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Acetaminophen liver injury is the most common cause of acute liver injury in the United States and several other countries. Diagnosis of acetaminophen-induced acute liver injury in the clinic is challenging due to the lack of validated and specific biomarkers. The following chapter provides an overview of recent advances evaluating candidate biomarkers in development for acetaminophen acute liver injury. Relationships of biomarkers to mechanisms of acetaminophen toxicity and their potential role in confirming the diagnosis and/or predicting evolving toxicity are addressed., (© 2020 Elsevier Inc. All rights reserved.)

Background & Aims: A rapid and reliable point-of-care assay to detect acetaminophen protein adducts in the serum of patients with acute liver injury could improve diagnosis and management. AcetaSTAT is a competitive immunoassay used to measure acetaminophen protein adducts formed by toxic metabolites in serum samples from patients. We compared the accuracy of AcetaSTAT vs high-pressure liquid chromatography with electrochemical detection (HPLC-EC; a sensitive and specific quantitative analytic assay) to detect acetaminophen protein adducts., Methods: We collected serum samples from 19 healthy individuals (no liver injury, no recent acetaminophen use), 29 patients without acetaminophen-associated acute liver injury, and 33 patients with acetaminophen-associated acute liver injury participating in the Acute Liver Failure Study Group registry. Each serum sample was analyzed by AcetaSTAT (reported as test band amplitude) and HPLC-EC (the reference standard). We also collected data on patient age, sex, weight, level of alanine aminotransferase on test day and peak values, concentration of acetaminophen, diagnoses (by site investigator and causality review committee), and outcome after 21 days. Differences between groups were analyzed using the Fisher exact test for categoric variables and the Kruskal-Wallis test or rank-sum test for continuous variables., Results: AcetaSTAT discriminated between patients with and without acetaminophen-associated acute liver injury; the median AcetaSTAT test band amplitude for patients with acetaminophen-associated acute liver injury was 584 (range, 222-1027) vs 3678 (range, 394-8289) for those without (P < .001). AcetaSTAT identified patients with acetaminophen-associated acute liver injury with 100% sensitivity, 86.2% specificity, a positive predictive value of 89.2%, and a negative predictive value of 100%. Results from AcetaSTAT were positive in 4 subjects who received a causality review committee diagnosis of non-acetaminophen-associated acute liver injury; HPLC-EC and biochemical profiles were consistent with acetaminophen-associated acute liver injury in 3 of these cases., Conclusions: The competitive immunoassay AcetaSTAT shows a high degree of concordance with HPLC-EC results in identifying patients with acetaminophen-associated acute liver injury. This rapid and simple assay could increase early detection of this disorder and aid clinical management., (Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Risk assessment for exposure to mixtures of drugs and pollutants relies heavily on in vitro characterization of their bioactivation and/or metabolism individually and extrapolation to mixtures assuming no interaction. Herein, we demonstrated that in vitro CYP2E1 metabolic activation of acetaminophen and styrene mixtures could not be explained through the Michaelis-Menten mechanism or any models relying on that premise. As a baseline for mixture studies with styrene, steady-state analysis of acetaminophen oxidation revealed a biphasic kinetic profile that was best described by negative cooperativity (Hill coefficient=0.72). The best-fit mechanism for this relationship involved two binding sites with differing affinities (Ks=830μM and Kss=32mM). Introduction of styrene inhibited that reaction less than predicted by simple competition and thus provided evidence for a cooperative mechanism within the mixture. Likewise, acetaminophen acted through a mixed-type inhibition mechanism to impact styrene epoxidation. In this case, acetaminophen competed with styrene for CYP2E1 (Ki=830μM and Ksi=180μM for catalytic and effector sites, respectively) and resulted in cooperative impacts on binding and catalysis. Based on modeling of in vivo clearance, cooperative interactions between acetaminophen and styrene resulted in profoundly increased styrene activation at low styrene exposure levels and therapeutic acetaminophen levels. Current Michaelis-Menten based toxicological models for mixtures such as styrene and acetaminophen would fail to detect this concentration-dependent relationship. Hence, future studies must assess the role of alternate CYP2E1 mechanisms in bioactivation of compounds to improve the accuracy of interpretations and predictions of toxicity., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Acetaminophen (APAP)-induced liver injury is the leading cause of acute liver failure in many countries. This study determined the extent of liver protein sulfhydryl depletion not only in whole liver homogenate but also in the zonal pattern of sulfhydryl depletion within the liver lobule. A single oral gavage dose of 150 or 300 mg/kg APAP in B6C3F1 mice produced increased serum alanine aminotransferase levels, liver necrosis, and glutathione depletion in a dose-dependent manner. Free protein sulfhydryls were measured in liver protein homogenates by labeling with maleimide linked to a near infrared fluorescent dye followed by SDS-polyacrylamide gel electrophoresis. Global protein sulfhydryl levels were decreased significantly (48.4%) starting at 1 hour after the APAP dose and maintained at this reduced level through 24 hours. To visualize the specific hepatocytes that had reduced protein sulfhydryl levels, frozen liver sections were labeled with maleimide linked to horseradish peroxidase. The centrilobular areas exhibited dramatic decreases in free protein sulfhydryls while the periportal regions were essentially spared. These protein sulfhydryl-depleted regions correlated with areas exhibiting histopathologic injury and APAP binding to protein. The majority of protein sulfhydryl depletion was due to reversible oxidation since the global- and lobule-specific effects were essentially reversed when the samples were reduced with tris(2-carboxyethy)phosphine before maleimide labeling. These temporal and zonal pattern changes in protein sulfhydryl oxidation shed new light on the importance that changes in protein redox status might play in the pathogenesis of APAP hepatotoxicity.

The role of protein glutathionylation in acetaminophen (APAP)-induced liver injury was investigated in this study. A single oral gavage dose of 150 or 300 mg/kg APAP in B6C3F1 mice produced increased serum alanine aminotransferase and aspartate aminotransferase levels and liver necrosis in a dose-dependent manner. The ratio of GSH to GSSG was decreased in a dose-dependent manner, suggesting that APAP produced a more oxidizing environment within the liver. Despite the increased oxidation state, the level of global protein glutathionylation was decreased at 1 h and continued to decline through 24 h. Immunohistochemical localization of glutathionylated proteins showed a complex dynamic change in the lobule zonation of glutathionylated proteins. At 1 h after APAP exposure, the level of glutathionylation decreased in the single layer of hepatocytes around the central veins but increased mildly in the remaining centrilobular hepatocytes. This increase correlated with the immunohistochemical localization of APAP covalently bound to protein. Thereafter, the level of glutathionylation decreased dramatically over time in the centrilobular regions with major decreases observed at 6 and 24 h. Despite the overall decreased glutathionylation, a layer of cells lying between the undamaged periportal region and the damaged centrilobular hepatocytes exhibited high levels of glutathionylation at 3 and 6 h in all samples and in some 24-h samples that had milder injury. These temporal and zonal pattern changes in protein glutathionylation after APAP exposure indicate that protein glutathionylation may play a role in protein homeostasis during APAP-induced hepatocellular injury.

To identify novel factors or mechanisms that are important for the resistance of tissues to chemical toxicity, we have determined the mechanisms underlying the previously observed increases in resistance to acetaminophen (APAP) toxicity in the lateral nasal gland (LNG) of the male Cyp2g1-null/Cyp2a5-low mouse. Initial studies established that Cyp2a5-null mice, but not a newly generated strain of Cyp2g1-null mice, were resistant to APAP toxicity in the LNG; therefore, subsequent studies were focused on the Cyp2a5-null mice. Compared with the wild-type (WT) male mouse, the Cyp2a5-null male mouse had intact capability to metabolize APAP to reactive intermediates in the LNG, as well as unaltered circulating levels of APAP, APAP-GSH, APAP-glucuronide, and APAP-sulfate. However, it displayed reduced tissue levels of APAP and APAP-GSH and increased tissue levels of testosterone and salivary androgen-binding protein (ABP) in the LNG. Furthermore, we found that ABP was able to compete with GSH and cellular proteins for adduction with reactive metabolites of APAP in vitro. The amounts of APAP-ABP adducts formed in vivo were greater, whereas the amounts of APAP adducts formed with other cellular proteins were substantially lower, in the LNG of APAP-treated male Cyp2a5-null mice compared with the LNG of APAP-treated male WT mice. We propose that through its critical role in testosterone metabolism, CYP2A5 regulates 1) the bioavailability of APAP and APAP-GSH (presumably through modulation of the rates of xenobiotic excretion from the LNG) and 2) the expression of ABP, which can quench reactive APAP metabolites and thereby spare critical cellular proteins from inactivation.

In overdose the analgesic/antipyretic acetaminophen (APAP) is hepatotoxic. Toxicity is mediated by initial hepatic metabolism to N-acetyl-p-benzoquinone imine (NAPQI). After low doses NAPQI is efficiently detoxified by GSH. However, in overdose GSH is depleted, NAPQI covalently binds to proteins as APAP adducts, and oxygen/nitrogen stress occurs. Toxicity is believed to occur by mitochondrial dysfunction. Manganese superoxide dismutase (MnSOD) inactivation by protein nitration has been reported to occur during other oxidant stress-mediated diseases. MnSOD is a critical mitochondrial antioxidant enzyme that prevents peroxynitrite formation within the mitochondria. To examine the role of MnSOD in APAP toxicity, mice were treated with 300 mg/kg APAP. GSH was significantly reduced by 65% at 0.5 h and remained reduced from 1 to 4 h. Serum alanine aminotransferase did not significantly increase until 4 h and was 2290 IU/liter at 6 h. MnSOD activity was significantly reduced by 50% at 1 and 2 h. At 1 h, GSH was significantly depleted by 62 and 80% at nontoxic doses of 50 and 100 mg/kg, respectively. No further GSH depletion occurred with hepatotoxic doses of 200 and 300 mg/kg APAP. A dose response decrease in MnSOD activity was observed for APAP at 100, 200, and 300 mg/kg. Immunoprecipitation of MnSOD from livers of APAP-treated mice followed by Western blot analysis revealed nitrated MnSOD. APAP-MnSOD adducts were not detected. Treatment of recombinant MnSOD with NAPQI did not produce APAP protein adducts. The data indicate that MnSOD inactivation by nitration is an early event in APAP-induced hepatic toxicity.

Although considered safe at therapeutic doses, at higher doses, acetaminophen produces a centrilobular hepatic necrosis that can be fatal. Acetaminophen poisoning accounts for approximately one-half of all cases of acute liver failure in the United States and Great Britain today. The mechanism occurs by a complex sequence of events. These events include: (1) CYP metabolism to a reactive metabolite which depletes glutathione and covalently binds to proteins; (2) loss of glutathione with an increased formation of reactive oxygen and nitrogen species in hepatocytes undergoing necrotic changes; (3) increased oxidative stress, associated with alterations in calcium homeostasis and initiation of signal transduction responses, causing mitochondrial permeability transition; (4) mitochondrial permeability transition occurring with additional oxidative stress, loss of mitochondrial membrane potential, and loss of the ability of the mitochondria to synthesize ATP; and (5) loss of ATP which leads to necrosis. Associated with these essential events there appear to be a number of inflammatory mediators such as certain cytokines and chemokines that can modify the toxicity. Some have been shown to alter oxidative stress, but the relationship of these modulators to other critical mechanistic events has not been well delineated. In addition, existing data support the involvement of cytokines, chemokines, and growth factors in the initiation of regenerative processes leading to the reestablishment of hepatic structure and function.

Acetaminophen (APAP)-induced liver toxicity occurs with formation of APAP-protein adducts. These adducts are formed by hepatic metabolism of APAP to N-acetyl-p-benzoquinone imine, which covalently binds to hepatic proteins as 3-(cystein-S-yl)-APAP adducts. Adducts are released into blood during hepatocyte lysis. We previously showed that adducts could be quantified by high-performance liquid chromatography with electrochemical detection following proteolytic hydrolysis, and that the concentration of adducts in serum of overdose patients correlated with toxicity. The following study examined the pharmacokinetic profile and clinical associations of adducts in 53 adults with acute APAP overdose resulting in acute liver failure. A population pharmacokinetic analysis using nonlinear mixed effects (statistical regression type) models was conducted; individual empiric Bayesian estimates were determined for the elimination rate constant and elimination half-life. Correlations between clinical and laboratory data were examined relative to adduct concentrations using nonparametric statistical approaches. Peak concentrations of APAP-protein adducts correlated with peak aminotransferase concentrations (r = 0.779) in adults with APAP-related acute liver failure. Adducts did not correlate with bilirubin, creatinine, and APAP concentration at admission, international normalized ratio for prothrombin time, or reported APAP dose. After N-acetylcysteine therapy, adducts exhibited first-order disappearance. The mean elimination rate constant and elimination half-life were 0.42 +/- 0.09 days(-1) and 1.72 +/- 0.34 days, respectively, and estimates from the population model were in strong agreement with these data. Adducts were detected in some patient samples 12 days post-ingestion. The persistence and specificity of APAP-protein adducts as correlates of toxicity support their use as specific biomarkers of APAP toxicity in patients with acute liver injury.

In studies designed to simulate a clinical observation in which an individual became tolerant to normally lethal doses of acetaminophen (APAP), mice were pretreated with increasing doses of APAP for 8 days and challenged on day 9 with normally supralethal doses of APAP. These animals developed minimal hepatotoxicity after a challenge dose with a fourfold increase in LD50 to 1,350 mg/kg. The pretreatment regimen resulted in hepatic changes including: centrilobular localization of 3-(cysteine-S-yl)APAP protein adducts, selective down-regulation of cytochrome P4502E1 (CYP2E1) and CYP1A2 that produced the toxic metabolite, N-acetyl-p-benzoquinone imine, higher levels of reduced glutathione (GSH), centrilobular inflammation, and a fourfold increase in hepatocellular proliferation. The protection against the lethal APAP doses afforded by pretreatment is secondary to these changes and to the associated regional shift in the bioactivation of the APAP challenge dose from centrilobular to periportal regions where CYP2E1 is not found, protective GSH is more abundant, and where cell-proliferative responses are better able to sustain repair. This shift in APAP bioactivation results in less-intense covalent binding that is more diffuse and spread uniformly throughout the hepatic lobe, most likely contributing to protection by delaying the early onset of liver injury that has been generally associated with centrilobular localization of the adducts. Intervention of APAP pretreatment-induced cell division in mice with colchicine left them resistant to a 500-mg/kg (normally lethal) dose of APAP, but unable to survive a 1,000-mg/kg APAP challenge dose. The data demonstrate multiple mechanistic components to the protection afforded by APAP pretreatment. Whereas metabolic and physiological changes not dependent on cell proliferation are adequate to protect against 500 mg/kg APAP, these changes plus a potentiated cell-proliferative response are necessary for protection against the supralethal 1,000-mg/kg APAP dose. Furthermore, the data document an uncoupling of the traditional association between covalent binding and toxicity, and suggest that the assessment of toxicity following repeated or chronic APAP exposure must consider altered drug interactions and parameters besides those historically used to assess acute APAP overdose.

The hepatotoxicity of the analgesic acetaminophen has been previously attributed to metabolic activation by cytochrome P450 to the reactive intermediate N-acetyl-p-benzoquinone imine. At therapeutic doses this species is detoxified by reaction with glutathione; however, following a hepatotoxic dose, liver glutathione levels are depleted and the metabolite covalently binds primarily to cysteine groups on proteins as 3-(cystein-S-yl)acetaminophen adducts. Altered function of critical proteins has been postulated to be the mechanism of hepatotoxicity. Covalent binding has been studied by both radiochemical methods and immunochemical methods. Utilizing Western blot analysis with an antiserum which recognizes acetaminophen we have previously shown that covalent binding occurs on a number of proteins in various hepatic fractions. In an effort to better understand the role of covalent binding in the toxicity, others have studied the non-hepatotoxic isomer 3'-hydroxyacetanilide. Administration of large doses of radiolabeled acetaminophen or 3'-hydroxyacetanilide resulted in similar levels of covalent binding to proteins. To better understand the role of covalent binding in toxicity we have administered mice 3'-hydroxyacetanilide and acetaminophen, and analyzed liver fractions for protein adducts using anti-3-(cystein-S-yl)acetaminophen and anti-arylacetamide antisera in Western blot assays. Analysis of liver fractions from acetaminophen-treated mice, with both antisera showed, as has been previously reported, that acetaminophen covalently binds to a number of hepatic proteins. In liver from 3'-hydroxyacetanilide-treated mice, covalent adducts were detected with an anti-arylacetamide antiserum only. A major 3'-hydroxyacetanilide protein adduct was observed in microsomes at 50 kDa. Minor adducts were observed at 47 kDa in microsomes and 56 kDa in cytosol. 3'-Hydroxyacetanilide protein adducts were not observed in the 10,000 x g pellet. Densitometric analysis of a time course of 3'-hydroxyacetanilide protein adducts indicated that peak levels of the 50 kDa microsomal protein adduct occurred at 1 h and subsequently decreased.

Acetaminophen-induced hepatotoxicity is believed to be mediated by covalent binding of the reactive metabolite N-acetyl-p-benzoquinone imine to essential proteins in liver. It has been shown that the primary reaction of this metabolite with hepatic proteins is the formation of 3-(cysteine-S-yl)-acetaminophen adducts. The importance of covalent binding to other amino acids that may be formed by reaction of N-acetyl-p-benzoquinone imine with protein is unclear. Previously, we developed immunochemical assays for the acetaminophen cysteine adducts by immunizing animals with the conjugate 3-(N-acetylcystein-S-yl)acetaminophen-keyhole limpet hemocyanin, wherein the carboxyl group of the N-acetyl-cysteine moiety was coupled to amino groups on the protein. A very sensitive and specific immunochemical assay was developed for acetaminophen specifically bound to cysteine groups on protein [3-(cystein-S-yl)acetaminophen protein adducts]. Analysis of protein adducts indicated that after toxic doses, acetaminophen covalently bound at high levels to cysteine residues on a relatively small number of hepatic proteins. In the present work, a new antiacetaminophen antiserum was prepared by immunizing mice with 4-acetamidobenzoic acid coupled to keyhole limpet hemocyanin. Competitive ELISA data indicate that the resulting antiserum has excellent recognition of acetaminophen and related arylacetamide derivatives. Using this new antiserum, Western blot analyses of liver proteins from acetaminophen-intoxicated mouse livers were performed and compared with similar assays using the anti-3-(cystein-S-yl)acetaminophen antiserum. Visual and densitometric analyses of the Western blots indicate that the two antisera detect the same primary acetaminophen protein adducts; however, minor differences in the intensity of certain bands were observed. These differences may represent either differences in antibody accessibility to 3-(cystein-S-yl)acetaminophen adducts or differences in the proportion of acetaminophen bound to cysteine vs. binding to other amino acids.

Chronic acetaminophen (APAP) toxicity poses a difficult diagnostic challenge to the clinician. Signs and symptoms are nonspecific and no currently available laboratory study can confirm APAP as the causative agent of hepatic injury. In this study an antigenic biomarker for APAP toxicity was used to confirm the diagnosis of APAP-induced hepatic failure in two children with chronic APAP toxicity. APAP that has been metabolized to N-acetyl-benzoquinone imine (NAPQI) reacts with cellular proteins to form 3-(cystein-S-yl)-APAP protein adducts (3-Cys-A). Serum from both patients was submitted for quantitation of 3-Cys-A by a competitive inhibition enzyme-linked immunosorbent assay (ELISA). Concentrations of 3-Cys-A in the two patients were 1.97 and 2.77 nmol/mg protein, which are similar to concentrations found in adults with hepatic injury secondary to an overdose of APAP. Individuals with no exposure to APAP have no detectable 3-Cys-A in serum. It was concluded that 3-Cys-A is a useful marker of APAP intoxication after long-term ingestion of APAP when total dose and time course of ingestion are uncertain, and may prove to be a useful clinical and investigative tool in the study of APAP intoxication.

Acetaminophen hepatotoxicity is associated with its biotransformation to the reactive metabolite N-acetyl-p-benzoquinone imine that binds to protein. Two forms of cytochrome P450, CYP2E1 and CYP1A2, have been implicated as primarily responsible for the bioactivation. To determine the relative contributions of these P450's, overnight fasted male NMRI mice were pretreated with 10 ml of 50% v/w propylene glycol/kg or fluvoxamine (10 mg/kg) at -80 and -20 min. relative to acetaminophen dosing to inhibit CYP2E1 and CYP1A2, respectively. Mice were sacrificed at 0.5 or 4 hr after a hepatotoxic dose of acetaminophen (300 mg/kg). Propylene glycol or propylene glycol plus fluvoxamine, but not fluvoxamine alone protected against acetaminophen hepatotoxicity as indicated by abolished increase in serum alanine aminotransferase activity, less depletion of hepatic glutathione and lower liver:body weight ratios. Propylene glycol inhibited the activity of CYP2E1 as indicated by 84% reduction in the clearance of 3 mg/kg dose of chlorzoxazone, whereas fluvoxamine inhibited the activity of CYP1A2 as indicated by 40% reduction in the clearance of a 10 mg/kg dose of caffeine. For this animal model, the data are consistent with the notion that hepatoxicity is associated with bioactivation of acetaminophen by CYP2E1 but not by CYP1A2.

The effect of acetaminophen (APAP) dose on the cytochrome P450s responsible for its bioactivation was examined in control mice and mice treated with acetone to induce CYP2E1, or beta-napthaflavone to induce CYP1A2. In non-induced mice, 150 mg/kg APAP caused minimal hepatotoxicity and loss of CYP2E1- but not CYP1A2-dependent activity. In contrast, 400 mg/kg APAP was hepatotoxic and diminished both CYP2E1 and CYP1A2 activities. In acetone-pretreated mice, the 150 and 400 mg/kg APAP doses caused similar depletion of CYP2E1 activity and similar levels of covalent binding of APAP to liver proteins. In beta-napthaflavone-pretreated mice, CYP1A2 activity was decreased only by the high dose of APAP, and covalent binding was > 2-fold higher at the high APAP dose. The data indicate CYP2E1 is important in the bioactivation of APAP at the low dose with little additional contribution at the high dose, whereas CYP1A2 contributes more to the bioactivation and toxicity APAP at high doses.

The human hepatoma cell line, HepG2, retains many cellular functions often lost by cells in culture. This research examined the constitutive bioactivation of acetaminophen and P450-dependent activity in microsomes from HepG2 cells and the effect of 0.1% acetone pretreatment on these activities. Low levels of acetaminophen bioactivation, P450 IIE1 activity, and P450 IA1-IA2 activity were demonstrated in non-induced HepG2 microsomes. Acetone increased acetaminophen bioactivation and IIE1-dependent metabolism but not P450 IA1-IA2-dependent activity. Thus, HepG2 cells may provide an in vitro model for assessing human xenobiotic metabolism of acetaminophen and other drugs.

Propylene glycol (PG) decreases the hepatotoxicity of acetaminophen (APAP). To elucidate the mechanism for this response, we measured the effect of PG on the in vitro metabolism of APAP by subcellular liver fractions from 6-10 week-old male B6C3F1 mice. The fractions were assayed for their ability to bioactivate APAP to N-acetyl-p-benzoquinone imine, which was trapped as APAP-glutathione conjugates or APAP-protein adducts, and for dimethyl-nitrosamine-N-demethylase (DMN), 4-nitrophenol hydroxylase (4-NPOH), and phenacetin-O-deethylase (PAD) activities. Activity in the crude mitochondrial-rich (10,000 x g pellet) fraction was low and PG had no effect. PG inhibited DMN and 4-NPOH, indicators of IIE1-dependent activity, and the formation of APAP-glutathione conjugates and APAP-protein adducts in both heavy (15,000 x g pellet) and light (100,000 x g pellet) microsomes. PAD, a measure of IA2-dependent activity, was not inhibited. These data demonstrate that PG selectively inhibits IIE1 activity, including the bioactivation of APAP, and implicates this as the mechanism for PG-mediated protection of APAP hepatotoxicity in mice.

Acetaminophen overdose causes severe hepatotoxicity in humans and laboratory animals, presumably by metabolism to N-acetyl-p-benzoquinone imine: and binding to cysteine groups as 3-(cystein-S-yl)acetaminophen-protein adduct. Antiserum specific for the adduct was used immunohistochemically to demonstrate the formation, distribution, and concentration of this specific adduct in livers of treated mice and was correlated with cell injury as a function of dose and time. Within the liver lobule, immunohistochemically demonstrable adduct occurred in a temporally progressive, central-to-peripheral pattern. There was concordance between immunohistochemical staining and quantification of the adduct in hepatic 10,000g supernate, using a quantitative particle concentration fluorescence immunoassay. Findings include: 1) immunochemically detectable adduct before the appearance of centrilobular necrosis, 2) distinctive lobular zones of adduct localization with subsequent depletion during the progression of toxicity, 3) drug-protein binding in hepatocytes at subhepatotoxic doses and before depletion of total hepatic glutathione, 4) immunohistochemical evidence of drug binding in the nucleus, and 5) adduct in metabolically active and dividing hepatocytes and in macrophagelike cells in the regenerating liver.

The hepatotoxicity of acetaminophen correlates with the formation of 3-(cystein-S-yl)acetaminophen protein adducts. Using a sensitive and specific immunochemical assay, we quantitated the formation of these protein adducts in liver fractions and serum after administration of a hepatotoxic dose of acetaminophen (400 mg/kg) to B6C3F1 mice. Adducts in the cytosolic fraction increased to 3.6 nmol/mg protein at 2 hr and then decreased to 1.1 nmol/mg protein by 8 hr. Concomitant with the decrease in adducts in the cytosol, 3-(cystein-S-yl)acetaminophen protein adducts appeared in serum and their levels paralleled increases in serum alanine aminotransferase. Microsomal protein adducts peaked at 1 hr (0.7 nmol/mg protein) and subsequently decreased to 0.2 nmol/mg at 8 hr. The 4000 g pellet (nuclei, plasma membranes, and cell debris) had the highest level of adducts (3.5 nmol/mg protein), which remained constant from 1 to 8 hr. Evaluation of fractions purified from a 960 g pellet indicated that the highest concentration of 3-(cystein-S-yl)acetaminophen protein adducts was located in plasma membranes and mitochondria; peak levels were 10.3 and 5.1 nmol/mg respectively. 3-(Cystein-S-yl)acetaminophen protein adducts were detected in nuclei only after enzymatic hydrolysis of the proteins. The localization of high levels of 3-(cystein-S-yl)acetaminophen protein adducts in plasma membranes and mitochondria may play a critical role in acetaminophen toxicity.

The hepatotoxicity of acetaminophen is believed to be mediated by the metabolic activation of acetaminophen to N-acetyl-p-benzoquinone imine which covalently binds to cysteinyl residues on proteins as 3-(cystein-S-yl)acetaminophen adducts. The formation of these adducts in hepatic protein correlates with the hepatotoxicity. In this study, the formation of 3-(cystein-S-yl)acetaminophen adducts in specific cellular proteins was investigated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and detected using affinity-purified antisera specific for 3-(cystein-S-yl)acetaminophen adducts on immunoblots. These techniques were used to investigate the liver 10,000g supernatant and serum from B6C3F1 mice that received hepatotoxic doses of acetaminophen. More than 15 proteins containing 3-(cystein-S-yl)acetaminophen adducts were detected in the liver 10,000g supernatant. The most prominent protein containing 3-(cystein-S-yl)acetaminophen adducts in the hepatic 10,000g supernatant had a relative molecular mass of 55 kDa. Serum proteins containing 3-(cystein-S-yl)acetaminophen adducts had molecular masses similar to those found in the liver 10,000g supernatant (55, 87, and approximately 102 kDa). These data, combined with our previous findings describing the temporal relationship between the appearance of 3-(cystein-S-yl)acetaminophen adducts in protein in the serum and the decrease in the levels of 3-(cystein-S-yl)acetaminophen adducts in protein in the liver, suggested that liver adducts were released into the serum following lysis of hepatocytes. The temporal relationship between the formation of specific adducts and hepatotoxicity in mice following a hepatotoxic dose of acetaminophen was examined using immunoblots of mitochondria, microsomes, cytosol, and plasma membranes. Hepatotoxicity indicated by serum alanine aminotransferase levels was increased at 2 and 4 hr after dosing. The cytosolic fraction contained numerous proteins with 3-(cystein-S-yl)acetaminophen adducts, the most intensely stained of which was a 55-kDa protein. 3-(Cystein-S-yl)acetaminophen adducts were detected in the 55-kDa liver protein 30 min after dosing and prior to the development of significant toxicity. Examination of gels suggested that maximal levels of immunochemically detectable adducts in the 55-kDa protein occurred at 1-2 hr, with a decrease in intensity 4 hr after dosing. The presence of 3-(cystein-S-yl)acetaminophen adducts in proteins prior to hepatotoxicity suggests a threshold for adduct formation in the development of toxicity. Protein in microsomes which contained 3-(cystein-S-yl)acetaminophen adducts ranged in molecular weight from 38 to approximately 106 kDa. The major proteins containing 3-(cystein-S-yl)acetaminophen adducts in the mitochondria had molecular masses of 39, 50, 68, and 79 kDa.(ABSTRACT TRUNCATED AT 400 WORDS)

Administration of acetaminophen (500 mg/kg) to male B6C3F1 mice resulted in alterations of pancreatic beta cell ultrastructure. These alterations were characterized by pronounced intercellular spaces, cytoplasmic vacuolization, damaged membranes of cytoplasm, secretory granules, and other organelles, and pyknotic nuclei with disrupted membranes. Concomitant with these changes, acetaminophen also caused increaes in serum insulin concentrations from 24 microU/ml at 0 time to 160 microU/ml at 8 hr and increases in serum alanine aminotransferase (ALT) concentrations from 42 to 13,279 U/liter, which indicated hepatic damage. Quantitation of 3-(cystein-S-yl)acetaminophen adducts in hepatic 10,000g supernatant protein using a particle concentration fluorescence immunochemical assay indicated a positive correlation between binding and the occurrence of the hepatotoxicity consistent with what has been previously reported; however, 3-(cystein-S-yl)acetaminophen protein adducts were not detected in pancreatic 10,000g supernatant. Immunohistochemical analysis of the liver and pancreas from acetaminophen-treated mice revealed acetaminophen-protein adducts in the centrilobular regions of the liver but not in the pancreatic islets. Doses of 100 and 200 mg/kg produced no evidence of hepatotoxicity and no increase in serum insulin; 300 mg/kg and higher doses produced both hepatotoxicity and increased serum insulin concentrations. A comparison of the time course for the increase in serum levels of ALT and insulin following a toxic dose of acetaminophen indicated that the increase in ALT preceded the increase in insulin. Thus the hepatotoxicity of acetaminophen correlates with the formation of 3-(cystein-S-yl)acetaminophen protein adducts in liver, which supports the concept that this toxicity is mediated by the reactive metabolite N-acetyl-p-benzoquinone imine; however, the toxicity of acetaminophen to beta cells in the pancreas is apparently not mediated by this mechanism.

The hepatotoxicity of acetaminophen may be mediated by the reactive metabolite N-acetyl-p-benzoquinone imine which binds covalently to protein primarily as 3-(cystein-S-yl)acetaminophen. We have developed an avidin biotin-amplified competitive enzyme-linked immunosorbent assay to detect protein-bound acetaminophen. This assay utilizes antisera from rabbits immunized with 3-(N-acetyl-L-cystein-S-yl)acetaminophen coupled via the carboxyl group to primary amino groups on the protein keyhole-limpet hemocyanin. The competitive enzyme-linked immunosorbent assay utilizes metallothionein derivatized with N-acetyl-p-benzoquinone imine (acetaminophen-bound metallothionein) and quantitation was obtained by competition of acetaminophen-derivatives for a limited amount of antibody in the presence of excess solid phase acetaminophen-bound metallothionein. Synthetic 3-(N-acetyl-L-cystein-S-yl)acetaminophen, acetaminophen bound to mouse 9,000 X g supernatant, 100,000 X g supernatant, microsomes, as well as acetaminophen-bound metallothionein were inhibitory. The 50% inhibition for 3-(N-acetyl-L-cystein-S-yl)acetaminophen was 110 fmol/well. In contrast, free acetaminophen was 6200 times less efficient as an inhibitor. The mean 50% inhibition for protein-bound acetaminophen was 2.89 pmol/well. The utility of the method to detect acetaminophen-protein adducts in biological samples was confirmed by detection of NADPH-dependent binding of acetaminophen to microsomal proteins.

The oxidation of acetaminophen to N-acetyl-p-benzoquinone imine and its subsequent binding to protein sulfhydryl groups may be important in the observed toxicity of acetaminophen. An avidin biotin-amplified competitive enzyme-linked immunosorbent assay has been developed which utilizes solid phase acetaminophen bound to metallothionein and antiserum obtained from rabbits that were immunized with 3-(N-acetyl-L-cystein-S-yl)acetaminophen covalently bound to keyhole-limpet hemocyanin. Over 25 synthetic analogs of acetaminophen conjugates and structurally similar compounds have been ranked relative to their ability to compete in the avidin biotin-amplified enzyme-linked immunosorbent assay. The most effective inhibitor was 3-(N-acetyl-L-cystein-S-yl)acetaminophen, which had an observed 50% inhibitory concentration of 120 fmol/well. Approximately 6200-fold higher concentrations of unbound acetaminophen and 5.2 x 10(6)-fold higher concentrations of N-acetyl-L-cysteine were required for comparable inhibition. It was demonstrated with acetaminophen analogs that the hydroxyl group and the N-acetyl moiety of acetaminophen were important in epitope recognition. A 5000-fold decrease in detection was observed when the analog did not contain the hydroxyl group or when the N-acetyl moiety was replaced with a hydroxyl substituent. Recognition by antibody was also dependent upon the stereochemistry of the analogs. The 50% inhibitory concentration for 3-(L-cystein-S-yl)acetaminophen was 2300 fmol/well, whereas a 25-fold higher concentration of 3-(D-cystein-S-yl)acetaminophen was required for 50% inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)

Using a recently developed enzyme-linked immunosorbent assay specific for 3-(cystein-S-yl)acetaminophen adducts we have quantitated the formation of these specific adducts in liver and serum protein of B6C3F1 male mice dosed with acetaminophen. Administration of acetaminophen at doses of 50, 100, 200, 300, 400 and 500 mg/kg to mice resulted in evidence of hepatotoxicity (increase in serum levels of alanine aminotransferase and aspartate aminotransferase) at 4 hr in the 300, 400 and 500 mg/kg treatment groups only. The formation of 3-(cystein-S-yl)acetaminophen adducts in liver protein was not observed in the groups receiving 50, 100 and 200 mg/kg doses, but was observed in the groups receiving doses above 300 mg/kg of acetaminophen. Greater levels of adduct formation were observed at the higher doses. 3-(Cystein-S-yl)acetaminophen protein adducts were also observed in serum of mice receiving hepatotoxic doses of acetaminophen. After a 400 mg/kg dose of acetaminophen, 3-(cystein-S-yl)acetaminophen adducts in the liver protein reached peak levels 2 hr after dosing. By 12 hr the levels decreased to approximately 10% of the peak level. In contrast, 3-(cystein-S-yl)acetaminophen adducts in serum protein were delayed, reaching a sustained peak 6 to 12 hr after dosing. The dose-response correlation between the appearance of serum aminotransferases and 3-(cystein-S-yl)acetaminophen adducts in serum protein and the temporal correlation between the decrease in 3-(cystein-S-yl)acetaminophen adducts in liver protein and the appearance of adducts in serum protein are consistent with a hepatic origin of the adducts detected in serum protein.(ABSTRACT TRUNCATED AT 250 WORDS)