Ib), which represents a logical precursor to 3-hydroxyacetaminophen (3-OHAPAP) (26),or the C-1-centered caged radical (IC)which, following recombination and elimination of acetamide (3, 6 ) , yields p-benzoquinone (BZQ) as end product (36). Thus, according to this scheme, all of the known products
Chem. Res. Toxicol., Vol. 3, No. 3, 1990 209
Metabolic Actioation of Acetaminophen
1* 24012
222/4
1 150
250
350
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04 1
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J
I
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mi
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HpO
3-OH-APAP
Figure 6. Proposed scheme for the cytochrome P-450 dependent metabolism of MAP. Initial abstraction of a single electron from the substrate is viewed as taking place at the phenolic OH group to give the oxygen-centered radical la. Recombination of this caged radical species with iron-bound oxygen affords the ferry1 hydroperoxide (11) which, in turn, collapses to form NAPQI. Analogous recombination processes involving the (carbon-centered) resonance forms of Ia, viz., caged radicals Ib and IC,lead to the generation of 3-OH-APAP and BZQ, respectively. In the representations of the various intermediates depicted in this scheme, the fifth (thiolate) ligand to the heme iron is included to provide electron balance in the redox reactions. Structures enclosed in brackets represent hypothetical intermediates which have not been isolated or trapped in studies of APAP metabolism.
m
I
m
I.
Figure 5. Representative electron impact mass spectra, recorded under GC-MS conditions, of the silyl derivatives obtained from thioether conjugates of APAP present in the excreta of mice dosed with (A) [180]APAP,(B) [2Hl]APAP,and (C) [2H2]APAP.For details, see text. of metabolic oxidation of APAP may be accounted for by invoking one-electron abstraction from a single site on the APAP molecule, viz., the phenolic oxygen. This hypothetical scheme, which is fully consistent with the stable isotope labeling data and lack of deuterium isotope effect (45) observed in the present study, gains further support from the recent work of Van der Straat et al. (31) and Koymans et al. (36),who have shown respectively that close approach of the phenolic OH group of APAP to the active site perferryl oxygen of purified cytochrome P-450 isozymes appears to be a prerequisite for turnover of substrate to NAPQI and that the APAP phenoxy radical
is the most energetically favored radical of APAP from ab initio calculations. An argument against this unified mechanism, however, derives from work by Harvison et al. (26) who have reported recently that incubation of APAP with different purified isozymes of cytochrome P-450 results in the production of 3-OH-APAP and APAP-GS in different ratios. This finding was interpreted to indicate that these two metabolites of APAP do not stem from a common precursor. However, whether differences in the local environment of the APAP phenoxy radical imposed by alterations in the cytochrome P-450 apoprotein structure might influence the distribution and/or rates of recombination of the various resonance forms of the APAP phenoxy radical, and thereby control the ratio of products generated during metabolism of APAP, would appear to be a key issue in this regard, resolution of which will have to await the results of further experimentation and theoretical calculations. Nevertheless, integrated schemes such as that depicted in Figure 6, in addition to their intrinsic appeal, are consistent with developing views of the mechanism of cytochrome P-450 catalyzed oxidation reactions and highlight further the probable importance of sequential one-electron transfers
210 Chem. Res. Toxicol., Vol. 3, No. 3, 1990
in processes considered formally to result from the removal of a pair of electrons (48).
Acknowledgment. We thank Dr. Sidney Nelson (Department of Medicinal Chemistry, University of Washington) for generous gifts of oxygen-18- and deuteriumlabeled analogues of APAP, and for valuable discussions during the course of this work. We are also grateful to Mr. William Howald of the same Department for assistance with the mass spectrometric analyses. One of us (K.-J.H.) thanks AB Hassle for support during a leave of absence at the University of Washington. These studies were supported by a research grant (DK 30699) from the National Institutes of Health, which is gratefully acknowledged. Registry No. APAP, 103-90-2; APAP-SG, 64889-81-2; APAP-CyS, 53446-10-9; APAP-NACyS, 52372-86-8; NAPQI, 50700-49-7; cytochrome P-450, 9035-51-2.
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