Raymond B. Birge

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.

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.

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.

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.

Selective protein arylation by acetaminophen and 2,6-dimethylacetaminophen in cultured hepatocytes from phenobarbital-induced and uninduced mice: Relationship to cytotoxicity☆

To evaluate the mechanistic importance of covalent binding in acetaminophen (APAP)-induced hepatotoxicity, we compared the effects of 2,6-dimethylacetaminophen (2,6-DMA) to those of APAP in primary cultures of mouse hepatocytes. Immunochemical analysis of electrophoretically separated proteins has shown that the majority of covalent binding after a cytotoxic dose of APAP occurs on two major bands of 44 and 58 kD (Bartolone et al., Biochem Pharmacol 36: 1193-1196, 1987). At equimolar concentrations, 2,6-DMA bound proteins only 15% as extensively as did APAP and was not cytotoxic in hepatocytes from uninduced mice. However, when the hepatocytes were obtained from phenobarbital-induced mice, APAP administration resulted in increased protein arylation and a more rapid onset of cytotoxicity. Furthermore, in the cells from phenobarbital-induced mice, 2,6-DMA not only resulted in increased binding but also in overt cytotoxicity. Since our affinity-purified anti-APAP antibody did not cross-react with 2,6-DMA, a new antibody specific for 2,6-DMA was prepared and, after affinity purification, was used to detect 2,6-DMA protein adducts by Western blotting. Results indicated that, in hepatocytes from both phenobarbital-induced and non-induced mice, the binding of 2,6-DMA was also highly selective with the most prominent target being the 58 kD cytosolic protein. However, by contrast to APAP, only minimal binding to the 44 kD protein was detected after 2,6-DMA treatment. Although several additional protein adducts were increased in treated cells from phenobarbital-induced mice, the 58 kD protein was clearly the most prominently arylated target associated with both APAP and 2,6-DMA cytotoxicity. These data suggest that both the specificity of covalent binding as well as the extent of binding to the major targets may play an important role in the ensuing toxicity.

A comparison of proteins S-thiolated by glutathione to those arylated by acetaminophen☆

This study was designed to evaluate whether the same proteins that irreversibly bind reactive electrophiles of drugs also bind glutathione (GSH) under oxidative conditions. Specifically, proteins that can be arylated by acetaminophen were compared to those that form glutathione-protein mixed disulfides (PSSG) after incubation with diamide. Data are presented which suggest that both GSH and acetaminophen bind to a subset of N-ethylmaleimide (NEM)-reactive protein thiols. To evaluate the pattern of proteins that bind GSH, PSSGs were formed in vitro by incubating cytosolic proteins with GSH and diamide. A sensitive procedure was developed in which PSSGs were first reduced with 0.1 mM dithiothreitol (DTT), and the newly exposed protein thiols were labeled with either [3H]NEM (for quantitative analysis) or with fluorescein-5-maleimide (for visual detection). Acetaminophen binding was achieved by incubating cytosolic proteins in vitro with the reactive acetaminophen metabolite, N-acetyl-p-benzoquinoneimine (NAPQI). Proteins from both assays were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose for Western blot analysis. Acetaminophen binding was detected by immunoblotting with an affinity-purified antibody against acetaminophen, and PSSGs were visualized using anti-fluorescein antibodies. In both instances, binding to proteins was observed to be selective. A comparison of the proteins modified by GSH binding with those that bind acetaminophen indicates that the major cytosolic acetaminophen-binding protein of 58 kDa may also be modified by glutathiolation under oxidative conditions.

The arylation of microsomal membrane proteins by acetaminophen is associated with the release of a 44 kDa acetaminophen-binding mouse liver protein complex into the cytosol

When analyzed by Western blotting with affinity purified antibodies against acetaminophen, proteins of molecular weight 44 and 58 kDa appear to be the major macromolecular targets in livers of mice administered hepatotoxic concentrations of acetaminophen. In this study, we have examined the characteristics and biochemical properties of the 44 kDa acetaminophen-binding protein in mouse liver. Data are presented which indicate that the 44-kDa protein is the earliest detectable protein targeted by acetaminophen; 30 min after acetaminophen administration in vivo, the binding to the 44 kDa protein is primarily localized in the microsomal fraction. After 1 hr, the 44 kDa acetaminophen-binding protein can be detected in both the microsomes and the cytosol. Extractions of microsomes with Triton X-114 or 1 M NaCl suggests that the acetaminophen-bound 44-kDa protein behaves as a peripheral membrane protein associated with the endoplasmic reticulum by ionic interactions. The cytosolic and microsomal 44-kDa proteins possess similar biochemical properties; both exist natively as components of a protein complex of greater than 200 kDa and both consist of two major isovariants with isoelectric points of 7.0 and 7.1 on two-dimensional gels. When N-acetyl-p-benzoquinone imine, the reactive metabolite of acetaminophen, is incubated with cytosolic or microsomal fractions from control liver, targeting of a 44-kDa protein is only observed in the microsomes. However, when acetaminophen is activated in an NADPH-regenerating microsomal system in vitro, some of the microsomal 44-kDa protein complex can be solubilized and released into the cytosol. Thus, acetaminophen administration can alter the subcellular distribution of at least one protein target in the cell.

In Vivo and in Vitro Evidence for in Situ Activation and Selective Covalent Binding of Acetaminophen (APAP) in Mouse Kidney

Acetaminophen (APAP, N-acetyl-p-aminophenol) is a widely used analgesic and antipyretic which, at high doses, causes acute hepatic centrilobular necrosis in man and a variety of laboratory animals (Proudfoot and Wright, 1970; Boyer and Rouff, 1971; Mitchell et al., 1973). In addition, acute renal proximal tubular necrosis following APAP has been reported in man (Kleinman et al., 1980; Cobden et al., 1982; Kher and Makker, 1987; Davenport and Finn, 1988). A similar lesion has been described in the Fischer rat (McMurtry et al., 1978; Newton et al., 1983) andthe CD-1 mouse (Placke et al, 1987) but the APAP metabolite responsible for the toxicity is different between species. In the rat, APAP is enzymatically deacetylated to paminophenol, a potent nephrotoxicant whose activation is independent of cytochrome P450 (Crowe et al., 1979; Calder et al., 1979; Newton et al., 1982; 1985a; 1985b). By contrast, enzymatic deacetylation of APAP is not required in the mouse but instead activation of intact APAP by cytochrome P450 appears to mediate nephrotoxicity (Bartolone et al, 1989; Emeigh Hart et al., 1989a; 1989b). Since hepatic metabolism of APAP is similarly dependent on cytochrome P450 (Mitchell et al, 1973), nephrotoxicity and renal adduct accumulation could arise from transport of a livergenerated metabolite or adduct to the kidney and not from in situ metabolism of APAP. The purpose of the present study was to determine, using renal proximal tubule (RPT) cell suspensions and immunohistochemistry, if the mouse kidney could generate such adducts in situ.

Post-Treatment Protection with Piperonyl Butoxide Against Acetaminophen Hepatotoxicity is Associated with Changes in Selective but Not Total Covalent Binding

Acetaminophen (APAP) induced hepatic centrilobular necrosis has been associated with cytochrome P-450-mediated generation of an electrophilic, reactive metabolite which covalently binds to cellular macromolecules as glutathione becomes depleted (Jollow, et al., 1973; Mitchell, et al., 1973a; 1973b; Potter, et al., 1973; 1974). Covalent binding has been well-correlated with the incidence and severity of liver necrosis and prior cytochrome P450 inhibition blocks both covalent binding and the atotoxicity (Jollow, et al., 1973; Potter, et al., 1973, 1974; Mitchell, et al., 1973a). In addition, administration of the cytochrome P-450 inhibitor, piperonyl butoxide (Pip B), 2 hrs after APAP, the time of maximal covalent binding (Jollow,et al., 1973, Ginsberg and Cohen, 1985) reduced the severity of liver damage (Brady, et al., 1988). The present study demonstrates that Pip B’s post-treatment protection is associated with alterations in selective protein arylation by APAP without a change in total covalent binding.