Chem. Res. Toxicol. 1989,2, 41-45
41
Characterization of Glutathione Conjugates of Reactive Metabolites of 3’-Hydroxyacetanilide, a Nonhepatotoxic Positional Isomer of Acetaminophen Mohamed S. Rashed and Sidney D. Nelson* Department of Medicinal Chemistry, BG-20, Uniuersity of Washington, Seattle, Washington 98195 Received August 3, 1988
3’-Hydroxyacetanilide (AMAP) is a nonhepatotoxic regioisomer of acetaminophen (APAP) t h a t nonetheless does form reactive metabolites which bind t o hepatic proteins. Because differences in the nature of reactive metabolites formed from AMAP and APAP may explain differences in their propensity t o cause hepatotoxicity, characterization of the reactive metabolites of AMAP was undertaken. The naturally occurring sulfhydryl-containing tripeptide glutathione (GSH) was used t o trap the reactive metabolites. Four mono-GSH conjugates and one di-GSH conjugate of oxidative AMAP metabolites were characterized by ‘H NMR and soft ionization (LSIMS or FAB) mass spectral techniques, as well as by comparison of liquid chromatographic and spectral characteristics with synthetic standards. Two isomeric mono-GSH conjugates of 2-acetamidohydroquinone (2-AcHQ) are formed as well as a bis-GSH conjugate. A mono-GSH conjugate of 3’,4’-dihydroxyacetanilide(3-OH-APAP) also was formed. Thus, these GSH conjugates most likely arise by reaction of GSH with 2-acetamido-p-benzoquinone(2-APBQ) and 4-acetamido-o-benzoquinone(4-AOBQ), respectively, as oxidation products of the known AMAP metabolites 2-AcHQ and 3-OH-APAP. Finally, a GSH conjugate of 3’-methoxy-4’-hydroxyacetanilide (3-OMe-APAP) was detected in bile of mice administered AMAP. This conjugate probably arises by oxidation of 3-OMe-APAP, another known metabolite of AMAP. T h e imine (MAPQI), was synpresumed oxidation product, N-acetyl-3-methoxy-p-benzoquinone thesized and found t o react with GSH to give the same GSH conjugate as that detected in bile and in incubations of 3-OMe-APAP with mouse liver microsomes plus GSH. These results indicate t h a t reactive metabolites of AMAP are similar to N-acetyl-p-benzoquinone imine (NAPQI), the major toxic metabolite of APAP, in their reactions with GSH to form arylthioether adducts.
Introduction One mechanism that has been proposed as an initiating event in the pathogenesis of hepatic necrosis caused by large doses of the widely used analgesic-antipyretic, acetaminophen (APAP) is covalent binding of a reactive oxidative metabolite(s) to hepatocellular proteins (1). Although several lines of evidence support this hypothesis (2-5),results of other studies are at variance with it (6-9). A particularly interesting compound that may provide some insight into the role that covalent binding plays in APAP-mediated acute lethal cell injury is a regioisomer, 3’-hydroxyacetanilide (AMAP). AMAP binds to hepatic proteins of hamsters almost as extensively as APAP but does not cause hepatic injury (10).This isomer also does not cause hepatic injury in mice (ll),yet it covalently binds to mouse liver microsomal proteins more extensively than APAP (12). The results of recent investigations (12,13) show that two diphenolic acetanilides, 2-acetamidohydroquinone (2-AcHQ) and 3-hydroxyacetaminophen (3-OH-APAP),are primary metabolites of AMAP formed in mice. Evidence also has been presented that 2-AcHQ is oxidized further to 2-acetamido-p-benzoquinone(APBQ) in vitro by mouse liver microsomes (12,14). The results of the work presented herein support this finding, and in addition they indicate that 3-OH-APAP and its subsequent metabolite, 3-methoxyacetaminophen (3-OMe-APAP), are oxidized *Author to whom correspondence should be sent.
both in vitro by mouse liver microsomes and in vivo in mice to an o-quinone and a quinone imine, respectively, that form glutathione (GSH) conjugates.
Experimental Procedures Materials. Materials and their sources were as follows: silver(1) oxide, 4-nitroguaiacol, acetic anhydride, potassium nitrosodisulfonate (Fremy’s salt), and AMAP, from Aldrich; APAP, bovine serum albumin, NADPH, and GSH, from Sigma; [ringU-’4CJAPAP and [ring-U-14C]AMAP,from Pathfinder Labs, St. Louis, MO; Aquasol-2 scintillant, from New England Nuclear; male Swiss-Webster mice (16-25 g), from Charles River Labs, Boston, MA. All HPLC solvents were passed through a 0.45-rm nylon-66 membrane prior to use. HPLC analyses were performed on an LKB Model 2152-25D dual-pump instrument equipped with an LKB Model 2151 variable-wavelength UV detector used a t 254 or 280 nm. Fractions were collected directly into scintillation vials either manually or with an LKl3 Redirac fraction collector. Liquid scintillation counting was performed on a Packard Tri-Carb 2000CA instrument. Counts were automatically corrected for quenching by the external standard channels ratio method. ‘H NMR spectra were recorded on a Varian VXR-300 spectrometer, as the solvent and 2,2-dimethyl-2-silapentane5-sulfonic with 2Hz0 acid (DSS) as internal standard. LSIMS spectra were performed on a Kratos MS-505 mass spectrometer. FAB mass spectra were recorded on a VG 70-SEQ mass spectrometer. Synthesis. 2-AcHQ (12),3-OH-APAP (15),3-OMeAPAP (15), 4-AOBQ (16),and 2-APBQ (14) were synthesized as previously described. N-Acetyl-3-methoxy-p-benzoquinone imine (MAPQI) was synthesized by adding Ag20 (510 mg, 0.22 “01) to a solution of 3-OMe-APAP (181mg, 0.1 mmol) in 50 mL of dry chloroform.
0893-228~/89/2702-0041$01.50/0 0 1989 American Chemical Society
Rashed and Nelson
42 Chem. Res. Toxicol., Vol. 2, No. 1, 1989 The reaction was stirred for 25 min a t room temperature. Activated charcoal was added and the mixture filtered through Celite. The chloroform was removed under reduced pressure to yield a semisolid which solidified on cooling (165 mg, 92%). The quinone imine was further purified by sublimation (70-75 "C a t 0.03 mmHg) to give yellow crystals, mp 89-90 "C. 'H NMR (300 MHz; Cs2H6)6 6.38 (dd, 1 H, J 6 , 5 = 10.0 Hz and J6,2 = 2.0 Hz), 6.04 (d, 1H, J5,6 = 10.0 Hz), 5.57 (d, 1 H, J2,6 = 2.0 Hz), 2.85 (s, 3 H), 1.93 ( s , 3 H; EI/MS (direct probe) m / z 179 (M)", 181 (M + 2)'+, 164 (M - CH3)'+, 139 (M 2 - C2H20)", 137 (M - CzH20)", 136 (M - CH,CO)'+; UV (CHCI,) A, (E) 236 nm (7898), 274 nm (18600), 356 nm (3056). Anal. Calcd for C9HBN0$ C, 60.33; H, 5.06; N, 7.82. Found: C, 60.72, H, 5.03, N, 7.48. General Procedure for Synthesis of the GSH Conjugates. In a 5-mL Reacti-vial, GSH (0.12 mmol) was dissolved in 2.5 mL of deionized water and sealed under nitrogen. The quinone or quinone imine (0.1 mmol) was dissolved in 1 mL of acetonitrile (HPLC grade) and injected gradually through the septum of the vial under nitrogen. The reaction mixture was stirred for 30 min and acidified with formic acid to pH 2.0 and the acetonitrile removed by using a gentle stream of nitrogen. The resulting aqueous solutions were frozen a t -20 "C until HPLC purification was performed. Following lyophilization the powders obtained were stored immediately a t -20 "C. For NMR purposes, the powders were dissolved in 2H20and lyophilized once more. 'H NMR and LSIMS Data for the Synthetic Glutathione Adducts. (A) 2-Acetamido-5-(glutat hion-S-yl)-1,4-hydroquinone and 2-Acetamido-6-(glutathion-S-yl)-l,4-hydroquinone. 'H NMR b 7.12 (s, 1 H), 7.03 (s, 1H), 6.95 (d, 1 H, J38,5, = 3.0 Hz), 6.90 (d, 1 H, J5,,,, = 3.0 Hz), 4.50-4.42 (m, 2 H), 3.73 (t, 2 H), 3.69-3.55 (m, 4 H), 3.44-3.32 (m, 2 H), 3.26-3.18 (m, 2 H), 2.45-2.40 (m, 4 H), 2.20, 2.19 (2 s, 6 H), 2.08 (q, 4 H). LSIMS m / z 473 (M H)', 495 (M + Na)+. (B) 2-Acetamidobis(glutathion-S-y1)-l,4-hydroquinone. 'H NMR b 7.37 (s, 1 H), 4.34-4.31 (m, 2 H), 3.75-3.71 (br t, 2 H), 3.60 (d, 4 H), 3.35 (dd, 2 H), 2.45-2.42 (m, 4 H), 2.22 (s, 3 H), 2.18-2.06 (m, 4 H). LSIMS m / z 778 (M H)', 800 (M + Na)'. (C) 3-(Glutathion-S-yl)-5-acetamidocatechol. 'H NMR 6 7.00 (d, 1 H, J = 2.3 Hz), 6.98 (d, 1 H, J = 2.3 Hz), 4.54-4.46 (m, 1H), 3.73 (t,1H), 3.65-3.59 (m, 2 H), 3.42-3.34 (dd, 1H), 3.28-3.20 (dd, H), 2.47-2.40 (m, 2 H), 2.20 (s, 3 H), 2.11-2.01 (m). LSIMS m / z 473 (M + H)', 495 (M + Na)+. (D) 3-(Glutathion-S-yl)-5-methoxyacetaminophen.'H NMR d 7.08 (d, 1 H, J = 2.0 Hz), 7.04 (d, 1 H, J = 2.0 Hz), 4.48-4.35 (m, 1H), 3.87 (s, 3 H), 3.75 (t, 1H), 3.60 (dd, 2 H), 3.40 (dd, 1 H), 3.25 (dd, 1 H), 2.46-2.40 (m, 2 H), 2.15 (s, 3 H), 2.08 ( q , 2 H). FABMS m / z 487 (M + H)+, 509 (M + Na)', 358 (M H - 129)'. Methods. Microsomes were prepared from the livers of male Swiss-Webster mice (16-25 g) which had been induced with phenobarbital sodium (0.1% solution as drinking water for 5 days). Protein content of microsomal preparations was determined by a modification of the method of Lowry et al. (17). Incubation Conditions. Incubations contained either [14C]AMAP(1.0 mM, sp act. 0.62 mCi/mmol) or 3-OMe-APAP (1.0 mM), microsomes (2 mg/mL), and NADPH (1mM) in a final volume of 3 mL of 0.1 M potassium phosphate buffer, pH 7.4. Some incubations also contained glutathione (2 mM). All incubations were carried out for 20 min in a shaking water bath a t 37 OC. The incubations were terminated by addition of 1.0 mL of HCl (6 M). The precipitated proteins were removed by centrifugation (lOOOg, 15 min) and used for covalent binding determination (18). The supematants were filtered through nylon-66 filters (0.45 pm) and analyzed for metabolites by HPLC. Formation of Glutathione Adducts in Vivo. A group of ten phenobarbital-pretreated Swiss-Webster mice were dosed with [14C]AMAP(600 mg/kg ip, sp act. 20 pCi/mol) in warm saline (0.5 mL). Four hours after administration of the drug, the mice were sacrificed and their gall bladders excised into a cup and punctured. The pooled bile was filtered through nylon-66 filter, and aliquots were analyzed by HPLC and scintillation counting of 1-min fractions. HPLC Conditions. Analyses and purifications were performed on a 6-pm Ultrasphere Cle column (10 mm X 25 cm) protected with a 5-pm Ultrasphere precolumn. Three solvent systems were employed for HPLC work as follows:
I 1
I
128AUFS
+
+
+
+
10
20
Time (min) Figure 1. HPLC chromatogram of reaction mixture of 2-APBQ and GSH. Column used was a 5-pm ultrasphere ODS (25 cm X 4.6 mm). The mobile phase consisted of solvent A [0.1 M sodium acetate adjusted to pH 4.0 with glacial acetic acid/acetonitrile (99:l v/v)] and solvent B (acetonitrile). A linear gradient was employed from 0% B to 10% B at 30 min with a flow rate of 1.0 mL/min. (A) System I. This system was used for quantitation of metabolites and conjugates from microsomal incubations. The mobile phase consisted of solvent A [2% acetonitrile and 98% 0.1 M KHzPOl (pH of buffer adjusted to 2.5 with 85% phosphoric acid)] and solvent B (acetonitrile). A linear gradient was employed from 0% to 40% B over 40 min a t a flow rate of 2.5 mL/min. Reverse gradient to 100% A was accomplished in 20 min. Under these conditions the following retention times were obtained for the synthetic standards: 3-OH-APAP, 13.5 min; 2-AcHQ; 14.2 min; glutathione conjugate of 4-AOBQ, 14.8 min; glutathione conjugates of 2-APBQ, coelute at 15.5 min. (B) System 11. This system was used for quantitation of the bis-glutathione conjugate of APBQ. The system was operated isocratically with water/acetonitrile/glacial acetic acid (96:2:2) v/v) at a flow rate of 3.0 mL/min. The bis-glutathione conjugate eluted at 12.5 min. (C) System 111. This system was used for purification of the synthetic glutathione adducts and analysis of bile samples. The mobile phase consisted of solvent A [water/acetonitrile/glacial acetic acid (96:2:2 v/v)] and solvent B (acetonitrile). A linear gradient was employed form 0% to 20% B over 40 min, and to 40% B at 50 min at a flow rate of 2.0 mL/min. Return to 100% A was affected by a reverse gradient for 15 min. Under these conditions the following retention times were obtained: glutathione conjugate of 4-A0BQ1 24.0 min; glutathione conjugates of 2-APBQ, coelute a t 25.0 min; glutathione conjugate of MAPQI, 32.5 min.
Results Synthetic GSH Conjugates. The reaction of 2-APBQ with GSH resulted in the formation of four major products, the reduction product, 2-AcHQ (peak 2, Figure l),and three GSH conjugates that were identified by 'H NMR and LSIMS following HPLC purification. Two mono-GSH conjugates that coeluted in HPLC systems 1-111 were slightly resolved (peaks 3 and 4, Figure 1) by using a different system (legend of Figure 1). 'H NMR analysis (Figure 2) revealed that peaks 3 and 4 are two positional isomers, 2-acetamido-5-(glutathion-S-yl)-l,4-hydroquinone and 2-acetamido-6-(glutathion-S-yl)-1,4-hydroquinone. This regioisomeric mixture gave a protonated molecular ion at mlz 473 and sodium adduct ions a t mlz 495 in LSIMS. HPLC peak 1 (Figure 1) was determined to be a bis-GSH conjugate of 2-AcHQ. The 'H NMR spectrum showed one singlet in the aromatic region vs three methyl
Chem. Res. Toxicol., Vol. 2, No. 1, 1989 43
GSH Conjugates of 3'-Hydroxyacetanilide 0
0
I1
I1
COCHC S
I
CH,
Qt
I
HzN,
,CHCH$H,OC-N HOOC
/CH\
H CONCH&OOH QlU
75
70
45
40
35
30
g1u
20
25
PPm
Figure 2. 'H NMR of the regioisomeric conjugates of 2-AcHQ. Signals for the internal standard, DSS, appear at 2.9 ppm as indicated. The spectrum is truncated to eliminate the large signal from HOD.
10
20
Time (min)
Figure 4. HPLC analysis of supernatants of microsomal incubations of [14C]AMAPand GSH. Solvent system I was used and is described under Experimental Procedures.
OCH
Table I. Effect of GSH on Covalent Binding and Metabolism of AMAPa
0
(nmol/mg of protein)/20 min
COCl
metabolites -GSH +GSH covalent binding to microsomes 4.3 f 0.1 0.4 0.0 2-AcHQ 32.9 f 2.0 19.7 f 2.0 3-OH-APAP 19.7 f 1.3 16.5 f 0.6 mono-GSH conjugates of 2-AcHQ 12.9 f 0.9 bis-GSH conjugate of 2-AcHQ 2.8 f 0.2 mono-GSH conjugate of 3-OH-APAP 5.1 f 0.4
*
LL 710
"
415
4.0
total metabolites
56.9
57.4
"Results are means f SD ( N = 5). 3.5
310
215
2:0
PPm
'H NMR of 3-(glutathion-S-yl)-5-methoxyacetaminophen. See legend to Figure 2 for further comments. Figure 3.
protons of the acetamido group. In addition, the spectrum showed the characteristic aliphatic protons of GSH which integrated for 2 molecules of GSH per aromatic nucleus. This conclusion was supported by LSIMS data which showed a protonated molecular ion at mlz 778 and a sodium adduct ion at mlz 800. When 4-AOBQ was allowed to react with GSH, one major product was formed. 'H NMR analysis of the purified product showed that the material was 3-(glutathion-S-yl)-5-acetamidocatechol. When the novel quinone imine MAPQI was allowed to react with GSH, the products detected were 3-OMe-APAP and a mono-GSH conjugate. This conjugate was determined by 'H NMR and LSIMS to be 3-(glutathion-Syl)-5-methoxyacetaminophen(Figure 3). Enzymatic Formation of GSH Adducts of AMAP in Vitro. Incubation of [14C]AMAPwith liver microsomes and GSH resulted in the appearance of two radioactive peaks (c and d) on HPLC chromatograms (Figure 4). These were identified as the mono-GSH conjugates of 3-OH-APAP and 2-AcHQ, respectively, by cochromatography with the synthetic standards. These peaks were not present in incubations lacking GSH or NADPH. Whether GSH was present or not, the two previously reported diphenolic metabolites, 3-OH-APAP and 2-AcHQ, were detected (peaks a and b, respectively, Figure 4). The radioactivity associated with each peak was collected manually and measured by liquid scintillation counting. The
bis-GSH conjugate of 2-APBQ was also detected by using a different HPLC solvent system. The results of quantitation of AMAP metabolites are summarized in Table I. Covalent binding of AMAP in the presence and absence of GSH was determined in the same incubations, and results are reported in Table I. Addition of GSH to microsomal incubations of [14C]AMAPabolished NADPHdependent covalent binding and resulted in a significant decrease 03 C 0.05) in the apparent rates of formation of 3-OH-APAP and 2-AcHQ. The decrease in both cases can be accounted for by the formation of the corresponding GSH conjugates. Presumably, if GSH is not present, a portion of each quinone that is formed is reduced back to the corresponding diphenolic metabolite. Formation of GSH Adducts of AMAP in Mice. Administration of [14C]AMAPto mice results in the appearance of radioactive peaks in their bile that had the same HPLC retention times as GSH conjugates of 3-OHAPAP and 2-AcHQ (peaks a and b, respectively, Figure 5). These peaks cochromatographed with synthetic standards. Radioactivity associated with peak a (7% of recovered radioactivity) was collected and subjected to LSIMS analysis. It showed a protonated molecular ion at mlz 473. A major radioactive peak c (14% of recovered radioactivity) eluted later in the chromatogram (Figure 5). LSIMS analysis of the purified material gave a strong protonated molecular ion a t mlz 487, and two additional signals a t mlz 509 and 525 indicative of the sodium and potassium adduct ions, respectively. The mass difference between this material and that of the mono-GSH conjugates of 3-OH-APAP and 2-AcHQ (MW = 472) suggested that this
44
Rashed and Nelson
Chem. Res. Toricol., Vol. 2, No. 1, 1989
I
C
I
b 10
20
30
40
Time (min) Figure 5. HPLC analysis of bile samples collected from mice 4 h after the administration of 600 mg/kg [14C]AMAP.Solvent system I11 was used and is described under Experimental Procedures.
compound was a GSH conjugate of 3-OMe-APAP. The synthesis of the authentic GSH conjugate supported this conclusion. HPLC analysis of supernatants from incubations of 3-OMe-APAP with hepatic microsomes, NADPH, and GSH also showed a peak that cochromatographed with the material isolated from bile and with the synthetic standard.
Discussion In a previous report by Roberts et al. (10) it was suggested that the lack of hepatotoxicity of AMAP resides in the nature of reactive intermediateb) of AMAP vs APAP. Since AMAP is a m-hydroxyacetanilide, it cannot be directly oxidized to a quinone imine as is thought to be the case for its p-hydroxyacetanilide isomer, APAP. Since NAPQI is a relatively unstable compound that readily reacts with reducing agents as well as nucleophiles, it cannot be detected directly in vivo. Its formation, however, was supported by the isolation of 3-(glutathion-S-yl)acetaminophen from bile of rats administered APAP (19). In the present study we provide evidence for the formation of three different reactive intermediates as a result of AMAP oxidative metabolism. Both 0-and p-quinones are formed by further oxidation of 3-OH-APAP and 2AcHQ. These intermediates, in the presence of GSH, are trapped as their thioether adducts. The data in Table I clearly indicate that essentially all of the P-450-dependent covalent binding in vitro is due to species that are readily trapped by GSH. In addition, the rate of formation of the diphenolic metabolites is decreased significantly in the presence of GSH. The decrease in both covalent binding and diphenolic metabolites can be accounted for by the formation of the glutathione adducts, in that there is no significant difference between the total amount of the substrate metabolized in the absence and presence of GSH. The reason for the decrease in rates of formation of the diphenolic metabolites is not altogether clear. However, on the basis of previous work with 2-AcHQ and APBQ (14), we believe that, in the absence of GSH, NADPH may serve to reduce the quinones that result from oxidation of the diphenolic metabolites back to the diphenols. The detection of a bis-GSH conjugate of 2-AcHQ in microsomal incubations indicates that the corresponding mono-GSH adducts may be oxidized further, either en-
zymatically or through autoxidation, to quinones or quinone imines of the mono-GSH adducts that undergo conjugation with another molecule of GSH to give the bis adduct. Although GSH conjugates of 2-AcHQ apparently are formed at approximately three times the rate of GSH conjugates of 3-OH-APAP in mouse liver microsomal incubations, the major GSH conjugates in the bile of mice treated with AMAP were those derived from 3-OH-APAP. There are several possible reasons for this apparent anomaly. First, 2-AcHQ formed in vivo might be a better substrate for other competing pathways, such as sulfation and glucuronidation. Second, 2-AcHQ may be less easily oxidized in the cell than 3-OH-APAP, or its oxidation product may be more easily reduced than that of 3-OHAPAP. Finally, GSH conjugates of 2-AcHQ may be further oxidized more easily to quinones that covalently bind to cellular macromolecules and thereby do not leave the cell. The results of the study in vivo show that the GSH conjugates of both quinone and quinone imine metabolites of AMAP are formed in mice and excreted in the bile. These conjugates are formed sequentially from products of oxidation of AMAP to dihydroxylated metabolites and either their further oxidation to quinones (2-APBQ and 4-AOBQ) or methylation followed by oxidation to a quinone imine (MAPQI). Two of the proximate metabolites of AMAP, 3-OH-APAP and 3-OMe-APAP, also are metabolites of APAP (15). Both metabolites were tested for their ability to cause hepatotoxicity in mice (15), and 3OH-APAP was essentially nonhepatotoxic. 3-OMe-APAP was about as potent a hepatotoxin as APAP, but not enough of this metabolite is likely formed from AMAP to cause hepatoxicity, though this remains to be determined. The results of the studies in vitro and in vivo clearly indicate that both APAP and AMAP form quinone and quinone imine metabolites that bind to protein thiol groups. Therefore, the difference in ability to cause hepatotoxicity by APAP and AMAP is either (1)unrelated to their covalent protein binding or (2) related to the subcellular location of the binding, reactivity with specific proteins, and/or intrinsic binding characteristics of the individual reactive metabolites to proteins. Work is in progress to examine these various possibilities. Acknowledgment. This work was supported by NIH Grant GM25418 (S.D.N.). Registry No. AMAP, 621-42-1; 3-OMe-APAP-GSH, 11865559-7; MAPQI, 118630-02-7;5-GSH-2-AcHQ,118629-99-5;6-GSH2-AcHQ, 118630-00-5;bis(GSH)-2-AcHQ,118655-58-6;3-GSH5-acetamidocatechol,118630-01-6.
References (1) Jollow, D. J., Mitchell, J. R., Potter, W. Z., Davis, D. C., Gillette, J. R., and Brodie, B. B. (1973) Acetaminophen-induced hepatic necrosis. 11. Role of covalent binding in uiuo. J.Pharmacol. Exp. Ther. 187, 195-202. (2) Smith, C. V., and Mitchell, J. R. (1985) Acetaminophen hepatotoxicity in vivo is not accompanied by oxidant stress. Biochem. Biophys. Res. Commun. 133, 329-336. (3) Porubek, D. J., Rundgren, M., Harvison, P. J., Nelson, S. D., and MoldBus, P. (1987) Investigation of mechanisms of acetaminophen toxicity in isolated rat hepatocytes with the acetaminophen analogues 3,5-dimethylacetaminophenand 2,6-dimethylacetaminophen. Mol. PharmacoE31, 647-653. (4) Roberts, D. W., Pumford, N. R., Potter, D. W., Benson, R. W., and Hinson, J. A. (1987) A sensitive immunochemical assay for acetaminophen-protein adducts. J . Pharmacol. Exp. Ther. 241,
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( 5 ) Bartalone, J. B., Sparks, K., Cohen, S. D., and Khairallah, E. A.
(1987) Immunochemical detection of acetaminophen-bound liver proteins. Biochem. Pharmacol. 36, 1193-1196.
GSH Conjugates of 3’-Hydroxyacetanilide (6) Devalia, J. L., Ogilvie, R. C., and McLean, A. E. (1982) Dissociation of cell death from covalent binding of paracetamol by flavones in a hepatocyte system. Biochem. Pharmacol. 31, 3745-3749. (7) Rosen, G. M., Singletary, W. V., Jr., Rauckman, E. J., and Killingberg, P. G. (1983) Acetaminophen hepatotoxicity: an alternative mechanism. Biochem. Pharmacol. 32, 2053-2059. (8) Gerson, R. J., Casini, A., Gilfor, D., Serrone, A., and Farber, J. L. (1985) Oxygen-mediated cell injury in the killing of cultured hepatocytes by acetaminophen. Biochem. Biophys. Res. Commun. 126, 1129-1137. (9) Tee, L. B. G., Boobis, A. R., Hugett, A. C., and Davies, D. S. (1986) Reversal of acetaminophen toxicity in isolated hamster hepatocytes by dithiothreitol. Toxicol. Appl. Pharmacol. 83, 294-3 14. (10) Roberts, S. A., and Jollow, D. J. (1979) Acetaminophen structure-toxicity studies: in vivo covalent binding of a nonhepatotoxic analog, 3-hydroxyacetanilide. Fed. Proc. 38,426 (Abstract). 1) Nelson, E. B. (1980) The pharmacology and toxicology of meta-substituted acetanilide. I. Acute toxicity of 3-hydroxyacetanilide in mice. Res. Commun. Chem. Pathol. Pharmacol. 28, 447-456. 2) Streeter, A. J., Bjorge, S. M., Axworthy, D. B., Nelson, S. D., and Baillie, T. A. (1984) The microsomal metabolism and site of
Chem. Res. Toxicol., Vol. 2, No. 1, 1989 45 covalent binding to protein of 3’-hydroxyacetanilide, a non-hepatotoxic positional isomer of acetaminophen. Drug Metab. Dispos. 12,565-576. (13) Hamilton, M., and Kissinger, P. T. (1986) The metabolism of 2- and 3-hydroxyacetanilide. Drug. Metab. Dispos. 14, 5-12. (14) Streeter, A. J., and Baillie, T. A. (1985) 2-Acetamido-p-benzoquinone: a reactive arylating metabolite of 3’-hydroxyacetanilide. Biochem. Pharmacol. 34,2871-2876. (15) Forte, A. J., Wilson, J. M., Slattery, J. T., and Nelson, S. D. (1984) The formation and toxicity of catechol metabolites of acetaminophen in mice. Drug Metab. Dispos. 12, 484-491. (16) Ansell, M. F., Bignold, A. F., Gosden, A. F., Leslie, V. J., and murrlay, R. A. (1971) The Diels-Alder reactions of o-benzoquinones with acyclic dienes. J . Chem. SOC.C, 1414-1422. (17) Lowry, 0. H., Rosebrough, N. J., Fan, A. L., and Randall, R. J. (1951) Protein measurement with the Fohn phenol reagent. J. Biol. Chem. 193, 265-275. (18) Streeter, A. J., Dahlin, D. C., Nelson, S. D., and Baillie, T. A. (1984) The covalent binding of acetaminophen to protein. Evidence for cysteine residues as major sites of arylation in uitro. Chem.-Biol. Interact. 48, 349-366. (19) Hinson, J. A., Monks, T. J., Hong, M., Highet, R. J., and Pohl, L. R. (1982) 3-(Glutathion-S-yl)acetaminophen:a biliary metabolite of acetaminophen. Drug Metab. Dispos. 10, 47-50.
