Acetaminophen-associated hepatic injury: evaluation of acetaminophen protein adducts in children and adolescents with acetaminophen overdose.

Acetaminophen protein adducts (APAP adducts) were quantified in 157 adolescents and children presenting at eight pediatric hospitals with the chief complaint of APAP overdose. Two of the patients required liver transplantation, whereas all the others recovered spontaneously. Peak APAP adducts correlated with peak hepatic transaminase values, time-to-treatment with N-acetylcysteine (NAC), and risk determination per the Rumack-Matthews nomogram. A population pharmacokinetic analysis (NONMEM) was performed with post hoc empiric Bayesian estimates determined for the elimination rate constants (k(e)), elimination half-lives (t(1/2)), and maximum concentration of adducts (C(max)) of the subjects. The mean (+/-SD)k(e) and half-life were 0.486 +/- 0.084 days(-1) and 1.47+/- 0.30 days, respectively, and the C(max) was 1.2 (+/-2.92) nmol/ml serum. The model-derived, predicted adduct value at 48 h (Adduct 48) correlated with adductC(max), adduct T(max), Rumack-Matthews risk determination, peak aspartate aminotransferase (AST), and peak alanine aminotransferase (ALT). The pharmacokinetics and clinical correlates of APAP adducts in pediatric and adolescent patients with APAP overdose support the need for a further examination of the role of APAP adducts as clinically relevant and specific biomarkers of APAP toxicity.

Acetaminophen-induced hepatotoxicity in mice lacking inducible nitric oxide synthase activity.

We recently reported that nitrotyrosine and acetaminophen (APAP)-cysteine protein adducts colocalize in the hepatic centrilobular cells following a toxic dose of APAP to mice. Whereas APAP-adducts are formed by reaction of the metabolite N-acetyl-p-benzoquinone imine with cysteine, nitrotyrosine residues are formed by reaction of tyrosine with peroxynitrite. Peroxynitrite is formed from nitric oxide (NO) and superoxide. This manuscript examines APAP (300 mg/kg) hepatotoxicity in mice lacking inducible nitric oxide synthase activity (NOS2 null or knockout mice; C57BL/6-Nos2(tm1Lau)) and in the wildtype mice. In a time course the ALT levels in the exposed NOS2 null mice were approximately 50% of the wildtype mice; however, histological examination of liver sections indicated similar levels of centrilobular hepatic necrosis in both wild-type and NOS2 null mice. Serum nitrate plus nitrite levels (NO synthesis) were identical in saline-treated NOS2 null and wild-type mice (53 +/- 2 microM). APAP increased NO synthesis in wild-type mice only. The increases paralleled the increases in ALT levels with peak levels of serum nitrate plus nitrite at 6 h (168 +/- 27 microM). In wild-type mice hepatic tyrosine nitration was greatly increased relative to saline treated controls. Tyrosine nitration increased in NOS2 null mice also, but the increase was much less. APAP increased hepatic malonaldehyde levels (lipid peroxidation) in NOS2 null mice only. The results suggest the presence of multiple pathways to APAP-mediated hepatic necrosis, one via nitrotyrosine, as in the wild-type mice, and another that is not dependent upon inducible nitric oxide synthase activity, but which may involve increased superoxide.

Elevation of serum interleukin 8 levels in acetaminophen overdose in children and adolescents.

BACKGROUND: Elevations of inflammatory cytokines have been reported in animal models of acetaminophen (INN, paracetamol) toxicity. In addition, interleukin 8, a chemokine, has been found to be elevated in toxin-associated hepatic disease (ie, acute alcoholic hepatitis). The purpose of this study was to measure serum cytokine levels in children and adolescents with acetaminophen overdose and to evaluate relationships between cytokine elevation and hepatotoxicity. METHODS: Serum levels of tumor necrosis factor alpha, interleukin 1beta, interleukin 6, interleukin 8, and interleukin 10 were measured by ELISA in children and adolescents (n = 35) with acetaminophen overdose. Peak cytokine levels were examined relative to biochemical evidence of hepatocellular injury, nomogram risk assessment, and prothrombin time. RESULTS: Five patients had aspartate aminotransferase or alanine aminotransferase levels >1000 IU/L, and 4 patients had aspartate aminotransferase or alanine aminotransferase levels > or =100 IU/L and < or =1000 IU/L. No elevations of tumor necrosis factor alpha or interleukin 1beta were detected. Peak interleukin 8, but not interleukin 6 or interleukin 10, correlated with hepatotoxicity (Mann-Whitney exact test, P <.001). The peak interleukin 8 level was greater in patients at high risk by the nomogram combined with those presenting at >15 hours, as compared with other patients (Mann-Whitney U test, P <.01). The interleukin 8 level peaked before aspartate aminotransferase or alanine aminotransferase in 5 of the 9 patients with hepatotoxicity. In addition, interleukin 8 concentrations of >20 pg/mL were associated with peak prothrombin time values (Mann-Whitney exact test, P <.015). CONCLUSIONS: Interleukin 8 elevation in patients with acetaminophen hepatotoxicity corresponds with other common clinical measures that are predictive of hepatocellular injury. Further study is warranted to evaluate possible mechanistic relationships between inflammatory cytokines and acetaminophen hepatotoxicity in children and adults.

Measurement of acetaminophen-protein adducts in children and adolescents with acetaminophen overdoses. .

Acetaminophen-protein adducts are biomarkers of acetaminophen toxicity present in the centrilobular region of the liver of laboratory animals following the administration of toxic doses of acetaminophen. These biomarkers are highly specific for acetaminophen-induced hepatic injury and correlate with hepatic transaminase elevation. The objective of this prospective, multicenter study was to evaluate the clinical application of the measurement of acetaminophen-protein adducts in pediatric acetaminophen overdose patients. Serum samples were obtained from 51 children and adolescents with acetaminophen overdose at the time of routine blood sampling for clinical monitoring. Six subjects developed "severe" hepatotoxicity (transaminase elevation > 1,000 IU/L), and 6 subjects had transaminase elevation of 100 to 1,000 IU/L. Acetaminophen-protein adducts were detected in the serum of only 1 study subject, a patient with marked transaminase elevation (> 6,000 IU/L) and high risk for the development of hepatotoxicity according to the Rumack nomogram. While this study provides further support for the occurrence of covalent binding of acetaminophen to hepatic protein in humans following acetaminophen overdose, the detection of acetaminophen-protein adducts in serum with the current methodology requires significant biochemical evidence of hepatocellular injury.

Vascular and hepatocellular peroxynitrite formation during acetaminophen toxicity: role of mitochondrial oxidant stress.

Peroxynitrite may be involved in acetaminophen-induced liver damage. However, it is unclear if peroxynitrite is generated in hepatocytes or in the vasculature. To address this question, we treated C3Heb/FeJ mice with 300 mg/kg acetaminophen and assessed nitrotyrosine protein adducts as indicator for peroxynitrite formation. Vascular nitrotyrosine staining was evident before liver injury between 0.5 and 2 h after acetaminophen treatment. However, liver injury developed parallel to hepatocellular nitrotyrosine staining between 2 and 6 h after acetaminophen. The mitochondrial content of glutathione disulfide, as indicator of reactive oxygen formation determined 6 h after acetaminophen, increased from 2.8 +/- 0.6% in controls to 23.5 +/- 5.1%. A high dose of allopurinol (100 mg/kg) strongly attenuated acetaminophen protein-adduct formation and prevented the mitochondrial oxidant stress and liver injury after acetaminophen. Lower doses of allopurinol, which are equally effective in inhibiting xanthine oxidase, were not protective and had no effect on nitrotyrosine staining and acetaminophen protein adduct formation. In vitro experiments showed that allopurinol is not a direct scavenger of peroxynitrite. We conclude that there is vascular peroxynitrite formation during the first 2 h after acetaminophen treatment. On the other hand, reactive metabolites of acetaminophen bind to intracellular proteins and cause mitochondrial dysfunction and superoxide formation. Mitochondrial superoxide reacts with nitric oxide to form peroxynitrite, which is responsible for intracellular protein nitration. The pathophysiological relevance of vascular peroxynitrite for hepatocellular peroxynitrite formation and liver injury remains to be established.

Evaluation of occult acetaminophen hepatotoxicity in hospitalized children receiving acetaminophen. Pediatric Pharmacology Research Unit Network.

The safety of repeated doses of acetaminophen in ill children with the potential of reduced glutathione stores has been questioned. This study measured hepatic transaminases in children and adolescents (n=100) who received > or = 6 therapeutic doses of acetaminophen over a 48-hour period of hospitalization. Acetaminophen-protein adducts were measured in a cohort of subjects with hepatic transaminase elevation (n=8) and in those (n=10) receiving concurrent drug therapy with agents that induce the cytochrome P450 enzymes involved in acetaminophen metabolism. Acetaminophen-protein adducts were not detected in this cohort of 18 subjects. Based on this pilot study, the routine use of acetaminophen at therapeutic doses in ill, hospitalized children and adolescents appears safe.

Oxidant stress in rat liver after lipopolysaccharide administration: effect of inducible nitric-oxide synthase inhibition.

The role of inducible nitric-oxide synthase (iNOS) in lipopolysaccharide (LPS)-induced hepatic oxidant stress was evaluated using the iNOS inhibitor L-iminoethyl-lysine (L-NIL). Male rats were divided into three groups. One group received LPS (Salmonella minnesota) 2 mg/kg i.v. A second group received LPS plus L-NIL (3 mg/kg i.p.) at the time of LPS administration followed by a second dose 3 h later. A third group received saline i.v. At 6 h, blood and liver tissue were collected. Serum nitrate/nitrite (metabolic products of nitric oxide) levels were increased from 5.4 +/- 1.5 nmol/ml in the saline group to 360 +/- 48 nmol/ml in the LPS group (n = 5). Values for the LPS + L-NIL group were significantly reduced to 35 +/- 7 nmol/ml. Tissue malondialdehyde levels were increased from 0.20 +/- 0.02 nmol/mg (n = 4) in the saline group to 0.41 +/- 0.03 nmol/mg (n = 4) in the LPS group. L-NIL significantly reduced the values in the LPS group to 0.29 +/- 0.02 nmol/mg (n = 4). 4-Hydroxynonenal-protein adducts levels were increased 3.6-fold by LPS treatment as compared with saline. L-NIL significantly reversed the levels to 1.6-fold (n = 4). Intracellular GSH levels were decreased from 8.49 +/- 0.64 nmol/mg (n = 4) in the saline group to 5.63 +/- 0.51 nmol/mg in the LPS group (n = 7). L-NIL significantly increased the levels in the LPS group to 7.04 +/- 0.46 nmol/mg (n = 7). These data indicate that LPS-induced nitric oxide generation can result in oxidant stress in the liver, and that inhibitors of iNOS may offer some protection in LPS-induced hepatic toxicity.

Western blot analysis for nitrotyrosine protein adducts in livers of saline-treated and acetaminophen-treated mice.

The hepatic centrilobular necrosis produced by the analgesic/antipyretic acetaminophen correlates with metabolic activation of the drug leading to its covalent binding to protein. However, the molecular mechanism of the toxicity is not known. Recent immunohistochemical analyses using an antinitrotyrosine antiserum indicated that nitrotyrosine protein adducts co-localized with the acetaminophen-protein adducts in the centrilobular cells of the liver. Nitration of proteins is believed to occur by peroxynitrite, a substance formed by the rapid reaction of superoxide with nitric oxide. Nitric oxide and superoxide may be formed by activated Kupffer cells or by other cells. Because we were unable to successfully utilize the commercial antiserum in Western blot analyses of liver fractions, we developed a new antiserum. With our antiserum, liver fractions from saline-treated control and acetaminophen-treated mice were successfully analyzed for nitrated proteins. The immunogen for this new antiserum was synthesized by coupling 3-nitro-4-hydroxybenzoic acid to keyhole limpet hemocyanin. A rabbit immunized with this adduct yielded a high titer of an antiserum that recognized BSA nitrated with peroxynitrite. Immunoblot analysis of nitrated BSA indicated that nitrotyrosine present in a protein sample could be easily detected at levels of 20 pmoles. Immunohistochemical analyses indicated that nitrotyrosine protein adducts were detectable in the centrilobular areas of the liver. Immunoblot analysis of liver homogenates from both saline-treated and acetaminophen-treated mice (300 mg/kg) indicate that the major nitrotyrosine protein adducts produced have molecular weights of 36 kDa, 44 kDa, and 85 kDa. The 85-kDa protein stained with the most intensity. The hepatic homogenates of the acetaminophen- treated mice showed significantly increased levels of all protein adducts.

Pretreatment of mice with macrophage inactivators decreases acetaminophen hepatotoxicity and the formation of reactive oxygen and nitrogen species.

Hepatotoxic doses of acetaminophen to mice produce not only acetaminophen-protein adducts in the centrilobular cells of the liver, but nitrotyrosine-protein adducts in the same cells, the site of the necrosis. Nitration of tyrosine occurs with peroxynitrite, a species formed by reaction of nitric oxide (NO.) with superoxide (O2. -). Because NO. and O2.- may be produced by activated Kupffer cells and/or infiltrated macrophages, we pretreated mice with the macrophage inactivators/depeleters gadolinium chloride (7 mg/kg, intravenously [iv]) or dextran sulfate (10 mg/kg, iv) 24 hours before administration of acetaminophen (300 mg/kg). Mice treated with acetaminophen plus gadolinium chloride, or acetaminophen plus dextran sulfate, had significantly less evidence of hepatotoxicity as evidenced by lower serum alanine transaminase (ALT) levels (28 +/- 1 IU/L and 770 +/- 240 IU/L, respectively) at 8 hours compared with acetaminophen (6,380 +/- 408 IU/L). Analysis of hepatic homogenates for acetaminophen-protein adducts at 2 hours, a time of maximal covalent binding and before hepatocyte lysis, indicated that these pretreatments did not decrease covalent binding. Western blot analysis for the macrophage marker protein F4/80 in homogenates revealed not only the expected decrease by the macrophage inactivators/depleters, but also an apparent increase in acetaminophen-only-treated mice. At 8 hours nitrotyrosine-protein adducts were present in the acetaminophen-only-treated mice, but not in the acetaminophen plus gadolinium chloride-treated mice, or acetaminophen plus dextran sulfate-treated mice. High levels of heme-protein adducts, a measure of oxidative stress, were detected in livers of the 8 hour acetaminophen-only-treated mice. These data suggest that acetaminophen hepatotoxicity is mediated by an initial metabolic activation and covalent binding, and subsequent activation of macrophages to form O2.-, NO., and peroxynitrite. Nitration of tyrosine correlates with toxicity.

Deferoxamine delays the development of the hepatotoxicity of acetaminophen in mice.

The hepatotoxicity of acetaminophen is conventionally ascribed to metabolism by CYP450 to N-acetyl-p-benzoquinone imine and covalent binding to proteins. We investigated a potential role for oxidative stress by determining the effect of the ferric chelator deferoxamine (Desferal) on acetaminophen (paracetamol)-induced hepatotoxicity in mice. Administration of deferoxamine (75 mg/kg) 1 h after a toxic dose of acetaminophen (300 mg/kg) significantly delayed the development of the toxicity without altering covalent binding. In saline-treated mice serum ALT was 18 +/- 2 IU/l. In acetaminophen-treated mice serum alanine aminotransferase (ALT) was 779 +/- 271 at 2 h, 7421 +/- 552 IU/l at 4 h, 5732 +/- 523 IU/l at 8 h, and 5984 +/- 497 IU/l at 24 h. In acetaminophen plus deferoxamine-treated mice, serum ALT was 80 +/- 10 at 2 h, 472 +/- 74 IU/l at 4 h, 2149 +/- 597 IU/l at 8 h, and 5766 +/- 388 at 24 h. Deferoxamine at 1 h after acetaminophen did not decrease serum ALT at 12 h; however, deferoxamine at 1 and 4 h, or deferoxamine at 1 h plus N-acetylcysteine at 4 h to replete hepatic glutathione, decreased the toxicity from 5625 +/- 310 IU/l to 3436 +/- 546 IU/l and 3003 +/- 282 IU/l, respectively. Deferoxamine plus N-acetylcysteine at 1.25 h after acetaminophen was more effective at decreasing the 24 h toxicity than N-acetylcysteine alone. In acetaminophen treated mice, higher doses of deferoxamine (150-300 mg/kg) at 1 h greatly increased the observed hepatotoxicity at 4 h in a dose responsive manner, but deferoxamine alone was nontoxic.

Nitrotyrosine-protein adducts in hepatic centrilobular areas following toxic doses of acetaminophen in mice.

Treatment of mice with a toxic dose of acetaminophen (300 mg/kg, ip) significantly increased hepatotoxicity at 4 h, as evidenced by histological necrosis in the centrilobular areas of the liver, and increased serum levels of alanine aminotransferase (ALT) (from 8 +/- 1 IU/L in saline-treated mice to 3226 +/- 892 IU/L in the acetaminophen-treated mice). Serum levels of nitrate plus nitrite (a marker of nitric oxide synthesis) were also increased from 62 +/- 8 microM in saline-treated mice to 110 +/- 14 microM in acetaminophen-treated mice (P < 0.05). Regression analysis of serum ALT levels to serum nitrate plus nitrite levels in individual mice revealed a positive, linear relationship between serum ALT levels and serum nitrate plus nitrite levels with a correlation coefficient of 0.9 (P < 0.05). The y intercept value (nitrate plus nitrite level) was 63 +/- 15 microM. Immunohistochemical analysis of liver sections from acetaminophen-intoxicated mice using an anti-3-nitrotyrosine antibody indicated tyrosine nitration in the proteins of the centrilobular cells. Tyrosine nitration has been shown to occur by peroxynitrite, a reactive intermediate formed by an extremely rapid reaction of nitric oxide and superoxide and a species which also has hydroxyl radical-like activity. Analysis of liver sections using an anti-acetaminophen antiserum indicated the centrilobular cells also contained acetaminophen-protein adducts, a reaction of the metabolite N-acetyl-p-benzoquinone imine with cysteine residues on proteins. These data are consistent with acetaminophen metabolic activation leading to increased synthesis of nitric oxide and superoxide and to peroxynitrite as an important intermediate in the toxicity.

Immunochemical comparison of 3'-hydroxyacetanilide and acetaminophen binding in mouse liver.

The hepatotoxicity of the analgesic acetaminophen is believed to be mediated by covalent binding to critical proteins. Radiolabeled 3'-hydroxyacetanilide, a regioisomer of acetaminophen, covalently binds to proteins at levels similar to those of acetaminophen, but without toxicity. Covalent binding has recently been detected by Western blot to a 50-kDa microsomal protein that comigrated with CYP2E1 and was accompanied by a loss of the CYP2E1 activity. However, radiolabel studies previously indicated that a significant amount of the radiolabel is lost during electrophoresis. In the present study, 3'-hydroxyacetanilide covalent binding was detected immunohistochemically in liver using an anti-acetaminophen antiserum. 3'-Hydroxyacetanilide (1000 mg/kg, ip) administration to mice resulted in panlobular immunostaining in liver, with the single layer of hepatocytes surrounding the central veins having the greatest intensity of staining. Staining was most intense at 1 hr and somewhat decreased at 3 and 6 hr. In contrast, immunochemical staining indicated that covalent binding of acetaminophen (250 mg/kg, ip) was confined to the centrilobular hepatocytes, the area of the ensuing necrosis. Cobaltous chloride pretreatment decreased the total intensity of the panlobular immunostaining following 3'-hydroxyacetanilide. The CYP2E1 inhibitor diallyl sulfide decreased the intensity of immunostaining in the central vein area only. Western blot analysis indicated diallyl sulfide also eliminated binding to the microsomal 50-kDa protein. These data are consistent with centrilobular binding of 3'-hydroxyacetanilide, mediated in part by CYP2E1, and panlobular binding, mediated by other P450 enzymes.

The acetaminophen regioisomer 3'-hydroxyacetanilide inhibits and covalently binds to cytochrome P450 2E1.

3'-Hydroxyacetanilide has been previously studied as a nontoxic regioisomer of the analgesic acetaminophen (4'-hydroxyacetanilide). The radiolabeled derivative has been shown to covalently bind to liver proteins at levels similar to that observed with hepatotoxic doses of radiolabeled acetaminophen with no evidence of hepatic damage. Using an anti-arylacetamide antiserum the primary protein adduct detected following administration of 3'-hydroxyacetanilide (300 and 600 mg/kg) to mice was a 50 kDa microsomal protein that co-migrated with cytochrome P450 2E1. Cytochrome P450 2E1 enzyme activity (p-nitrophenol hydroxylase) was decreased by 79% in the mice treated with 3'-hydroxyacetanilide (600 mg/kg). Incubation of 3'-hydroxyacetanilide with hepatic microsomes resulted in a time dependent 47% decrease in cytochrome P450 2E1 activity. Pre-incubation of acetaminophen with microsomes did not result in covalent binding to the cytochrome P450 nor was there a decrease in p-nitrophenol hydroxylase activity. These data suggest that 3'-hydroxyacetanilide covalently binds to cytochrome P450 2E1 with preferential loss of activity.

Selective protein covalent binding and target organ toxicity.

Protein covalent binding by xenobiotic metabolites has long been associated with target organ toxicity but mechanistic involvement of such binding has not been widely demonstrated. Modern biochemical, molecular, and immunochemical approaches have facilitated identification of specific protein targets of xenobiotic covalent binding. Such studies have revealed that protein covalent binding is not random, but rather selective with respect to the proteins targeted. Selective binding to specific cellular target proteins may better correlate with toxicity than total protein covalent binding. Current research is directed at characterizing and identifying the targeted proteins and clarifying the effect of such binding on their structure, function, and potential roles in target organ toxicity. The approaches employed to detect and identify the tartgeted proteins are described. Metabolites of acetaminophen, halothane, and 2,5-hexanedione form covalently bound adducts to recently identified protein targets. The selective binding may influence homeostatic or other cellular responses which in turn contribute to drug toxicity, hypersensitivity, or autoimmunity.

Covalent binding of xenobiotics to specific proteins in the liver.

Chemicals that cause toxicity though a direct mechanism, such as acetaminophen, covalently bind to a select group of proteins prior to the development of toxicity, and these proteins may be important in the initiation of the events that lead to the hepatotoxicity. Disruption of the cell is measured by release of intracellular proteins such as alanine aminotransferase and occurs late in the time course following a hepatotoxic dose of a direct toxin. Prior to this disruption, there appears to be a large number of proteins covalently modified by a reactive metabolite. There are at least two possible mechanisms that may cause the toxicity. First, some critical protein is a target of the reactive metabolite. Disruption of the enzymatic function (or a critical pathway for a regulatory protein) may lead directly to cell death. With the direct hepatotoxin acetaminophen, there is a decrease in the activity of several of the early target proteins, but how this disruption of critical proteins leads to the toxicity is still unclear. The early targets appear to be proteins with accessible nucleophilic sulfhydryl groups, and usually the target has a high concentration of the protein within the cell. It is possible that the binding to some of these proteins represents a detoxification protecting more critical targets within the cell. A second mechanism for the direct toxicity is that more and more proteins become targets in the time course following administration of a direct toxin, and eventually the cells machinery is overwhelmed. The cell can then no longer function, or there is a disruption the redox balance within the cell due to the decreased function of numerous proteins. In contrast to the direct-acting toxins, the chemical-protein conjugates that initiate toxicity through an activation of the immune system appear to have a limited number of target proteins and are localized within one subcellular fraction. Halothane produces adducts almost exclusively in the microsomal fraction, and these adducts appear to be limited to selective proteins with high concentrations in this fraction. The substitution level is an important factor in the development of an immune response. Halothane hepatitis patients' antibodies primarily recognize proteins with a high substitution level. For halothane and diclofenac, the proteins are accessible to the immune system through exposure on the plasma membrane. Trichloroethylene binds primarily to a 50-kDa microsomal protein, and preliminary evidence has been presented which indicates that a trichloroethylene-protein conjugate is released into the blood following exposure, where contact with the immune system can occur. In order to elicit an immune response the immune system requires multiple exposure to the chemical-protein conjugates. With halothane hepatitis and with diclofenac hepatitis, as well as occupational and environmental exposure to trichloroethylene, there are multiple exposures leading to repeat presentation of the protein adducts to the immune system; this situation is not generally found with acetaminophen overdose patients. In summary, direct toxicants such as acetaminophen covalently bind to selected targets which may be critical to the development of hepatotoxicity, and they later form adducts with numerous proteins which may overwhelm the cell's capacity to maintain homeostasis, leading to loss of vital function and cell death (Fig.3). In contrast, indirect toxicants that elicit an immune-mediated toxicity such as halothane, and possibly diclofenac and trichloroethylene, appear to have a limited number of protein targets with a high substitution level, and the immune system is exposed repeatedly to the modified proteins.

Comparison of covalent binding of acetaminophen and the regioisomer 3'-hydroxyacetanilide to mouse liver protein.

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.

Covalent binding of acetaminophen to N-10-formyltetrahydrofolate dehydrogenase in mice.

The analgesic acetaminophen is frequently used as a model chemical to study hepatotoxicity; however, the critical mechanisms by which it produces toxicity within the cell are unknown. It has been postulated that covalent binding of a toxic metabolite to crucial proteins may inhibit vital cellular functions and may be responsible for, or contribute to, the hepatotoxicity. To further understand the importance of covalent binding in the toxicity, a major cytosolic acetaminophen-protein adduct of 100 kDa has been purified by a combination of anion exchange chromatography and preparative electrophoresis. N-Terminal and internal amino acid sequences of peptides from the purified 100-kDa acetaminophen-protein adduct were found to be homologous with the deduced amino amino acid sequence from the cDNA of N-10-formyltetrahydrofolate dehydrogenase. Antiserum specific for N-10-formyltetrahydrofolate dehydrogenase and acetaminophen react in a Western blot with the purified 100-kDa acetaminophen-protein adduct. Administration of a toxic dose of acetaminophen (400 mg/kg) to mice resulted in a 25% decrease in cytosolic N-10-formyltetrahydrofolate dehydrogenase activity at 2 hr. The covalent binding of acetaminophen to proteins such as N-10-formyltetrahydrofolate dehydrogenase and the subsequent decreases in their enzyme activity may play a role in acetaminophen hepatotoxicity.

Acetaminophen-induced hepatotoxicity. Analysis of total covalent binding vs. specific binding to cysteine.

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.

Mechanism of acetaminophen-induced hepatotoxicity: covalent binding versus oxidative stress.

The hepatotoxicity of acetaminophen is believed to be mediated by the reactive metabolite N-acetyl-p-benzoquinone imine; however, the mechanism by which this metabolite produces the toxicity is unknown. The metabolite, which is both an electrophile and an oxidizing agent, may covalently bind to critical proteins, or it may initiate oxidative damage. We have previously developed a Western blot assay for detection of acetaminophen covalently bound to protein and have reported the relationship between covalent binding and the development of hepatotoxicity. Recently, we developed a Western blot assay for protein aldehyde formation, which may occur via the reactive oxygen species, the hydroxyl radical. In this paper, we have compared covalent binding to protein aldehyde formation. Toxic doses of acetaminophen (400 mg/kg) were administered to mice, and the mice were subsequently killed at 0, 1, 2, 4, and 6 h. Since the oxidizing agent FeSO4 has been reported to potentiate lipid peroxidation when administered with acetaminophen, other mice received FeSO4 (100 mg/kg) plus acetaminophen. Compared to saline-treated control mice, acetaminophen treatment significantly increased serum alanine aminotransferase levels, an index of hepatotoxicity, at 4 and 6 h, but not at 1 or 2 h. Acetaminophen plus FeSO4 treatment of mice significantly increased serum alanine aminotransferase levels at 2, 4, and 6 h compared to controls. Levels of alanine aminotransferase in serum of acetaminophen plus ferrous sulfate-treated mice were higher at 4 and 6 h than those of acetaminophen-treated mice, but not significantly different. FeSO4 alone did not increase alanine aminotransferase levels. Western blot assays revealed that acetaminophen did not cause an increase in protein aldehydes over control at any time, nor did acetaminophen plus FeSO4; however, FeSO4 alone increased the intensity of staining of the immunoblot for protein aldehydes over control at all times after 0 time. Acetaminophen-protein adducts were detected in acetaminophen- and acetaminophen plus FeSO4-treated mice. In vitro experiments indicated that FeSO4 plus tert-butyl hydroperoxide in the presence of bovine serum albumin increased protein aldehyde formation. Inclusion of acetaminophen in the incubation mixture inhibited protein oxidation of bovine serum albumin in a concentration dependent manner. The data indicate that acetaminophen quenches protein oxidation, presumably by reacting with the hydroxyl radical. These data are consistent with the theory that acetaminophen covalent binding is the primary mechanism of toxicity and argue against a role for protein oxidation in acetaminophen hepatotoxicity.

Glutamate dehydrogenase covalently binds to a reactive metabolite of acetaminophen.

The mechanism of the hepatotoxicity of the analgesic acetaminophen is believed to be mediated by covalent binding to protein; however, critical targets which effect the toxicity are unknown. It has been shown that mitochondrial respiration in vivo is inhibited in mice as early as 1 h following a hepatotoxic dose of acetaminophen, and it is postulated that covalent binding to critical mitochondrial proteins may be important. A time course of mitochondrial proteins stained with anti-acetaminophen in an immunoblot detected two major adducts of 50 and 67 kDa as early as 30 min after a hepatotoxic dose of acetaminophen in mice. To further understand the role of covalent binding to mitochondrial proteins and acetaminophen hepatotoxicity, we have purified and identified a 50 kDa mitochondrial protein which becomes covalently bound to a reactive metabolite of acetaminophen. An N-terminal sequence of the 50 kDa adduct was 100% homologous with the deduced amino acid sequence of glutamate dehydrogenase. In addition, the purified protein was immunochemically reactive with rat liver anti-glutamate dehydrogenase. Enzyme activity of glutamate dehydrogenase was significantly decreased in mice 1 h following hepatotoxic treatment with acetaminophen. These data suggest that acetaminophen hepatotoxicity may in part be mediated by covalent binding to glutamate dehydrogenase.