Mary K. Bruno

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.

Exaggerated hepatotoxicity of acetaminophen in mice lacking tumor necrosis factor receptor-1. Potential role of inflammatory mediators.

Transgenic mice with a targeted disruption of the tumor necrosis factor receptor 1 (TNFR1) gene were used to analyze the role of TNF-alpha in pro- and anti-inflammatory mediator production and liver injury induced by acetaminophen. Treatment of wild-type mice with acetaminophen (300 mg/kg) resulted in centrilobular hepatic necrosis. This was correlated with expression of inducible nitric oxide synthase (NOS II) and nitrotyrosine staining of the liver. Expression of macrophage chemotactic protein-1 (MCP-1), KC/gro, interleukin-1beta (IL-1beta), matrix metalloproteinase-9 (MMP-9), and connective tissue growth factor (CTGF), inflammatory mediators known to participate in tissue repair, as well as the anti-inflammatory cytokine, interleukin-10 (IL-10), also increased in the liver following acetaminophen administration. TNFR1(-/-) mice were found to be significantly more sensitive to the hepatotoxic effects of acetaminophen than wild-type mice. This was correlated with more rapid and prolonged induction of NOS II in the liver and changes in the pattern of nitrotyrosine staining. Acetaminophen-induced expression of MCP-1, IL-1beta, CTGF, and MMP-9 mRNA was also delayed or reduced in TNFR1(-/-) mice relative to wild-type mice. In contrast, increases in IL-10 were more rapid and more pronounced. These data demonstrate that signaling through TNFR1 is important in inflammatory mediator production and toxicity induced by acetaminophen.

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.

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 alterations in the patterns of newly synthesized proteins by acetaminophen and its dimethylated analogues in primary cultures of mouse hepatocytes

Alterations in protein synthesis following exposure to and recovery from hepatotoxic doses of acetaminophen (APAP) and its analogues, 3,5-dimethyl acetaminophen (3,5-DMA) and 2,6-dimethyl acetaminophen (2,6-DMA), were investigated in primary cultures of mouse hepatocytes. The rates of protein synthesis decreased within 4 hr after administration of 10 mM APAP and occurred after significant depletion of intracellular glutathione and covalent binding of APAP to proteins, but preceded the leakage of lactate dehydrogenase into the media. The inhibition of protein synthesis was reversible only if APAP exposure did not exceed 8 hr. Electrophoretic analysis of 35S-labeled proteins by one-dimensional SDS-PAGE revealed two consistent alterations in the patterns of newly synthesized proteins. First was a progressive diminution in the de novo synthesis of a protein migrating at approximately 58 kDa (p58). This was observed with APAP (10 mM) and 3,5-DMA (5 mM) but not with 2,6-DMA (10 mM). If exposure to APAP exceeded 8 hr, the biosynthesis of this protein was not only further decreased but was also no longer detectable during the recovery period. The second major alteration was an increase in the relative rate of biosynthesis of a 32-kDa protein (p32) following exposure and recovery from APAP and 3,5-DMA but not 2,6-DMA. Exposure to heme or arsenite induced the synthesis of a protein of similar molecular weight but did not result in the inhibition of p58 biosynthesis. The fact that the reactive metabolites of both APAP and 3,5-DMA, but not 2,6-DMA, possess oxidative properties suggests that the alterations in the synthesis of p32 and p58 may be related to an oxidative component induced by these compounds.

Selective Alterations in the Profiles of Newly Synthesized Proteins by Acetaminophen (APAP) and its Dimethylated Analogues: Relationship to Oxidative Stress

Acetaminophen (N-acetyl-p-aminophenol, APAP), one of the most widely used analgesic, antipyretic drugs currently available, when taken in excess of therapeutic doses can be activated by cytochrome P-450 to a highly reactive metabolite, Nacetylbenzoquinoneimine (NAPQI) (Dahlin, et al., 1984). NAPQI has been characterized as a strong electrophile and a potent oxidizing agent (Blair, et al., 1980) and both properties can lead to adverse effects on cellular metabolism (Albano, et al., 1985; Porubek, et al., 1987; Birge, et al., 1988). In order to more effectively evaluate the mechanisms of action of APAP and their physiological consequences, it becomes crucial not only to identify the early metabolic events that are altered, but also whether the functional impairments can be restored or have become irreversible.

Detection of covalent binding.

Immunochemical detection of xenobiotics covalently bound to cellular proteins can provide information about toxic mechanism and is more specific than the alternative radiochemical studies. Both immunoblotting and immunohistochemical methods are used to pinpoint the target protein(s) and to identify the tissue targets. Also included in this unit are protocols for synthesizing artificial antigens, immunizing suitable host species, and using noncompetitive and competitive ELISA assays to characterize the antibodies produced.