AMAP,the alleged non-toxic isomer of acetaminophen,is toxic in rat and human liver

N-acetyl-meta-aminophenol (AMAP) is generally considered as a non-toxic regioisomer of the well-known hepatotoxicant acetaminophen (APAP). However, so far, AMAP has only been shown to be non-toxic in mice and hamsters. To investigate whether AMAP could also be used as non-toxic analog of APAP in rat and human, the toxicity of APAP and AMAP was tested ex vivo in precision-cut liver slices (PCLS) of mouse, rat and human. Based on ATP content and histomorphology, APAP was more toxic in mouse than in rat and human PCLS. Surprisingly, although AMAP showed a much lower toxicity than APAP in mouse PCLS, AMAP was equally toxic as or even more toxic than APAP at all concentrations tested in both rat and human PCLS. The profile of proteins released into the medium of AMAP-treated rat PCLS was similar to that of APAP, whereas in the medium of mouse PCLS, it was similar to the control. Metabolite profiling indicated that mouse PCLS produced the highest amount of glutathione conjugate of APAP, while no glutathione conjugate of AMAP was detected in all three species. Mouse also produced ten times more hydroquinone metabolites of AMAP, the assumed proximate reactive metabolites, than rat or human. In conclusion, AMAP is toxic in rat and human liver and cannot be used as non-toxic isomer of APAP. The marked species differences in APAP and AMAP toxicity and metabolism underline the importance of using human tissues for better prediction of toxicity in man.     

Use of a systems model of drug-induced liver injury (DILIsym) to elucidate the mechanistic differences between acetaminophen and its less-toxic isomer,AMAP, in mice

Acetaminophen (APAP) has been used as a probe drug to investigate drug-induced liver injury (DILI). In mice, 3′-hydroxyacetanilide (AMAP), a less-toxic isomer of APAP, has also been studied as a negative control. Various mechanisms for the divergence in toxicological response between the two isomers have been proposed. This work utilized a mechanistic, mathematical model of DILI to test the plausibility of four mechanistic hypotheses. Simulation results were compared to an array of measured endpoints in mice treated with APAP or AMAP. The four hypotheses included: (1) quantitative differences in drug metabolism profiles as a result of different affinities for the relevant enzymes; (2) differences in the amount of reactive metabolites produced due to cytochrome P450 (CYP450) inhibition by the AMAP reactive metabolites; (3) differences in the rate of conjugation between the reactive metabolites and proteins; (4) differences in the downstream effects or potencies of the reactive metabolites on vital components within hepatocytes. The simulations did not support hypotheses 3 or 4 as the most likely hypotheses underlying the difference in hepatoxic potential of APAP and AMAP. Rather, the simulations supported hypotheses 1 and 2 (less reactive metabolite produced per mole of AMAP relative to APAP). Within the simulations, the difference in reactive metabolite formation was equally likely to have occurred from differential affinities for the relevant drug metabolism enzymes or from direct CYP450 inhibition by the AMAP reactive metabolite. The demonstrated method of using simulation tools to probe the importance of possible contributors to toxicological observations is generally applicable across species.     

Hepatic protein arylation, glutathione depletion, and metabolite profiles of acetaminophen and a non-hepatotoxic regioisomer, 3'-hydroxyacetanilide, in the mouse

The metabolism and disposition of acetaminophen (APAP) and a non-hepatotoxic regioisomer, 3'-hydroxyacetanilide (AMAP), were investigated in the mouse using 14C-labeled analogues. Covalent binding of metabolites of both compounds was observed on the order of 1 nmol/mg tissue protein. AMAP binding was much higher than that of APAP at 1 hr, but by 24 hr, AMAP binding was significantly lower than that of APAP. APAP binding peaked at 3 hr and did not decrease significantly thereafter. Despite the high early levels of covalent binding, AMAP was not as effective in causing glutathione depletion as was APAP. This was reflected in the urinary metabolite profiles of the two compounds. Approximately twice as much APAP was cleared through thioether conjugation compared to AMAP, based on an analysis of urinary metabolites. These results and results of other studies suggest that electrophilic metabolites of AMAP are more reactive than those of APAP, and do not diffuse as far from their site of formation, which may spare some critical target proteins from damage.     

Comparative cytotoxic effects of N-acetyl-p-benzoquinone imine and two dimethylated analogues

N-acetyl-p-benzoquinone imine (NAPQI), a reactive metabolite of acetaminophen, has previously been shown to be toxic to hepatocytes freshly isolated from rat liver [Mol. Pharmacol. 28:306-311 (1985)] NAPQI arylates and oxidizes cellular thiols, and either one or both reactions may be important in the pathogenesis of cytotoxicity. Two dimethylated analogues of NAPQI, N-acetyl-3,5-dimethyl-p-benzoquinone imine (3,5-diMeNAPQI) and N-acetyl-2,6-dimethyl-p-benzoquinone imine (2,6-diMeNAPQI), were prepared to determine whether one reaction might be more damaging to cells than the other. Of the three quinone imines, the least potent cytotoxin to rat hepatocytes was 3,5-diMeNAPQI. However, the cytotoxicity of 3,5-diMeNAPQI was markedly enhanced by pretreatment of cells with 1,3-bis-(2-chloroethyl)-N-nitrosourea, which inhibits glutathione reductase. Reactions of 3,5-diMeNAPQI with GSH, both chemically and in hepatocytes, indicated that this quinone imine primarily oxidized thiols. These findings were corroborated by results of covalent binding experiments, which showed that radiolabeled 3,5-diMeNAPQI bound only to a small extent to hepatocyte proteins. On the other hand, 2,6-diMeNAPQI, the most potent cytotoxin of the three quinone imines that was investigated bound extensively to hepatocyte proteins. In addition, 2,6-diMeNAPQI reacted with GSH, both chemically and in hepatocytes, to form significant amounts of GSSG. Reduction products of NAPQI and its dimethylated analogues were not important contributors to cytotoxicity or GSSG formation based on the following results: 1) the quinone imines did not increase oxygen consumption by hepatocytes nor did they lead to oxygen uptake in solution; 2) dicoumarol, an inhibitor of the reductase, DT-diaphorase, had no effect on cytotoxicity caused by the quinone imines. Evidence for the involvement of ipso-adducts of the quinone imines in their reactions with cellular thiols is provided by results of investigations on the effects of DTT on the metabolism, covalent protein binding, and cytotoxic effects of the quinone imines.     

Involvement of human cytochrome P450 2D6 in the bioactivation of acetaminophen.

Acetaminophen (APAP), a widely used analgesic and antipyretic agent, can cause acute hepatic necrosis in both humans and experimental animals when consumed in large doses. It is generally accepted that N-acetyl-p-benzoquinone imine (NAPQI) is the toxic, reactive intermediate whose formation from APAP is mediated by cytochrome P450. Several forms of P450 in humans, including 2E1, 1A2, 2A6, 3A4, have been shown to catalyze the oxidation of APAP to NAPQI. We now present evidence which demonstrates that human cytochrome P450 2D6 (CYP2D6) is also involved in the bioactivation of APAP. The formation of NAPQI from APAP by cDNA-expressed CYP2D6 was examined. K(m) and V(max) values were 1.76 mM and 3.02 nmol/min/nmol of P450, respectively, such that the efficiency of CYP2D6 in the conversion of APAP to NAPQI is approximately one-third of that of CYP2E1. The contribution of CYP2D6 to the total formation of NAPQI from APAP (1 mM) in human liver was investigated using quinidine (1 microm) as a CYP2D6-specific inhibitor, and varied from 4.5 to 22.4% among 10 livers, with an average at 12.6%. The correlation between the contribution of CYP2D6 to NAPQI formation in human liver microsomes and the CYP2D6 activity probed by the O-demethylation of dextromethorphan was studied, and found to be strong (r(2) = 0.85), and significant (P <.0001). Our findings indicate that CYP2D6, one of the major P450 isoforms in humans and also one of the pharmacogenetically important isoforms, may contribute significantly to the formation of the cytotoxic metabolite NAPQI, especially in CYP2D6 ultra-rapid and extensive metabolizers and at toxic doses of APAP when plasma APAP concentrations reach 2 mM or more.     

Characterization of glutathione conjugates of reactive metabolites of 3'-hydroxyacetanilide, a nonhepatotoxic positional isomer of acetaminophen

3'-Hydroxyacetanilide (AMAP) is a nonhepatotoxic regioisomer of acetaminophen (APAP) that nonetheless does form reactive metabolites which bind to hepatic proteins. Because differences in the nature of reactive metabolites formed from AMAP and APAP may explain differences in their propensity to cause hepatotoxicity, characterization of the reactive metabolites of AMAP was undertaken. The naturally occurring sulfhydryl-containing tripeptide glutathione (GSH) was used to trap the reactive metabolites. Four mono-GSH conjugates and one di-GSH conjugate of oxidative AMAP metabolites were characterized by 1H 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'-hydroxy-acetanilide (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. The presumed oxidation product, N-acetyl-3-methoxy-p-benzoquinone imine (MAPQI), was synthesized and found to 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.(ABSTRACT TRUNCATED AT 250 WORDS)     

Covalent modification and time-dependent inhibition of human CYP2E1 by the meta-isomer of acetaminophen

The hypothesis that N-acetyl-m-aminophenol (AMAP), the meta isomer of acetaminophen, will covalently bind to and inhibit human CYP2E1 in a time- and NADPH-dependent manner was investigated. Liquid chromatography/electrospray ionization-mass spectrometry analysis indicated that AMAP metabolites (i.e., AMAP*) selectively and covalently modified CYP2E1 apoprotein in a ratio of 1.4:1 (AMAP*/CYP2E1) in a reconstituted system. The deconvoluted spectra of CYP2E1 apoprotein from incubations containing NADPH and AMAP displayed mass shifts of 167.2 ± 7.1 and 334.4 ± 6.5 Da, suggesting the addition of one and two hydroxylated AMAP metabolites to CYP2E1, respectively. Mass shifts in cytochrome P450 reductase, cytochrome b(5), and heme from these samples were not observed. CYP2E1 inhibition by AMAP increased with time in the presence of NADPH; a reversible inhibition component was also observed. The results support a bioactivation process that involves formation of a hydroquinone metabolite that undergoes further oxidation to a quinone, which reacts with CYP2E1 nucleophilic residues. The data are consistent with evidence from previous studies that identified hydroxylated AMAP glutathione conjugates collected from mice and indicate that cysteine residues are the most likely sites for adduct formation. This study reports the first direct evidence of AMAP-derived hydroquinone metabolites bound to human CYP2E1.     

Role of NAD(P)H:quinone oxidoreductase 1 in clofibrate-mediated hepatoprotection from acetaminophen

Mice pretreated with the peroxisome proliferator clofibrate (CFB) are resistant to acetaminophen (APAP) hepatotoxicity. Whereas the mechanism of protection is not entirely known, CFB decreases protein adducts formed by the reactive metabolite of APAP, N-acetyl-p-benzoquinone imine (NAPQI). NAD(P)H:quinone oxidoreductase 1 (NQO1) is an enzyme with antioxidant properties that is responsible for the reduction of cellular quinones. We hypothesized that CFB increases NQO1 activity, which in turn enhances the conversion of NAPQI back to the parent APAP. This could explain the decreases in APAP covalent binding and glutathione depletion produced by CFB without affecting APAP bioactivation to NAPQI. Administration of CFB (500 mg/kg, i.p.) to male CD-1 mice for 5 or 10 days increased NQO1 protein and activity levels. To evaluate the capacity of NQO1 to reduce NAPQI back to APAP, we utilized a microsomal activating system. Cytochrome P450 enzymes present in microsomes bioactivate APAP to NAPQI, which binds the electrophile trapping agent, N-acetyl cysteine (NAC). We analyzed the formation of APAP–NAC metabolite in the presence of human recombinant NQO1. Results indicate that NQO1 is capable of reducing NAPQI. The capacity of NQO1 to amelioriate APAP toxicity was then evaluated in primary hepatocytes. Primary hepatocytes isolated from mice dosed with CFB are resistant to APAP toxicity. These hepatocytes were also exposed to ES936, a high affinity, and irreversible inhibitor of NQO1 in the presence of APAP. Concentrations of ES936 that resulted in over 94% inhibition of NQO1 activity did not increase the susceptibility of hepatocytes from CFB treated mice to APAP. Whereas NQO1 is mechanistically capable of reducing NAPQI, CFB-mediated hepatoprotection does not appear to be dependent upon enhanced expression of NQO1.     

Immunochemical analysis of acetaminophen covalent binding to proteins. Partial characterization of the major acetaminophen-binding liver proteins

A sensitive immunoassay for detecting acetaminophen (APAP) bound to proteins was developed using an affinity purified antibody directed against the N-acetylated end of the APAP molecule. Western blots of electrophoretically resolved liver proteins taken from mice given an hepatotoxic dose of APAP demonstrated that nearly 85% of the total detectable protein-bound APAP was covalently associated with proteins of 44 and 58 kD. Pretreatment of liver extracts with the sulfhydryl-specific reagent, N-ethylmaleimide (NEM), prior to derivatization with the reactive metabolite of APAP, N-acetyl-p-benzoquinone imine (NAPQI), greatly reduced immunochemically detectable APAP-protein adducts and indicated that the antibody detects protein-thiol conjugates of APAP. To investigate the basis of the binding selectivity in vivo, a variety of systems which yielded APAP-protein adducts were analyzed. Systems which activate APAP enzymatically, as in hepatocyte suspensions or in post-mitochondrial (S9) fractions fortified with an NADPH-regenerating system, resulted in a protein binding profile similar to that produced in vivo. Conversely, when extracts or cells were treated with chemically synthesized NAPQI, an alternative protein binding profile was obtained. Two-dimensional electrophoretic analysis of the reduced protein thiol (PSH) content of liver proteins using [3H]NEM labeling revealed that the 58 kD APAP-binding proteins were rich in PSH, whereas the major 44 kD binding protein had virtually no detectable PSH. Many PSH-rich proteins that were not arylated in vivo did bind NAPQI in vitro. However, the 44 kD proteins were not arylated when chemically synthesized NAPQI was added to homogenates or cell suspensions. The present data further suggest that, in addition to the amount and reactivity of free protein sulfhydryls, the cellular localization with respect to the cytochrome P-450 activation site may influence the susceptibility of proteins to NAPQI binding. These findings signal the need for caution in interpreting studies of APAP mechanisms that rely solely on NAPQI addition.     

Cytotoxic effects of polychlorinated biphenyl hydroquinone metabolites in rat hepatocytes

Polychlorinated biphenyls (PCBs) are persistent organic pollutants that exhibit various toxic effects in animals and exposed human populations. The molecular mechanisms of PCB toxicity have been attributed to the toxicological properties of its metabolites, such as hydroquinones, formed by cytochrome‐P‐450 oxidation. The effects of PCB hydroquinone metabolites towards freshly isolated rat hepatocytes were investigated. Hydroquinones can be oxidized to semiquinones and/or quinone metabolites. These metabolites can conjugate glutathione or can oxidize glutathione as a result of redox cycling. This depletes hepatocyte glutathione, which can inhibit cellular defence mechanisms, causing cell death and an increased susceptibility to oxidative stress. However in the following, glutathione‐depleted hepatocytes became more resistant to the hydroquinone metabolites of PCBs. This suggested that their glutathione conjugates were toxic and that there was a third type of quinone toxicity mechanism which involved a hydrogen peroxide‐accelerated autoxidation of the hydroquinones to form toxic electrophilic quinone and semiquinone–glutathione conjugates. Copyright © 2009 John Wiley & Sons, Ltd.     

Development and evaluation of an electrochemical method for studying reactive phase-I metabolites: correlation to in vitro drug metabolism

A reactive metabolite may react covalently with proteins or DNA to form adducts that ultimately may lead to a toxic response. Reactive metabolites can be formed via, for example, cytochrome P450-mediated phase 1 reactions, and in this study, we report the development and evaluation of an electrochemical method for generating reactive metabolites. Paracetamol was used as a test compound to develop the method. The stability of the electrochemically generated N-acetyl-p-benzoquinoneimine (NAPQI) from paracetamol was investigated at 37 degrees C at pH 5.0, 7.4, and 9.0. The highest stability of NAPQI was observed at pH 7.4. The reaction rate between NAPQI and glutathione (GSH) was studied with cyclic voltammetry. NAPQI reacted quantitatively with GSH within 130 ms. The reactivity of NAPQI toward other nucleophiles was investigated, and for the reaction with N-acetyltyrosine, a time-dependent formation of a conjugate with N-acetyltyrosine was observed from 0 to 4 min. The applicability of the method was evaluated with compounds that were able to form quinone imines (amodiaquine), quinones (3-tert-butyl-4-hydroxyanisole and p-cresol), imine methides (3-methylindole; trimethoprim), quinone methides (3,5-di-tert-butyl-4-hydroxytoluene), and nitrenium ions (clozapine). The compounds were oxidized in an analytical electrochemical cell, and the formed reactive metabolites were trapped with GSH. The samples were then analyzed by LC-MS and LC-MS/MS. For comparison, all compounds were incubated with GSH in rat and human liver microsomes, and the formation of GSH conjugates was compared with that observed by electrochemical oxidation. Furthermore, the electrochemical method was used to synthesize a GSH conjugate of clozapine, which made it possible to obtain structural information by NMR. In summary, a high degree of similarity was observed between the conjugates identified from electrochemical oxidation and GSH conjugates identified from incubation with liver microsomes. In conclusion, we have developed a method that is useful for studies on reactive metabolites and furthermore can be scaled up for the synthesis of GSH conjugates for NMR.     

Inhibition and activation of acetaminophen reactive metabolite formation by caffeine. Roles of cytochromes P-450IA1 and IIIA2.

Caffeine has previously been shown to diminish or potentiate acetaminophen (APAP) hepatotoxicity in rats, depending on induction state. To elucidate the P-450 forms involved in these divergent effects, rat liver microsomes, prepared after pretreatment with various inducers, were used to examine the effect of caffeine on N-acetyl-p-benzoquinone imine (NAPQI) formation. The addition of caffeine to incubations with 3-methylcholanthrene (MC)-induced microsomes resulted in a biphasic effect on the formation of NAPQI. A 43% decrease in NAPQI formation was observed as caffeine concentration was increased from 0 to 0.5 mM; however, NAPQI formation was accelerated as caffeine concentration increased, exceeding the control (no caffeine) value, at caffeine concentrations greater than 2.5 mM. Incubations with purified P-450IA1 showed that as caffeine concentration increased from 0 to 5 mM, a 50% inhibition was observed with no evidence of acceleration. In contrast to MC microsomes, the addition of caffeine to incubations with uninduced and phenobarbital-induced adult rat microsomes resulted in a marked (3- to 4-fold) acceleration of NAPQI formation with no evidence of inhibition. Caffeine (5 mM) also accelerated NAPQI formation in microsomes isolated from diabetic rats, but to a substantially lesser extent (120%), suggesting a modest (if any) effect on P-450IIE1, a form previously shown to form NAPQI from APAP. Interestingly, caffeine caused a 3- to 4-fold increase in NAPQI formation by juvenile male and female rat microsomes, but no activation was observed with adult female rat microsomes. These results suggested that caffeine activated a member of the cytochrome P-450IIIA subfamily.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dissociation of covalent binding from the oxidative effects of acetaminophen. Studies using dimethylated acetaminophen derivatives

The cytotoxic effects of 10 mM acetaminophen (APAP) in primary cultures of non-induced mouse hepatocytes are accompanied by depletion of intracellular glutathione (GSH), arylation of protein, and loss of protein sulfhydryl (PSH) groups. Investigation of the stoichiometry of the covalent binding and PSH loss after APAP exposure demonstrated a greater loss in PSH than could be accounted for by covalent binding to proteins and suggests that APAP exhibits both oxidative and arylative actions in cell culture. Subcellular fractionation revealed that the PSH oxidation induced by APAP was greatest in the microsomal fraction. Exposure of the hepatocytes to 10 mM 3,5-dimethyl-acetaminophen (3,5-DMA) or 2,6-dimethyl-acetaminophen (2,6-DMA) permitted dissociation of the oxidative and arylative properties of APAP. Even though treatment of cultured hepatocytes with 3,5-DMA did not result in covalent binding, there was a more rapid depletion of intracellular GSH, oxidation of PSH, and cytotoxicity compared to APAP. This investigation also provides the first evidence that the cytotoxic effects of both APAP and 3,5-DMA are accompanied by the formation of protein aggregates of high molecular weight that are not disulfide linked. The aggregates probably reflect the oxidative properties of these drugs and may be a mediator of their toxic effects. By contrast, 2,6-DMA, which did bind to cellular proteins and deplete GSH, did not lead to PSH loss, protein aggregation, or cytotoxicity. Since PSH oxidation and protein aggregation correlated well with cytotoxicity, these data suggest that the oxidative component of APAP and 3,5-DMA can play a significant role in eliciting cellular damage in cultured hepatocytes.     

Investigations of the N-hydroxylation of 3'-hydroxyacetanilide, a non-hepatotoxic positional isomer of acetaminophen

The hydroxamic acid of 3'-hydroxyacetanilide (AMAP) was synthesized to test the hypothesis that different reactive metabolites of AMAP and acetaminophen account for similarities in covalent binding of the two positional isomers to hepatic proteins, but for differences in their ability to cause hepatotoxicity. N-OH-AMAP was found to be a relatively stable hydroxamic acid, but it was not detected as a metabolite of AMAP formed in vitro by mouse liver microsomes or in urine of mice administered AMAP. Therefore, metabolites other than N-OH-AMAP must be responsible for covalent binding observed with AMAP to mouse liver proteins.     

Cytotoxic effects of N-acetyl-p-benzoquinone imine,a common arylating intermediate of paracetamol and N-hydroxyparacetamol

The cytotoxic effects of N-acetyl-p-benzoquinone imine (NAPQI), a postulated ultimate reactive metabolite of paracetamol (pHAA), was studied in suspensions of isolated rat hepatocytes. Incubation of cells for 10–300 min with 0.1–0.5 mM NAPQI led to concentration dependent cell damage. as determined by increased trypan blue exclusion, lactate dehydrogenase release and glutathione (GSH) depletion. NAPQI and N-hydroxyparacetamol (N-OH-pHAA), a postulated proximate metabolite of pHAA, caused cytotoxic effects in the same concentration range. In contrast, no toxic effects of pHAA (? 20 mM) could be demonstrated. With the short half-life of NAPQI, less than 0.5% of the NAPQI added is expected to be left in the incubation medium after a 2 min incubated period. Nevertheless, 10–120 min (depending on the concentration of NAPQI) elapsed before the cells responded with increased membrane permeability. Clearly, the initial damage caused by NAPQI must be followed by subsequent cellular steps before toxicity becomes apparent. The addition of N-acetylcysteine, GSH or ascorbate during the NAPQI exposure period fully protected the hepatocytes from NAPQI damage. Lesser effects were demonstrated when these agents were added after the 5 min NAPQI exposure period. The results presented in this study further support the hypothesis that NAPQI is the ultimate reactive formed from pHAA.     

Prevention of acetaminophen (APAP)-induced hepatotoxicity by leflunomide via inhibition of APAP biotransformation to N-acetyl-p-benzoquinone imine

Acetaminophen (APAP) is safe at therapeutic levels but causes liver injury via N-acetyl-p-benzoquinone imine (NAPQI)-induced oxidative stress when overdose. Recent studies indicated that mitochondrial permeability transition (mPT) plays a key role in APAP-induced toxicity and leflunomide (LEF) protects against the toxicity through inhibition of c-jun NH(2)-terminal protein kinase (JNK)-mediated pathway of mPT. It is not clearly understood if LEF also exerts its protective effect through inhibition of APAP bioactivation to the toxic NAPQI. The present work was undertaken to study the effect of LEF on the bioactivation of APAP to NAPQI. Mechanism-based inhibition incubations performed in mouse and human liver microsomes (MLM and HLM) indicated that inhibition of APAP bioactivation to NAPQI was observed in MLM but not in HLM. Furthermore, LEF but not its active metabolite, A77-1726, was shown to be the main inhibitor. When APAP and LEF were incubated with human recombinant P450 enzymes, CYP1A2 was found to be the isozyme responsible for the inhibition of APAP bioactivation. Species variation in CYP1A2 enzymes probably accounted for the different observations in our MLM and HLM studies. We concluded that inhibition of NAPQI formation is not a probable pathway that LEF protects APAP-induced hepatotoxicity in human.     

Gender- and Age-Specific Cytotoxic Susceptibility to Benzene Metabolites in Vitro

Benzene, a widely used compound, is a known carcinogen and hematopoietictoxicant. Several studies have shown gender and age differencesin the responses to benzene-induced hematotoxic-ity. It is notknown if these differences in response are due to age-or gender-associatedmetabolic differences or to age- or gender-associated differencesin the susceptibilities of the target cells. In order to addressthis issue, mouse colony-forming units-erythroid (CFU-e, anerythroid precursor cell particularly susceptible to benzenetoxicity) were cultured in the presence of either individualbenzene metabolites or binary mixtures of these metabolites.CFU-e were obtained from unexposed age-matched adult male andfemale (both virgin and pregnant) Swiss Webster (SW) mice andfrom SW male and female 16-day fetuses. The metabolites usedwere phenol, hydroquinone, catechol, benzoquinone, and trans,trans-muconic acid. The concentrations of the individual metabolitesused were 10, 20, and 40 µM. Binary mixtures of metaboliteswere prepared using the lowest concentrations of the individualmetabolites that caused cytotoxicity. These concentrations were10 µM for hydroquinone, catechol, and benzoquinone, and40 µM for phenol and muconic acid. In general, the CFU-efrom adult females (both virgin and pregnant) were more resistantto the toxic effects of the individual metabolites than CFU-efrom other subjects. CFU-e from adult males were more susceptibleto the cytotoxic effects of hydroquinone and benzoquinone thanCFU-e from other subjects and CFU-e from both male and femalefetuses were highly sensitive to the toxic effects of catechol.On the other hand, CFU-e from adult males were less susceptibleto the cytotoxic effects of catechol than CFU-e from other subjects.Similar results were observed with binary mixtures of metabolites.CFU-e from adult males were more susceptible to the binary mixturesthan CFU-e from virgin females and CFU-e from fetal males weremore susceptible than CFU-e from fetal females. In addition,CFU-e from fetuses were more resistant than CFU-e from adultsto the cytotoxic effects of those binary mixtures that did notcontain catechol. In contrast, binary mixtures containing catecholwere more toxic to fetal cells than to adult cells. These resultssuggest that differences in benzene hematotoxicity associatedwith gender and age may be due, at least in part, to intrinsicfactors at the level of the target cell rather than solely toage- or gender-related differences in the metabolism of benzene.     

Activation of acetaminophen-reactive metabolite formation by methylxanthines and known cytochrome P-450 activators.

Previous in vitro studies suggested that caffeine enhanced acetaminophen (APAP) oxidation to N-acetyl-p-benzoquinone imine (NAPQI) by selectively activating the male-specific constitutive cytochrome P-450IIIA2. Monomethylxanthine and dimethylxanthine analogs of caffeine (also metabolites) were studied for their potential effect to accelerate NAPQI formation in various preparations of rat liver microsomes. In contrast to caffeine, none of the mono- and dimethylxanthines (2.5 mM) activated P-450. Rather, the analogs either inhibited NAPQI formation or had no effect; 1-methylxanthine (2.5 mM) was the only compound which consistently inhibited (25-70%) APAP oxidation in all microsomal preparations. Thus, all three methyl groups appear to be required for P-450 activation by methylxanthines. Because of the highly selective activation effect of caffeine, it was of particular interest to determine whether other known P-450 activators could enhance APAP oxidation. Both acetone (400 mM) and flavone (50 microM) accelerated NAPQI formation in all microsomal preparations, whereas metyrapone caused only inhibition. Flavone (50 microM) caused a pattern of activation similar to that observed with 5 mM caffeine (maximal activation of 125-300%), except that NAPQI formation was increased approximately 40% by flavone in microsomes prepared from adult females, whereas no activation was caused by caffeine. Acetone yielded a pattern of P450 activation very different from that of either caffeine or flavone; the maximal degree of activation (3 times control) was observed in microsomes prepared from adult females. In contrast to caffeine and flavone, the degree of activation by acetone in microsomes prepared from juvenile animals was considerably lower (50%) than that observed in adult microsomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Contribution of acetaminophen-cysteine to acetaminophen nephrotoxicity II. Possible involvement of the gamma-glutamyl cycle

Acetaminophen (APAP) nephrotoxicity has been observed both in humans and research animals. Our recent investigations have focused on the possible involvement of glutathione-derived APAP metabolites in APAP nephrotoxicity and have demonstrated that administration of acetaminophen-cysteine (APAP-CYS) potentiated APAP-induced renal injury with no effects on APAP-induced liver injury. Additionally, APAP-CYS treatment alone resulted in a dose-responsive renal GSH depletion. This APAP-CYS-induced renal GSH depletion could interfere with intrarenal detoxification of APAP or its toxic metabolite N-acetyl-p-benzoquinoneimine (NAPQI) and may be the mechanism responsible for the potentiation of APAP nephrotoxicity. Renal-specific GSH depletion has been demonstrated in mice and rats following administration of amino acid gamma-glutamyl acceptor substrates for gamma-glutamyl transpeptidase (gamma-GT). The present study sought to determine if APAP-CYS-induced renal glutathione depletion is the result of disruption of the gamma-glutamyl cycle through interaction with gamma-GT. The results confirmed that APAP-CYS-induced renal GSH depletion was antagonized by the gamma-glutamyl transpeptidase (gamma-GT) inhibitor acivicin. In vitro analysis demonstrated that APAP-CYS is a gamma-glutamyl acceptor for both murine and bovine renal gamma-GT. Analysis of urine from mice pretreated with acivicin and then treated with APAP, APAP-CYS, or acetaminophen-glutathione identified a gamma-glutamyl-cysteinyl-acetaminophen metabolite. These findings are consistent with the hypothesis that APAP-CYS contributes to APAP nephrotoxicity by depletion of renal GSH stores through interaction with the gamma-glutamyl cycle.     

Susceptibility to acetaminophen (APAP) toxicity unexpectedly is decreased during acute viral hepatitis in mice

Acetaminophen (APAP) hepatotoxicity results from cytochrome P450 metabolism of APAP to the toxic metabolite, n-acetyl-benzoquinone imine (NAPQI), which reacts with cysteinyl residues to form APAP adducts and initiates cell injury. As APAP is commonly used during viral illnesses there has been concern that APAP injury may be additive to that of viral hepatitis, leading physicians to advise against its use in such patients; this has not been investigated experimentally. We infected C57BL/6 male mice with replication-deficient adenovirus to produce moderately severe acute viral hepatitis and observed that APAP doses that were hepatotoxic or lethal in control mice produced neither death nor additional increase in serum ALT when administered to infected mice at the peak of virus-induced liver injury. Moreover, the concentration of hepatic APAP-protein adducts formed in these mice was only 10% that in control mice. Protection from APAP hepatotoxicity also was observed earlier in the course of infection, prior to the peak virus-induced ALT rise. Hepatic glutathione limits APAP-protein adduct formation but glutathione levels were similar in control and infected mice. Cyp1a2 (E.C. 1.14.14.1) and Cyp2e1 (E.C. 1.14.13.n7) mRNA expression decreased by 3 days post-infection and hepatic Cyp2e1 protein levels were reduced almost 90% at 7 days, when adduct formation was maximally inhibited. In vitro, hepatocytes from virally infected mice also were resistant to APAP-induced injury but sensitive to NAPQI. Rather than potentiating APAP-induced liver injury, acute viral hepatitis in this model resulted in selective down-regulation of APAP metabolizing P450s in liver and decreased the risk of APAP hepatotoxicity.