Edward A. Khairallah

Acetaminophen-induced inhibition of hepatic mitochondrial respiration in mice☆

Morphological changes are observed in mitochondria early in the course of acetaminophen (APAP) hepatotoxicity. In order to determine if functional deficits also occur, this study examined the effect of APAP, in vivo and in vitro, on mitochondrial respiration in fasted, male CD-1 mice (3-4 months old). After a hepatotoxic dose of APAP (600 mg/kg, po), when glutamate was used as the respiratory substrate, state 3 respiration (ADP-stimulated) was inhibited and this was reflected in a decreased respiratory control ratio (RCR). In contrast, when succinate was the respiratory substrate, the decreased RCR was reflective of an increase in state 4 (resting) respiration. There was no detectable effect after a nonhepatotoxic dose of APAP (300 mg/kg, po). These APAP-induced respiratory effects and hepatotoxicity were prevented by piperonyl butoxide pretreatment, and were absent in 1- and 2-month-old mice, which are resistant to APAP-induced damage. Since the APAP-induced inhibition of mitochondrial respiration, in vivo, correlated with age-related and piperonyl butoxide-dependent differences in toxicity, the data suggest that the in vivo effects result, at least in part, from a mixed-function oxidase generated metabolite. In vitro, both state 3 and state 4 respiration, as well as the RCR, were inhibited by APAP in a concentration-dependent manner with glutamate as substrate. However, no effects were observed with succinate as substrate, thereby contrasting with results obtained following in vivo exposure. Therefore the in vitro effects of APAP are different from those observed in vivo and may result from a direct insult of the parent compound. These studies suggest that early alterations in mitochondrial function may be mechanistically important in APAP hepatotoxicity.

Selective Protein Arylation and Acetaminophen-Induced Hepatotoxicity

More than 20 years have passed since the early reports of acute hepatotoxicity with APAP overdose. During that period investigative research to discover the mechanism underlying the toxicity has been conducted in many species and strains of intact animals as well as in a variety of in vitro and culture systems. Such work has clarified the primary role of biotransformation and the protective role of GSH. Understanding the former provides explanations for the toxic interactions which may occur with alcohol or other xenobiotics, while understanding of the latter led to the development of antidotes for the treatment of acute poisoning. Acetaminophen (APAP)-induced hepatotoxicity: roles for protein arylation. Initiating events in toxicity require biotransformation of APAP to NAPQI followed by arylation of several important proteins with subsequent alteration of protein structure and function. The immediate consequence of the alterations is detectable in several organelles and these may represent multiple initiating events which are depicted as acting in concert to cause cell injury (large arrowheads). Arylation of cytosolic 58-ABP with subsequent translocation to the nucleus is depicted as a possible signaling mechanism for determining outcome at the cell or organ level (within dotted boundary). For simplicity NAPQIs potentials for oxidizing protein sulfhydryls and direct binding to DNA have been omitted. Significant light has also been shed on the biochemical and cellular events which accompany APAP-induced hepatotoxicity. However, such studies have not identified a unique mechanism of toxicity that is universally accepted. The recent identification of several protein targets which become arylated during toxicity--along with the findings that arylation of some of those target proteins results in loss of protein function--demonstrates that covalent binding does, indeed, have biological consequences and is not merely an indicator of the fleeting presence of reactive electrophiles. These observations further suggest that multiple independent insults to the cell may be involved in toxicity. it is now apparent that the concept of a multistage process that involves both initiation and progression events is appropriate for APAP toxicity, and it is unlikely that a unique initiating event will ever be identified. In light of recent findings it is more likely that a number of such cellular events occur very early after toxic overdosage, and that they collectively set in motion and perpetuate the biochemical, cellular, and molecular processes which will determine outcome. The importance of 58-ABP arylation with early, apparently selective, translocation to the nucleus remains to be elucidated. To date there is nothing to suggest that this represents an initiating event in toxicity. rather it is plausible that the translocation may play a role in signaling electrophile presence and in calling for cellular defense against electrophile insult. This is reflected in the hypothetical model presented in Fig. 3. Critical experimental testing of this model will advance our understanding of the cellular and molecular responses to toxic electrophile insult.

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.

Identification of a 54-kDa mitochondrial acetaminophen-binding protein as aldehyde dehydrogenase

The covalent binding of acetaminophen (APAP) to mitochondrial proteins has been postulated to alter the function of the organelle and contribute to the development of the hepatotoxicity upon APAP overdose. To identify the arylated proteins CD-1 mice were administered 600 mg/kg APAP and Western blots of mitochondrial proteins collected 4 hr after dosing were probed with anti-APAP antibodies. Five proteins of approximately 75, 60, 54, 44, and 33 kDa were detected on 1-D gels. Immunostaining of the 54-kDa protein was most intense. Mitochondria were subsequently fractionated into inner and outer membrane, matrix, and intermembrane space using digitonin, sonication, and differential centrifugation. The 54-kDa target was most highly enriched in the inner membrane fraction. On 2-D gels this 54-kDa band was resolved into three arylated proteins with pIs of 6.4, 6.6, and 7.1. The pI 7.1 protein was excised from 55 2-D gels, and, after tryptic digestion, the two best-resolved peptides were sequenced and found to be 100% identical to mitochondrial aldehyde dehydrogenase. Coincident with APAP covalent binding the specific activity of the enzyme decreased; by the time of maximal covalent binding at 4 hr after APAP, the activity was 60% of control. Since the enzyme is an abundant mitochondrial dehydrogenase, its decreased activity may contribute to the impaired mitochondrial function observed after APAP administration.

Purification, antibody production, and partial amino acid sequence of the 58-kDa acetaminophen-binding liver proteins

Immunochemical analysis of electrophoretically resolved liver proteins from mice administered hepatotoxic doses of acetaminophen has identified two proteins of 44 and 58 kDa as major targets for acetaminophen arylation. In the present study the 58-kDa acetaminophen-binding protein (58-ABP) was purified from mouse liver cytosol by gel permeation chromatography, preparative isoelectric focusing, and polyacrylamide gel electrophoresis. The acetaminophen adducts were visualized on immunoblots using affinity-purified anti-acetaminophen antibodies after each step of the purification. Gel permeation chromatography, under nondenaturing conditions, indicated that the protein is a monomer. Two-dimensional gel electrophoresis demonstrated that the 58-ABP consists of a cluster of four immunochemically reactive isoforms with isoelectric points ranging from 6.2 to 6.6. V-8 protease digestion of the isoforms suggested that they contained similar peptide fragments. The purified 58-ABP was utilized to produce polyclonal antibodies and to determine the amino acid composition and partial sequence of the protein. These antibodies revealed a protein cluster of similar molecular weight and isoelectric points in the cytosol of a human liver specimen. Amino acid analysis of the purified protein indicated that it contains eight cysteine residues (about 1.4% by weight). This low cysteine content raises the possibility that at hepatotoxic doses acetaminophen may also bind to non-thiol sites on the protein. The amino acid sequence of two cyanogen bromide/tryptic peptide fragments revealed that the major immunochemically detectable acetaminophen target in the cytosol is homologous to a selenium-binding protein which has been recently sequenced.

Immunohistochemical Localization of Acetaminophen in Target Tissues of the CD-1 Mouse: Correspondence of Covalent Binding with Toxicity

Administration of hepatotoxic doses of acetaminophen (APAP) to mice results in necrosis, not only of liver cells but of renal proximal tubules and bronchiolar and olfactory epithelium. In the liver, covalent binding is localized to the centrilobular hepatocytes which later undergo necrosis. This study was undertaken to compare the cellular distribution of bound APAP in all four major target tissues with that of cytochrome P4502E1 (a P450 isoenzyme commonly associated with APAP bioactivation), with emphasis on the cell types which later undergo necrosis. Tissues were collected from mice at selected times after APAP administration (600 mg/kg, po) and fixed by microwave irradiation for immunohistochemistry, or in formalin for histopathological study. Immunohistochemical localization of bound APAP was performed on 5-microns paraffin sections using an affinity-purified anti-APAP antibody. Similar tissues from naive mice were used for immunohistochemical localization of cytochrome P4502E1 (using a polyclonal sheep anti-P4502E1 antibody). Positive staining with both the anti-APAP and the anti-P4502E1 antibodies was similar in distribution, being present in the cell types which become damaged by APAP in all four target tissues. These results demonstrate that covalent binding and subsequent necrosis are localized in common with cytochrome P4502E1, suggesting that, as in the liver, toxicity in extrahepatic targets is also related to the ability of these tissues to activate APAP in situ.

Selective acetaminophen metabolite binding to hepatic and extrahepatic proteins: An in vivo and in vitro analysis

Acetaminophen (APAP) administration (600 mg/kg, po) to fasted male CD-1 mice resulted in cellular damage to liver, lung, and kidney. An affinity purified antibody against covalently bound APAP was used to identify APAP-protein adducts in microsomal and cytosolic extracts from these target organs. The proteins were resolved on SDS-PAGE, transblotted to nitrocellulose membranes, and analyzed immunochemically. Covalent binding of APAP to intracellular proteins was only observed in those organs which exhibited cellular damage; no APAP adducts were detected in tissues which did not undergo necrosis. In all target tissues the arylation of proteins was not random but highly selective with two adducts of 44 and 58 kDa accounting for the majority of the total APAP-bound proteins which were detected immunochemically. In addition, a third major APAP-protein adduct of 33 kDa was also observed in kidney cytosol. The severity of tissue damage and the amount of adducts present in these tissues could be significantly reduced when mice were pretreated with the mixed function oxidase inhibitor, piperonyl butoxide, prior to APAP dosing. Immunochemical analysis of plasma from APAP-treated animals indicated the presence of several protein adducts by 4 hr following drug administration. These adducts did not appear to be of plasma origin. Incubation of cytosolic proteins from liver, lung, kidney, spleen, brain, and heart with an APAP metabolite generating liver microsomal system demonstrated that the cytosolic 58-kDa protein target was native to all tissues tested. By contrast, the 58-kDa protein target did not appear to be endogenous to plasma since it was not detected when plasma was incubated in vitro with the liver microsomal system. These studies indicate that, although the 58-kDa proteins appear to be endogenous to both target and nontarget tissues, the 58-kDa APAP-protein adducts are detectable only in tissues which become damaged by APAP.

Acetaminophen hepatotoxicity: Correspondence of selective protein arylation in human and mouse liver in vitro, in culture, and in vivo

Human and mouse liver were exposed to an APAP-activating system, in vitro. Subsequent immunochemical analysis of electrophoretically separated proteins with an affinity-purified anti-APAP antibody indicated that when a cytosolic fraction from human liver was incubated with APAP, an NADPH-regenerating system, and mouse microsomes selective APAP binding occurred predominantly to proteins of approximately 38, 58, and 130 kDa. To evaluate whether similar proteins are targeted in situ, primary cultures of human hepatocytes were treated with 10 mM APAP for 4 hr prior to immunochemical analysis. APAP binding was again detected in protein bands of approximately 38, 58, and 130 kDa. In addition, selective binding was also noted to other cytosolic protein bands, e.g., approximately 52 and 62 kDa. For mouse liver, the majority of the binding, in vitro or in culture, was to proteins of approximately 44 and 58 kDa with lesser binding to proteins of approximately 33 and 130 kDa among others. By contrast, at the times monitored, little covalent binding was detected in the 44-kDa region in the human liver experiments. Most noteworthy was the finding that when the protein arylation patterns on liver samples from a human APAP fatality were compared to those from a mouse given a hepatotoxic dose of APAP, the binding patterns were similar to those detected after the in vitro and the culture experiments with mouse and human livers. Furthermore, an immunohistochemical analysis revealed that as with the mouse, APAP covalent binding in the human liver exhibited a distinct zonal pattern consistent with centrilobular binding. That APAP arylation of the 58- and 130-kDa proteins was observed in livers from both mice and humans suggests that the mouse provides a valid model for studying the mechanistic importance of covalent binding. Elucidation of the identities and functions of the common targeted proteins may clarify their toxicological significance.

Antidotal effectiveness of N-acetylcysteine in reversing acetaminophen-induced hepatotoxicity: enhancement of the proteolysis of arylated proteins

The post-arylative mechanisms by which N-acetylcysteine (NAC) reduces the severity of the hepatotoxicity induced by acetaminophen (APAP) were investigated in primary cultures of mouse hepatocytes. When administered at selected times immediately following removal of medium containing 10 mM APAP, 2.0 mM NAC was shown to restore glutathione levels through 16 hr of APAP pretreatment and to minimize the leakage of glutamate-oxaloacetate transaminase resulting from the first 8 hr of drug exposure. This temporal difference defined a critical period in which cells were responsive to NAC and permitted the investigation of potential post-arylative mechanisms of the antidote. In the absence of NAC during the recovery period, the cellular loss of covalently-bound APAP could be accounted for by the appearance of arylated proteins in the medium without any apparent degradation of APAP-bound proteins. By contrast, when NAC was present during the recovery period, there was a decrease in intracellular protein-bound APAP which could not be accounted for by that detected in the medium. Since during the recovery period the low residual intracellular concentration of APAP could not contribute significantly to any additional covalent binding in this system, NAC could not merely be acting as a nucleophilic trap for the reactive electrophile. Furthermore, NAC is not likely to dissociate covalently bound APAP from proteins. Hence, the overall decrease in covalent binding observed in cultures previously exposed to APAP for up to 8 hr must have arisen from an NAC-dependent enhancement of the degradation of the arylated proteins. However, after a more prolonged exposure to APAP, the ineffectiveness of NAC may have resulted from APAP-induced irreparable damage to the intracellular proteolytic system. These data suggest that the post-arylative efficacy of NAC may reside in the ability of the antidote to restore the functional capacity of the proteolytic system to rid the cells of arylated proteins.

Selective protein arylation and the age dependency of acetaminophen hepatotoxicity in mice

Male CD-1 mice 1, 1.5, 2, and 3 months old were given 600 mg of acetaminophen (APAP)/kg, po, and liver damage was assessed 12 hr later. The most severe hepatotoxicity was in 3-month-old mice, while the other age groups exhibited little damage. The onset of susceptibility to APAP hepatotoxicity did not correlate with the level of activity of the mixed-function oxidase system as assessed in vitro, since drug metabolizing capability was similar between 2- and 3-month-old mice. Through 4 hr after administration of APAP to 2- and 3-month-old mice in vivo, glutathione (GSH) depletion and both plasma and liver APAP concentrations were similar between ages. Additionally, 24 hr after dosing, 3-month-old mice excreted marginally more APAP-glucuronide conjugate and parent compound in urine than 2-month-old animals, while both age groups excreted similar amounts of the APAP-sulfate and GSH-derived conjugates. Even though the extent of binding of radioactive APAP to macromolecules at 4 hr was similar between 2- and 3-month-old animals, the pattern of immunochemically targetted cytosolic and microsomal proteins was different. Thus, in APAP exposure the extent of binding to specific proteins rather than the overall amount of covalent binding may be the critical determinant of the hepatotoxic response. In the present study, the age-related differences in susceptibility to APAP-induced hepatotoxicity were related to the differences in selective protein arylation.