Chem. Res. Toxicol. 1988,1, 47-52
47
Cytochrome P-450 Isozyme Selectivity in the Oxidation of Acetaminophen Peter J. Harvison,? F. Peter Guengerich,t Mohamed S. Rashed,t and Sidney D. Nelsont* Department of Medicinal Chemistry, BG-20, University of Washington, Seattle, Washington 98195, and Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University, Nashville, Tennessee 37232 Received October 2, 1987
Highly purified isozymes of cytochrome P-450 catalyzed the formation of 3-glutathion-Sylacetaminophen (GS-APAP) and 3-hydroxyacetaminophen (3-OH- APAP) from acetaminophen (APAP). A major isozyme from untreated male rats (P-45hT-A)catalyzed the formation of ca. 2.0 nmol/nmol of P-450/10 min of 3-OH-APAP and approximately 7.2 nmol of GS-APAP/nmol of P-450/10 min. Antibodies specific for cytochrome P - 4 5 h - A caused a decrease in the amounts of both metabolites produced in microsomal incubations. In contrast to these results, two other constitutive P-450 isozymes from rat liver, cytochrome P - 4 5 k - F and the female specific isozyme P-450"~-1,produced less of both oxidative metabolites. Moreover, they produced significantly more of the catechol metabolite than the glutathione conjugate. These results are in accord with the observation that male rats are more susceptible to acetaminophen hepatotoxicity than female rats. Isozymes induced by phenobarbital also produced more of the catechol than the glutathione conjugate. Conversely, the major isozyme induced by 8-naphthoflavone, cytochrome P-450@NF-B, produced a significantly greater amount of GS-APAP than 3-OH-APAP. When comparison was made to a major phenobarbital inducible form (cytochrome P-454~43)a definite isozyme specificity for the formation of the two metabolites was seen. The catechol was formed at rates of 2.21 and 0.53 nmol/nmol of P-450/10 min by cytochromes P-450pB-B and P-4508NF-B, respectively. On the other hand, cytochrome P-450pB-B produced 1.62 nmol/nmol of P-450/ 10 min of GS-ASAP versus 4.26 nmol/nmol of P-450/10 min for cytochrome P-450pNF-B. These results show that 3-OH-APAP and GS-APAP arise primarily from different intermediates. Furthermore, a reactive intermediate in the oxidation of APAP by cytochrome P-450, N acetyl-p-benzoquinone imine (NAPQI), was directly detected in single turnover experiments with cytochrome Pm450@NF-B, whereas the amount formed by cytochrome P-450pB-B was below the limits of detection for the assay.
I ntroductlon The mild analgesic and antipyretic agent, acetaminophen (APAP), is generally considered to be a safe drug. Overdoses, however, can result in severe hepatic damage in both man and experimental animals ( I ) . The mechanism of bioactivation of APAP has therefore been the subject of intense studies for a number of years. It is now widely accepted that cytochrome P-450 is responsible for the bioactivation of APAP in the liver (1-3). N-Acetyl-p-benzoquinoneimine (NAPQI) is believed to be the toxic, reactive intermediate produced from APAP by cytochrome P-450 (1-3), and it has been detected as a cytochrome P-450-derived product of APAP in incubations supported by cumene hydroperoxide (4). NAPQI has been prepared in crystalline form and does possess the chemical and toxicological characteristics expected for a reactive intermediate (4-8). Any NAPQI generated from a therapeutic dose of APAP is normally detoxified by reaction with glutathione (GSH) (9). In fact, 3-glutathion-S-ylacetaminophen(GS-APAP) has been directly detected in the bile of rats receiving
* Author to whom all correspondence should be sent. t University f
of Washington. Vanderbilt University.
APAP (10). Glutathione-derived metabolites have also been detected in the urine of hamsters ( I l ) , rats (12), and humans (13). In an overdose situation GSH becomes depleted, and a larger fraction of the NAPQI formed reacts with cellular proteins. A second type of oxidative metabolite of APAP is the catechol 3-hydroxyacetaminophen (3-OH-APAP)(14-15), and 3-methoxyacetaminophen is the major catechol metabolite found in vivo (15). The catechol metabolite is produced by cytochrome P-450 and exhibits little hepatotoxicity. It is possible that the structurally related metabolites GS-APAP and 3-OH-APAP could arise by reaction of NAPQI with glutathione or water, respectively. However, Hinson et al. (14)have shown by isotopic labeling experiments that 3-OH-APAP is not formed by reaction of NAPQI with water. Alternatively, both NAPQI and 3-OH-APAP could be derived from a common precursor such as a 3,4-epoxide (Figure 1). Evidence has been presented that is not entirely consistent with such a mechanism in that cobaltous chloride pretreatment proteds mice from APAP hepatotoxicity without affecting the production of 3-OH APAP (15). This finding might indicate that different cytochrome P-450 isozymes are involved in the formation of GS-APAP and 3-OH-APAP. However, cobaltous chloride has also been demonstrated to elevate GSH levels (16)and this point could complicate 0 1988 American Chemical Society
48 Chem. Res. Toxicol., Vol. 1, No. 1, 1988
F on
i
HYAc
0 on
J
6 3 on
HO
1
0
I
&& 0 .(
GOH HO
Figure. 1. Possible routes of metabolism of APAP to NAPQI and 3-OH-APAP through a common intermediate.
the interpretation of the experiments. Therefore, we investigated the metabolism of APAP by highly purified isozymes of cytochrome P-450. By using HPLC, we were able to quantify the amounts of GS-APAP and 3-OH-APAP produced by each isozyme. The results clearly support the hypothesis that NAPQI and the catechol are formed at different rates by different isozymes of cytochrome P-450 and that they derive from different intermediates. We also found that the reactive and toxic intermediate of APAP oxidation, NAPQI, is formed more rapidly by cytochrome P-4508NF-B than by cytochrome P-450PB-B.
Experlmental Procedures Materials. Materials and their sources were as follows: silver(1) oxide, Florisil, 4-nitrocatechol, acetic anhydride, from Adrich; APN,bovine serum albumin, NADPH, NADP, dilauroyl phosphatidyl choline, glutathione, glucose-6-phosphate, glucose-6-phoaphate dehydrogenase, from Sigma; [n'ng-U-14C]APAP, from Pathfiider Labs, St. Louis, MO; Aquasol-2-scintillant,from New England Nuclear; ultrapure dimethyl sulfoxide, from Alfa Products, Danvers, MA; and male Sprague-Dawley rats, from Charles River Labs, Boston, MA. All HPLC solvents were passed through a 0.45-pm nylon-66 membrane prior to use. HPLC analyses were performed on a Waters component system consisting of two Model 6000A solvent delivery systems, a Model U6K injector, a Model 660 solvent programmer, and a Model 440 UV absorbance detector (254-nm filter). Fractions were collected directly into scintillation vials by using an LKB Redirac fraction collector. Liquid scintillation counting was performed on a Beckman LS-7500 instrument. Counts were automatically corrected for quenching by using the external standard channels ratio method. NAPQI (6)and 3-OH-APAP (15) were s y n t h e s d d as previously described. GS-APAP was synthesized by reacting NAPQI and GSH in the presence of a phase transfer catalyst and base. To NAPQI (12 mg, 0.08 mmol) in chloroform (4 mL) was added a solution of GSH (122 mg, 0.4 mmol) and tetrabutylammonium bisulfate in NaOH (4 mL, 0.5 M). The reaction was stirred vigorously for 30 min at 37 OC. The aqueQus layer was separated and neutralized with HCl to pH 7.2 and then washed three times with two volumes of diethyl ether to remove any acetaminophen or polymeric material. The remaining aqueous fraction was then
Harvison e t al. eluted through a Sephadex LH-20 column with 15% aqueous methanol at a flow rate of 0.20 mL min-'. GS-APAP had an elution volume of 85-95 mL as determined by UV recording at 254 nm. Lyophilization gave a white powder of the sodium salt of GS-APAP that was recrystallized from 80% ethanol to yield 29 mg of off-white crystals, mp 218-221 "C. The material had HPLC and MS characteristics consistent with those reported previously (17). Methods. The cytochrome P-450 isozymes were isolated and purified as previously described (18). The isozymes are identified as follows: P-450UT.A, major isozyme from untreated male rats; P-450UT.F, minor isozyme from untreated male rats; P-450UT.I, major isozyme from untreated female rats; P-450pB.B, major isozyme from phenobarbital pretreated male rats; P-450pB.cand P-450pB.D, minor isozymes from phenobarbital pretreated male rats; P - 4 5 0 p ~ ~major . ~ , isozyme isolated from pregnenolone16a-carbonitrilepretreated rats; P-4508NF.B, major isozyme isolated from 8-naphthoflavone pretreated rats; and P-4501SF.G,major isozyme isolated from isosafrole pretreated rats. For comparison to other literature preparations, see ref 19. Antibodies specific for P-450UT.A were raised in female New Zealand white rabbits and purified by cross-adsorption vs immobilized female rat microsomes and were the same used in a study on hormonal regulation of the enzyme (20). Microsomes were prepared from the livers of male SpragueDawley rats. Protein content of microsomal preparations was determined by a modification of the method of Lowry et al. (21). Carbon monoxide difference spectra were used to determine cytochrome P-450 content in the microsomes (22). Incubation Conditions. Isozyme incubations contained the appropriate P-450 isozyme (1pM), purified rat liver NADPHcytochrome P-450 reductase (1.5 pM), dilauroylphosphatidylcholine (30 pM), NADPH (0.5 mM), glucose-6-phosphate(10 mM), glucose-6-phosphate dehydrogenase (1IU/mL), GSH (10 mM), and 14C-APAP(1mM, sp act. 1.5 mCi/mmol) in 1.0 mL of 0.05 M potassium phosphate buffer, pH 7.4. After a 3-min preincubation at 37 "C, reactions were initiated by addition of NADPH. The incubations were allowed to proceed for 10 min at 37 "C and terminated by freezing in dry ice-acetone. After thawing, the proteins were precipitated by the addition of ice-cold methanol (1mL) and centrifuged (2000g, 20 min). The cold carriers, GSAPAP and 3-OH-APAP,were added to each sample to give a final concentration of ca. 0.05 mM. After evaporation of the methanol under Nz (40 "C) the samples were lyophilized. The buffer salts were precipitated with methanol ( 5 mL) and removed by centrifugation. The organic solvent was evaporated under Nz, and the samples were stored at -80 "C prior to analysis by HPLC. HPLC analysis was performed on a Waters pBondapak C18 column (3.9 mm X 30 cm) with UV detection at 254 nm. The mobile phase consisted of 92% 0.075 M KHzP04(1%in acetic acid) and 8% methanol pumped isocratically at a flow rate of 1.0 mL/min. The column was flushed with 100% methanol between injections. Under these conditions the following retention times were obtained for synthetic standards: 3-OH-APAP, 7.0 min; APAP, 10.5 min; GS-APAP, 17 min. Fractions were collected at 0.5- or 1-min intervals bracketing the appropriate retention times of the metabolites, and the radioactivity was measured by liquid scintillation counting (15 mL of Aquasol-2). Conditions for the radiometric detection of NAPQI were as previously described ( 4 ) except that NADPH and NADPH-cytochrome P-450 reductase (each at concentrations of 1 pmol/pmol of cytochrome P-450) replaced cumene hydroperoxide and incubations were only allowed to proceed for 2 inin. The specific activity of 14C-APAPused for these experiments was 12.3 mCi/mmol. Microsomal Ilicubation Conditions. Microsomal incubations contained 14C-APAP(1.0 mM, sp act. 0.50 mCi/mmol), microsomes (2 mg/mL), glutathione (10 mM), NADP (0.33 mM), glucose-6-phosphate (9.2 mM), MgClz (6.7 mM), and glucose-6phosphate dehydrogenase (1.33 units/mL) in a final volume of 0.5 mL of 0.05 M sodium phosphate buffer, pH 7.4. Antibodies were added to give the following final concentrations: IgG anti UT-A (0,2,5,10,25 mg/nmol of P-450), and preimmune antibody (10 and 25 mg/nmol of P-450). Antibodies and microsomes from untreated male rats were preincubated at room temperatures for 20 min with shaking. GSH and the regenerating system were added and the incubations then preincubated for 3 min at 37 "C
Chem. Res. Toxicol., Vol. 1, No. 1, 1988 49 I
18-
!
Table I. Metabolism of Acetaminophen by Purified Cytochrome P-450Isozymes nmol of product/nmol of
II
P-450 isozymen UT-A UT-F UT-I PB-B PB-C PB-D PCN-E PNF-B ISF-G
P-450/10 minb 3-OH-APAP GS-APAP 7.19 i 0.96' 1.99 f. 0.18 1.15 i 0.19' 2.74 i 0.59 0.27 i 0.06 0.95 & 0.66 0.53 i 0.07' 2.21 f. 0.26 0.56 f 0.13 0.65 f 0.22 0.22 i 0.06 1.04 i 0.42 0.23 i 0.15 1.09 f 0.31 4.26 f 0.65' 1.62 f 0.18 2.49 i 0.06 2.32 i 0.50
"See Materials and Methods section for a description of the isozyme nomenclature and incubation conditions. * Results are means i SD (N = 3). CSignificantdifference between 3-OH-APAP and GS-APAP at p < 0.05. 1201
0
5
10
15
20
Time, min
Figure 2. HPLC chromatogramsof lyophilized and reconstituted incubations of 14C-APAP with cytochrome P-450UT-A and NADPH-cytochrome P-450 reductase in the presence (A) and absence (B)of NADPH. Samples were prepared and analyzed as described in Methods. The dashed line (- - -) indicates UV absorbance at 254 nm and the bar graphs indicate radioactivity collected. The times of elution based on UV absorbance were as follows: 3-OH-APAP,7.0 min; APAP, 10.5 min; GS-APAP, 17 min. in a Dubnoff Lab-Line shaking incubator. Substrate was added and the reaction allowed to proceed for 10 min at 37 "C. The incubations were terminated by addition of ice-cold methanol (1 mL) and subsequent chilling on ice. Samples were worked up in a manner similar to that described above for the isozyme incubations. After workup the samples were stored at -80 O C until analysis by HPLC (see above).
Results Studies with Purified Isozymes. Incubation of 14CAPAP with purified cytochrome P-450 and GSH resulted in the appearance of two major radioactive peaks on HPLC chromatograms (Figure 2A). These have been identified as 3-OH-APAP (7.0 min) and GS-APAP (17.0 min) by cochromatography with authentic synthetic standards. Radioactivity was associated with each peak as measured by liquid scintillation counting of column eluent. The GS-APAP peak was absent and the 3-OH-APAP peak diminished in size in control incubations in which NADPH was omitted (Figure 2B). The origin of the radioactivity eluting under the 3-OH-APAP peak in control incubations is unknown. However, correction was made for this interference. The results of incubations of 14C-APAPwith nine purified isozymes of cytochrome P-450 are summarized in Table I. Significantly different (p < 0.05) amounts of the two metabolites were produced by the constitutive isozymes, P-450UT.A and P-450UT.F. Cytochrome P-4501iT.A (male-specific) generated a 3.5-fold greater amount of GS-APAP than 3-OH-APAP. The opposite situation was obtained with the minor isozyme P-450UT.F in which the catechol metabolite predominated. The difference in production of the two metabolites by the female-specific constitutive isozyme, P-45OUTSI, was not statistically significant.
v ." I
B
0.20
0 00 0
5
10
15
20
25
30
gG (mginmol P450)
Figure 3. Effect of the addition of varying amounts of rabbit gamma globulins (gG) on the formation of GS-APAP ( 0 , O ) or 3-OH-APAP (m, 0)in incubations of liver microsomes prepared from untreated male Sprague-Dawley rats. Closed symbols are in the presence of IgG anti UT-A antibody, and open symbols are in the presence of preimmune antibody. There was no significant difference €or 3-OH-APAPformation in the presence of either IgG anti-UT-A or preimmune antibody. Therefore, the closed squares are superimposed on the open squares. All isozymes that were inducible by phenobarbital (P~ ~ O P BP- B - ~, ~ ~ P PB -- ~c ~, ~ P and B-D P-450p~~-E) , produced greater amounts of 3-OH-APAP than GS-APAP. This difference was significant (p < 0.05) with the exception of the two minor isozymes P-45opB.c and P-45QpB-D. Cytochrome P-45opB-B,the major phenobarbital-inducible form was by far the most active in the production of 3-OHAPAP. The opposite situation was obtained with the major P-naphthoflavone-inducibleisozyme, P-450Bm.B. In this case, a significantly greater amount (p < 0.05) of GS-APAP was produced than 3-OH-APAP (Table I). Roughly equal amounts of both metabolites were produced by cytochrome P-4501SF.G. The results of the studies on GS-APAP formation with cytochromes P-4508NF-B and P-450pB.B are consistent with the results of studies on the direct detection of NAPQI. The concentrations of NAPQI determined in four incubations of 14C-APAPwith cytochrome P-450Bm.Bwere (35 f 16) X M, whereas with cytochrome P-450pB-Bconcentrations of NAPQI were below the detection limits of the assay which were determined to be 5.7 x M by the propagation of errors method (23). Immunochemical Inhibition Studies. A series of incubations with microsomes from untreated rats were run in the presence of increasing concentrations of rabbit IgG anti UT-A or preimmune antibody. The relative amounts of GS-APAP and 3-OH-APAP produced in the presence of increasing concentrations of the antibodies are shown
50 Chem. Res. Toxicol., Vol. 1, No. 1, 1988
in Figure 3. A dramatic decrease (ca. 65%) in GS-APAP levels was noted as IgG anti UT-A antibody concentration increased from 0 to 25 mg/nmol of P-450. Increasing antibody concentration also suppressed production of 3-OH-APAP. Preimmune antibody had no effect on GSAPAP production up to 10 mg/nmol of P-450; above this concentration a slight decrease was noted. Production of 3-OH-APAP was decreased at all concentrations of preimmune antibody. We do not know the reason for this effect. However, 3-OH-APAP is rapidly oxidized to an orthoquinone in the presence of trace amounts of transition metals (unpublished results) which may have been present in the rather large amounts of preimmune antibody that were used.
Discussion It is now widely accepted that cytochrome P-450 exists as a family of isozymes with varying substrate specificities (24). The metabolic activation of APAP by purified forms of cytochrome P-450 isolated from rats pretreated with phenobarbital or p-naphthoflavone has been investigated (25). Using covalent binding of W-APAP to bovine serum albumin as an index of NAPQI formation, it was found that the isozyme induced by 0-naphthoflavone was highly active in catalyzing the metabolic activation of APAP. In contrast, the major isozyme induced by phenobarbital was nearly inactive in this regard. The ability of various cytochrome P-450 isozymes purified from rabbit liver to catalyze the formation of GS-APAP from APAP has been shown (26). Again, a definite isozyme selectivity was observed in that isozymes 4 and 6 (induced by p-naphthoflavone) and isozyme 3a (induced by ethanol) produced GS-APAP, whereas isozyme 2 (induced by phenobarbital) was relatively inactive in this regard.l Hayes et al. (27) investigated the influence of “phenobarbital-type” and “p-naphthoflavone-type”polychlorinated biphenyls (PCBs) on the toxicity of APAP to cultured rat hepatocytes.2 Both induction states potentiated toxicity. However, the cytochrome P-450 inhibitor SKF-525A [2-(diethylamino)ethyl 2,2-diphenylvalerate] inhibited cytotoxicity in cells induced by a “phenobarbital-type” PCB, but not in cells induced by a “8-naphthoflavone-type”inducer. On the other hand, a-naphthoflavon had the opposite effect, which suggested that one isozyme may preferentially activate acetaminophen. In the present study we have investigated the metabolism of acetaminophen using highly purified cytochrome P-450 isozymes from rat liver. The most interesting results are the following: (1)Relative ratios for the formation of 3-OH-APAP vs GS-APAP range from approximately 5 down to approximately 0.3 depending on the cytochrome P-450 isozyme. (2) Of the constitutive isozymes examined, the male-specific cytochrome P-45OUTSA formed GS-APAP about 25 times faster than the female specific cytochrome P-45OUT+This observation is in accord with the fact that male rats are more sensitive to the hepatotoxic effects of APAP than female rats (28). (3) Cytochrome P - 4 5 0 ~ ~ - * accounts for approximately half of the total cytochrome P-450 in male rat liver microsomes that oxidizes APAP (results based on antibody ~ t u d i e s ) . ~(4) The reactive The apparent structural orthologues of rabbit P-450 forms 3a, 4, and 6 in rats are P-450j, P-4501SF.~, and P-4508NF.B,respectively (34). Although an allusion to possible acetaminophen oxidation by rat P-450, has been made (35),experimental data has never been reported and we did not isolate and test this particular P-450. This designation of the types of inductive effects of the PCB’s is somewhat simplistic and the reader is referred to the study of Dannan e t al. (36).
Harvison et al.
metabolite of APAP, NAPQI, is formed at very low rates from phenobarbital-inducible isozymes from rat liver and significantly higher rates by p-naphthoflavone-inducible isozymes. This last conclusion is based on results obtained both by trapping NAPQI as its GSH conjugate and by directly detecting NAPQI in single turnover experiments with purified cytochromes P-450pBBand P-45OBwsB.These latter observations also are in agreement with the finding that 6-naphthoflavone is more effective than phenobarbital in enhancing the susceptibility of rats to acetaminophen hepatotoxicity (2). In summary, the results strongly suggest that NAPQI and 3-OH-APAP are primarily formed by different reactions that do not proceed through a common intermediate. The results obtained with the purified isozymes do not show a parallel isozyme selectivity for the production of the two metabolites (Table I). This is significant because the formation of 3-OH-APAP represents a detoxication pathway whereas the formation of NAPQI is a toxication pathway. Thus, the cytochrome P-450 isozyme distribution in any given animal or human may be an important factor in susceptibility to APAP-mediated liver damage. In fact, species differences in susceptibility to hepatotoxicity have been attributed to rates of conversion of APAP to NAPQI (29). Since there is little doubt that GS-APAP arises from direct conjugation of GSH with NAPQI, the question remains as to the origin of NAPQI and the catechol metabolite. A previous paper from this laboratory (25) proposed a mechanism whereby the production of both compounds could arise from a common intermediate. However, such a mechanism (shown in an abbreviated form in Figure 1)is not consistent with the results reported herein. Therefore, we propose that 3-OH-APAP arises either by an oxidation of the aromatic ring in radical abstraction or addition pathways that are consistent with current theories (30-32) or by initial oxidation of the phenolic group. In contrast, NAPQI must be formed via a pathway that does not involve the same intermediates, presumably initial N-oxidation (not N-oxygenation). Thus, isozyme selectivity for the two pathways may be dictated by the proximity of either the phenolic group or the acetamido group to the P-450 oxyheme. Mechanisms for the formation of 3-OH-APAP and NAPQI through distinct intermediates are shown in Figure 4. The pathways invoke one-electron abstraction from either the phenolic oxygen or the amide nitrogen followed by rapid recombination of the radical pair to form either the catechol or a cytochrome P-450 hemesite-bound hydroxamic acid. The latter intermediate can essentially dehydrate to generate NAPQI and a ferric state of the heme. Such a hypothesis is consistent with the results of experiments which demonstrate that the metabolism of The antibody inhibition experiment requires a caveat. Bandiera e t al. (38)have reported that some other forms of P-450 are immunochemically cross-reactive with a protein which appears to be identical with P - 4 5 0 ~ . & One of these proteins was designated P-450g and is malespecific (39); therefore cross-adsorption of anti-UT-A versus female rat microsomes might not remove anti-P-450g if such antibodies were present. Immunoblots of microsomes done with anti-P-45o“T.A detected only one band (18,37)and P-45Og was resolved from P-45ouT.A (P-450h) by electrophoresis (40). However, since P-45Og was not available and not used in direct electrophoretic comparisons, the absence of anti-P-450g in the antibody (anti-P-45hT.*) cannot be proven unambiguously. P-450g has low testosterone G&hydroxylase activity (40) and anti-P-450uT.Adid not inhibit this activity in microsomes (ZO),but the activity is probably overshadowed by the contribution P-450PCN.E(19). However, purified P-450UT.Awas devoid of testosterone G@-hydroxylaseactivity and we conclude that the acetaminophen oxidation is due to this enzyme. I t should also be pointed out, however, that some evidence for multiple forms of P - 4 5 0 ~ in ~ .rats ~ has been presented (39).
Chem. Res. Toxicol., Vol. 1, No. 1, 1988 51
Oxidation of Acetaminophen by P-450
“7“
Acknowledgment. This work was supported by NIH Grants GM32165 and GM25418 (S.D.N.) and ES01590 (F.P.G.).
--r---
S
S
References
O H-0
O H-NCOCH,
-
HNCOCH,
SH
Q
(1) Hinson, J. A. (1980) “Biochemicaltoxicology of acetaminophen”. Reu. Biochem. Toxicol. 2, 103-130. (2) Mitchell, J. R., Jollow, D. J.; Potter, W. Z., Davis, D. C., Gillette, J. R., and Brodie, B. B. (1973) ‘Acetaminophen-induced hepatic
OH
187, 185-194. (3) Corcoran, G. B., Mitchell, J. R., Vaishnav, Y. N., and Homing, E. C. (1984) “Evidence that acetaminophen and N-hydroxyacet-
necrosis. I. Role of drug metabolsm”. J.Pharmacol. Exp. Ther.
1 v SH
0.
COCH 3
’
+I$ /
HNCOCH,
I
HNCOCH,
OH
i Y’,-
1 “^f/‘
SH
SH
aminophen form a common arylating intermediate, N-acetyl-pbenzoquinone imine”. Mol. Pharmacol. 18, 536-542. (4) Dahlin, D. C., Miwa, G. T., Lu, A. Y. H., and Nelson, S. D. (1984) “N-acetyl-p-benzoquinoneimine: a cytochrome P-450-mediated oxidation product of acetaminophen”. Proc. Natl. Acad. Sci. U.S.A. 81, 1327-1331. (5) Blair, I. A., Boobis, A. R., Davies, D. J., and Cresp, T. M. (1980) “Paracetamol oxidation: synthesis and reactivity of N-acetyl-pbenzoquinone imine”. Tetrahedron Lett. 21, 4947-4950. (6) Dahlin, D. C., and Nelson, S. D. (1982) “Synthesis, decomposition kinetics, and preliminary toxicological studies of pure Nacetyl-p-benzoquinone imine, a proposed toxic metabolite of acetaminophen”. J . Med. Chem. 25, 885-886. (7) Holme, J. A., Dahlin, D. C., Nelson, S. D., and Dybing, E. (1984) “Cytotoxic effects of N-acetyl-p-benzoquinone imine, a common arylating intermediate of paracetamol and N-hydroxyparacetamol”. Biochem. Pharmacol. 33, 401-406. (8) Albano, E., Rundgren, M., Harvison, P. J., Nelson, S. D., and Moldeus, P. (1985) “Mechanisms of N-acetyl-p-benzoquinone imine cytotoxicity”. Mol. Pharmacol. 28, 306-311. (9) Mitchell, J. R., Jollow, D. J., Potter, W. Z., Gillette, J. R., and Brodie, B. B. (1973) “Acetaminophen-induced hepatic necrosis. IV. Protective role of glutathione”. J.Pharmacol. Exp. Ther. 187, 211-217. (10) 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. (11) Gemborys, M. W., and Mudge, G. H. (1981) “Formation and
disposition of the minor metabolites of acetaminophen in the hamster”. Drug Metab. Dispos. 9, 340-351. (12) Hart, S. J., Aguilar, M. I., Healey, K., Smail, M. C., and Calder, I. C. (1984) “Improved high-performance liquid chromatographic separation of urinary paracetamol metabolites using radially compressed columns”. J. Chromatogr. 306, 215-229. (13) Wilson, J. M., Slattery, J. T., Forte, A. J., and Nelson, S. D. (1982) “Analysis of acetaminophen metabolites in urine by highperformance liquid chromatography with UV and amperometric detection”. J. Chromatogr. 227, 453-462. (14) Hinson, J. A., Pohl, L. R., Monks, T. J., Gillette, J. R., and Guengerich, F. P. (1980) “3-Hydroxyacetaminophen: a microsomal metabolite of acetaminophen”. Drug Metab. Dispos. 8,
HNCOCH,
I
03 H
1 7 S
Ho6
289-294. (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) Sasame, H. A., and Boyd, M. R. (1978) “Paradoxical effects of
HNCOCH,
0
Figure 4. Schemes proposed to explain isozyme selectivity in the cytochrome P-450 mediated oxidation of APAP t o 3-OHAPAP (column to the left) and NAPQI (column to the right). The electron distribution and proton localization shown for the oxyheme sulfide system are hypothetical and are simply used to balance the iron coordination state and hydrogen transfer t o and from the substrate.
APAP by microsomes gives rise only to the two-electron oxidation products and not products of one-electron oxidation (33) and by the fact that several other arylamides that cannot dehydrate because of the lack of a p-hydroxy group do form stable hydroxamic acids (34). Furthermore, these stable hydroxamic acids are formed to the greatest extent by P-450 isozymes of the P-naphthoflavone-inducible class as is NAPQI.
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