Hepatotoxic Dose Of Acetaminophen Research Articles

Tropisetron Protects Against Acetaminophen-Induced Liver Injury via Suppressing Hepatic Oxidative Stress and Modulating the Activation of JNK/ERK MAPK Pathways.

Objectives. To investigate the protective effects of tropisetron on acetaminophen- (APAP-) induced liver injury in a mice model. Methods. C57BL/6 male mice were given tropisetron (0.3 to 10 mg/kg) 30 minutes before a hepatotoxic dose of acetaminophen (300 mg/kg) intraperitoneally. Twenty hours after APAP intoxication, sera alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, hepatic myeloperoxidase (MPO), malondialdehyde (MDA), glutathione (GSH), and superoxide dismutase (SOD) activities, and liver histopathological changes were examined. The MAP kinases were also detected by western blotting. Results. Our results showed that tropisetron pretreatment significantly attenuated the acute elevations of the liver enzyme ALT level, hepatic MPO activity, and hepatocytes necrosis in a dose-dependent manner (0.3–10 mg/kg) in APAP-induced hepatotoxicity mice. Tropisetron (1 and 3 mg/kg) suppressed APAP-induced hepatic lipid peroxidation expression and alleviated GSH and SOD depletion. Administration of tropisetron also attenuated the phosphorylation of c-Jun-NH2-terminal protein kinase (JNK) and extracellular signal-regulated kinase (ERK) caused by APAP. Conclusion. Our data demonstrated that tropisetron's hepatoprotective effect was in part correlated with the antioxidant, which were mediated via JNK and ERK pathways on acetaminophen-induced liver injury in mice.

The neuronal nitric oxide synthase inhibitor NANT blocks acetaminophen toxicity and protein nitration in freshly isolated hepatocytes

3-Nitrotyrosine (3NT) in liver proteins of mice treated with hepatotoxic doses of acetaminophen (APAP) has been postulated to be causative in toxicity. Nitration is by a reactive nitrogen species formed from nitric oxide (NO). The source of the NO is unclear. iNOS knockout mice were previously found to be equally susceptible to APAP toxicity as wildtype mice and iNOS inhibitors did not decrease toxicity in mice or in hepatocytes. In this work we examined the potential role of nNOS in APAP toxicity in hepatocytes using the specific nNOS inhibitor NANT (10µM)(N-[(4S)-4-amino-5-[(2-aminoethyl)amino]pentyl]-N′-nitroguanidinetris (trifluoroacetate)). Primary hepatocytes (1 million/ml) from male B6C3F1 mice were incubated with APAP (1mM). Cells were removed and assayed spectrofluorometrically for reactive nitrogen and oxygen species using diaminofluorescein (DAF) and Mitosox red, respectively. Cytotoxicity was determined by LDH release into media. Glutathione (GSH, GSSG), 3NT, GSNO, acetaminophen-cysteine adducts, NAD, and NADH were measured by HPLC. APAP significantly increased cytotoxicity at 1.5–3.0h. The increase was blocked by NANT. NANT did not alter APAP mediated GSH depletion or acetaminophen-cysteine adducts in proteins which indicated that NANT did not inhibit metabolism. APAP significantly increased spectroflurometric evidence of reactive nitrogen and oxygen formation at 0.5 and 1.0h, respectively, and increased 3NT and GSNO at 1.5–3.0h. These increases were blocked by NANT. APAP dramatically increased NADH from 0.5–3.0h and this increase was blocked by NANT. Also, APAP decreased the Oxygen Consumption Rate (OCR), decreased ATP production, and caused a loss of mitochondrial membrane potential, which were all blocked by NANT.

Lymphocyte Loss and Immunosuppression Following Acetaminophen-Induced Hepatotoxicity in Mice as a Potential Mechanism of Tolerance

Current evidence suggests that drug-induced liver disease can be caused by an allergic response (drug-induced allergic hepatitis, DIAH) induced by hepatic drug-protein adducts. The relatively low incidence of these reactions has led us to hypothesize that tolerogenic mechanisms prevent DIAH from occurring in most people. Here, we present evidence for the existence of one of these regulatory pathways. Following a hepatotoxic dose of acetaminophen in C57Bl/6 mice, lymphocyte loss that appeared to be due at least in part to apoptosis was noted in the spleen, thymus, and draining lymph nodes of the liver. There was no observable lymphocyte loss in the absence of hepatotoxicity. Acetaminophen-induced liver injury (AILI) also led to a functional suppression of the immune system as determined by the inhibition of a delayed-type hypersensitivity response to dinitrochlorobenzene. Further studies with adrenalectomized mice suggested a role for corticosterone in the depletion of lymphocytes following APAP-induced liver injury. In conclusion, these findings suggest that lymphocyte loss and immunosuppression following AILI may prevent subsequent occurrences of allergic hepatitis and possibly other forms of APAP-induced allergies induced by hepatic drug-protein adducts. Similar regulatory pathways may inhibit other hepatotoxic drugs from causing allergic reactions.

Measurement of Serum Acetaminophen–Protein Adducts in Patients With Acute Liver Failure

Acetaminophen toxicity is the most common cause of acute liver failure (ALF) in the United States and Great Britain, but may be underrecognized in certain settings. Acetaminophen-protein adducts are specific biomarkers of drug-related toxicity in animal models and can be measured in tissue or blood samples. Measurement of serum adducts might improve diagnostic accuracy in acute liver failure (ALF) patients. We measured serum acetaminophen-protein adducts using high-pressure liquid chromatography with electrochemical detection in coded sera of 66 patients with ALF collected prospectively at 24 US tertiary referral centers. Samples were included from 20 patients with well-characterized acetaminophen-related acute liver failure, 10 patients with ALF owing to other well-defined causes, 36 patients with ALF of indeterminate etiology, and 15 additional patients without ALF but with known acetaminophen overdose and minimal or no biochemical liver injury. Acetaminophen-protein adducts were detected in serum in 100% of known acetaminophen ALF patients and in none of the ALF patients with other defined causes, yielding a sensitivity and specificity of 100%. In daily serial samples, serum adducts decreased in parallel with aminotransferase levels. Seven of 36 (19%) indeterminate cases demonstrated adducts in serum suggesting that acetaminophen toxicity caused or contributed to ALF in these patients. Low adduct levels were present in 2 of 15 patients with acetaminophen overdose without significant liver injury. Measurement of serum acetaminophen-protein adducts reliably identified acetaminophen toxicity, and may be a useful diagnostic test for cases lacking historical data or other clinical information.

Protective effect of arabic gum against acetaminophen-induced hepatotoxicity in mice

Overdose of acetaminophen, a widely used analgesic drug, can result in severe hepatotoxicity and is often fatal. This study was undertaken to examine the effects of arabic gum (AG), which is commonly used in processed foods, on acetaminophen-induced hepatotoxicity in mice. Mice were given arabic gum orally (100 g l −1) 5 days before a hepatotoxic dose of acetaminophen (500 mg kg −1) intraperitoneally. Arabic gum administration dramatically reduced acetaminophen-induced hepatotoxicity as evidenced by reduced serum alanine (ALT) and aspartate aminotransferase (AST) activities. Acetaminophen-induced hepatic lipid peroxidation was reduced significantly by arabic gum pretreatment. The protection offered by arabic gum does not appear to be caused by a decrease in the formation of toxic acetaminophen metabolites, which consumes glutathione, because arabic gum did not alter acetaminophen-induced hepatic glutathione depletion. Acetaminophen increased nitric oxide synthesis as measured by serum nitrate plus nitrite at 4 and 6 h after administration and arabic gum pretreatment significantly reduced their formation. In conclusion, arabic gum is effective in protecting mice against acetaminophen-induced hepatotoxicity. This protection may involve the reduction of oxidative stress.

The nitric oxide donor, V-PYRRO/NO, protects against acetaminophen-induced hepatotoxicity in mice

The liver-selective nitric oxide (NO) donor, O 2 -vinyl 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate (V-PYRRO/NO), is metabolized by P-450 enzymes to release NO in the liver, and is shown to protect the liver from tumor necrosis factor α (TNF-α)-induced apoptosis and D-glactosamine/endotoxin–induced hepatotoxicity. This study was undertaken to examine the effects of V-PYRRO/NO on acetaminophen-induced hepatotoxicity in mice. Mice were given V-PYRRO/NO via osmotic pumps (1.8-5.4 mg/mL, 8 μL/h) 4 to 16 hours before a hepatotoxic dose of acetaminophen (600 mg/kg, intraperitoneally [ip]). V-PYRRO/NO administration dramatically reduced acetaminophen-induced hepatotoxicity in a dose- and time-dependent manner, as evidenced by reduced serum alanine aminotransferase (ALT) activity, reduced hepatic congestion, apoptosis, and improved hepatocellular pathology. The protection afforded by V-PYRRO/NO does not appear to be caused by a decrease in the formation of toxic acetaminophen metabolites, which consumes glutathione (GSH), because V-PYRRO/NO did not alter acetaminophen-induced hepatic GSH depletion. Acetaminophen-induced lipid peroxidation, as determined by the concentrations of 4-hydroxyalkenals (4-HNE) and malondialdehyde (MDA), was reduced significantly by V-PYRRO/NO treatment. Although pretreatment was most effective, administration of V-PYRRO/NO simultaneously with acetaminophen also was able to reduce acetaminophen hepatotoxicity. Genomic analysis of the liver samples 10 hours after acetaminophen intoxication showed the enhanced expression of genes associated with stress/oxidative stress, apoptosis/cell death, and DNA damage/repair. Acetaminophen-induced alterations in gene expression were attenuated significantly by V-PYRRO/NO. Real-time reverse-transcription polymerase chain reaction (RT-PCR) and Western-blot analysis confirmed microarray results. In conclusion, V-PYRRO/NO is effective in blocking acetaminophen-induced hepatotoxicity in mice. This protection may involve the reduction of oxidative stress, the inhibition of apoptosis, and possibly the maintenance of hepatic vasculature to prevent congestion. (H EPATOLOGY 2003;37:324-333.)

Differential Induction of Heme Oxygenase-1 in Macrophages and Hepatocytes during Acetaminophen-Induced Hepatotoxicity in the Rat: Effects of Hemin and Biliverdin

Heme oxygenase-1 (HO-1), also known as heat shock protein 32, has been shown to protect against oxidant-induced tissue injury. In the present studies, we analyzed expression of this enzyme in macrophages and hepatocytes following acetaminophen administration and its potential role in hepatotoxicity. Treatment of rats with a hepatotoxic dose of acetaminophen (1 g/kg, ip) resulted in a time-dependent induction of HO-1 in the liver. This was observed within 6 h of acetaminophen administration in both hepatocytes and macrophages. Hepatocytes were found to be more sensitive than macrophages to the effects of acetaminophen on HO-1. Up regulation of HO-1 in the liver following acetaminophen administration correlated with induction of ferritin and manganese superoxide dismutase (MnSOD). To determine if HO-1 was hepatoprotective, rats were pretreated with hemin (30 μmol/kg, ip), a potent inducer of the enzyme. Following hemin treatment, we observed a time-dependent increase in HO-1 protein in the liver and in serum bilirubin levels. Pretreatment of rats with hemin was found to prevent acetaminophen-induced hepatotoxicity, as measured histologically and biochemically by decreased serum transaminase levels. This was correlated with more rapid increases in expression of hepatic ferritin and MnSOD. Heme metabolism via HO-1 generates biliverdin, which is rapidly converted to bilirubin by biliverdin reductase. Pretreatment of rats with biliverdin (40 μmol/kg, ip) was also found to block acetaminophen-induced injury. These data suggest that HO-1 is an important component of antioxidant defense during acetaminophen-induced hepatotoxicity.

Expression Profiling of Acetaminophen Liver Toxicity in Mice Using Microarray Technology

Drug-induced hepatotoxicity causes significant morbidity and mortality and is a major concern in drug development. This is due, in large part, to insufficient knowledge of the mechanism(s) of drug-induced liver injury. In order to address this problem, we have evaluated the modulation of gene expression within the livers of mice treated with a hepatotoxic dose of acetaminophen (APAP) using high-density oligonucleotide microarrays capable of determining the expression profile of >11,000 genes and expressed sequence tags (ESTs). Significant alterations in gene expression, both positive and negative, were noted within the livers of APAP-treated mice. APAP-induced toxicity affected numerous aspects of liver physiology causing, for instance, >twofold increased expression of genes that encode for growth arrest and cell cycle regulatory proteins, stress-induced proteins, the transcription factor LRG-21, suppressor of cytokine signaling (SOCS)-2-protein, and plasminogen activator inhibitor-1 (PAI-1). A number of these and other genes and ESTs were detectable within the liver only after APAP treatment suggesting their potential importance in propagating or preventing further toxicity. These data provide new directions for mechanistic studies that may lead to a better understanding of the molecular basis of drug-induced liver injury and, ultimately, to a more rational design of safer drugs.

Inhibition of Carbamyl Phosphate Synthetase-I and Glutamine Synthetase by Hepatotoxic Doses of Acetaminophen in Mice

The primary mechanisms proposed for acetaminophen-induced hepatic necrosis should deplete protein thiols, either by covalent binding and thioether formation or by oxidative reactions such as S-thiolations. However, in previous studies we did not detect significant losses of protein thiol contents in response to administration of hepatotoxic doses of acetaminophenin vivo.In the present study we employed derivatization with the thiol-specific agent monobromobimane and separation of proteins by SDS–PAGE to investigate the possible loss of specific protein thiols during the course of acetaminophen-induced hepatic necrosis. Fasted adult male mice were given acetaminophen, and protein thiol status was examined subsequently in subcellular fractions isolated by differential centrifugation. No decreases in protein thiol contents were indicated, with the exception of a marked decrease in the fluorescent intensity, but not of protein content, as indicated by staining with Coomassie blue, of a single band of approximately 130 kDa in the mitochondrial fractions of acetaminophen-treated mice. This protein was identified by isolation and N-terminal sequence analysis as carbamyl phosphate synthetase-I (CPS-I) (EC 6.3.4.16). Hepatic CPS-I activities were decreased in mice given hepatotoxic doses of acetaminophen. In addition, hepatic glutamine synthetase activities were lower, and plasma ammonia levels were elevated in mice given hepatotoxic doses of acetaminophen. The observed hyperammonemia may contribute to the adverse effects of toxic doses of acetaminophen, and elucidation of the specific mechanisms responsible for the hyperammonemia may prove to be useful clinically. However, the preferential depletion of protein thiol content of a mitochondrial protein by chemically reactive metabolites generated in the endoplasmic reticulum presents a challenging and potentially informative mechanistic question.

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.

Cytochrome P4502E1 inhibition by propylene glycol prevents acetaminophen (paracetamol) hepatotoxicity in mice without cytochrome P4501A2 inhibition.

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.

Diquat- and acetaminophen-induced alterations of biliary efflux of iron in rats

The effects of diquat on the biliary efflux of nonheme iron in rats were studied as a means of examining the possible effects of diquat metabolism on hepatocellular iron metabolism and the association of altered iron metabolism with the initiation of acute hepatic necrosis. Administration of hepatotoxic doses (0.1 mol/kg) of diquat to male Fischer-344 rats increased biliary iron concentrations from 6 μM to more than 15 μM. However, increases in biliary efflux of iron were not observed during the first 60 min following exposure to diquat, despite the rapid increases in biliary glutathione disulfide concentrations, which increased maximally within 40 min. Biliary efflux of iron was not altered by diquat in Sprague-Dawley rats, which are resistant to hepatic necrosis in response to diquat, despite the marked oxidant stress responses observed in these animals. Conversely, hepatotoxic doses of acetaminophen (1500 mg/kg) caused significant decreases in biliary iron efflux. The rapid decreases in biliary iron caused by acetaminophen and the delay in diquat-induced iron efflux suggested the possibility that some fraction of the biliary iron was being excreted as reversibly formed GS-Fe 2+ chelates, with inhibition of export by glutathione disulfide (GSSG) in the case of diquat, or by 3-(glutathion- S-yl)acetaminophen (GS-AAP) in the case of the acetaminophen-treated animals. However, 50–200 mg/kg doses of acetaminophen showed little effect on biliary iron excretion despite producing biliary GS-AAP conjugate concentrations almost 1000 times the 6μM concentrations of iron, which would not appear to support the hypothesis of excretion of GS-Fe 2+ chelates. The data demonstrate a significant effect of diquat on hepatic iron metabolism in Fischer-344 rats, and the possible importance of this iron redistribution to reactive oxygen-mediated cell damage in vivo is indicated by the absence of similar responses in diquat-treated Sprague-Dawley rats.

A protective effect of glutathione-dextran macromolecular conjugates on acetaminophen-induced hepatotoxicity dependent on molecular size.

Glutathion (GSH) was covalently attached to dextrans with various molecular weights of 2, 5, 10, 40, and 70 kDa by the cyanogen bromide activation method. The conjugates obtained synthetically were white or pale yellowish powders containing 6-10% (w/w) of GSH. The average molecular weights of the conjugates were estimated to be larger and the molecular weight distribution was a little broader than that of each original dextran. The conjugates significantly stabilized GSH and liberated it gradually under physiological conditions (t1/2 = 0.99-1.6h). Mice depleted of GSH by treatment with buthionine sulfoximine, a potent inhibitor of gamma-glutamylcysteine synthetase, exhibited a significant increase in hepatic GSH level after intravenous injection of the conjugates. In mice given a hepatotoxic dose of acetaminophen, the survival rate increased progressively with coadministration of the conjugates, whereas a small improvement was found when free GSH was given. The conjugate of GSH attached to dextran with the molecular weight of 40 kDa exhibited the highest prophylactic effect on acetaminophen-induced hepatotoxicity in mice. The prolonged retention of the conjugates of larger molecular weight in the circulation would cause a higher hepatic accumulation. These results suggested that molecular size would be the most critical factor in the delivery of GSH, as a dextran conjugate, into the liver.

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 m m 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 m m) and 3,5-DMA (5 m m) but not with 2,6-DMA (10 m m). 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.

Acetaminophen-induced cytotoxicity in cultured mouse hepatocytes: Correlation of nuclear Ca 2+ accumulation and early DNA fragmentation with cell death

Hepatotoxic doses of acetaminophen cause widespread alkylation of liver and early loss of cytosolic Ca 2+ regulation. Although the precise location and target of lethal alkylation are not known, Ca 2+ accumulation is viewed as a possible link between cell alkylation and cell death. We have recently shown that Ca 2+ accumulates in the nucleus and that DNA fragments in vivo before the development of acetaminophen-induced necrosis in mice. The present study examined cultured hepatocytes for nuclear damage and its association with cell death in vitro. Positive results would argue for two key points. (1) Nonparenchymal cell damage does not explain DNA fragmentation induced by acetaminophen in vivo. (2) A chemical that causes necrosis can produce DNA damage considered characteristic of apoptosis. Hepatocytes from NIH Swiss mice were isolated by collagenase perfusion, cultured in Williams' E medium for 24 hr, and exposed to acetaminophen. Cytotoxicity was assessed by lactate dehydrogenase leakage and release of [ 3H]adenine from a prelabeled nucleotide pool. Genomic DNA fragmentation was assessed quantitatively by colorimetric analysis and qualitatively by agarose gel electrophoresis. Acetaminophen caused DNA damage from 1–4 hr onward and produced significant release of lactate dehydrogenase and [ 3H]adenine nucleotides at later times. Agarose gel electrophoresis revealed a “ladder” of DNA fragments characteristic of Ca 2+-mediated endonuclease activation. Cytotoxicity correlated with nuclear Ca 2+ accumulation ( r > 0.895, p < 0.05) and with percentage DNA fragmentation ( r > 0.835, p < 0.05). Nuclear changes in vitro generally reproduced those observed in vivo. Collectively, these findings demonstrate that nuclear Ca 2+ accumulation and DNA fragmentation appear as early events that correlate directly with later cytotoxicity. These changes may contribute to acetaminophen-induced injury leading to cell death in vitro and necrosis in vivo.

Effect of ascorbic acid esters on hepatic glutathione levels in mice treated with a hepatotoxic dose of acetaminophen

Acetaminophen (APAP) with or without ascorbyl stearate (AS) or ascorbyl palmitate (AP) was administered by gavage to male Swiss-Webster mice at a dose of 600 mg/kg for each chemical. The biochemical markers of hepatotoxicity, serum transaminases (serum glutamate pyruvate transaminase [SGPT], serum glutamate oxaloacetic transaminase [SGOT]) and serum isocitrate dehydrogenase (SICD) activities were monitored after APAP and APAP + AP or AS dosing. There were significant reductions in serum transaminase and SICD activities in the APAP- + ascorbate ester-treated animals as compared to APAP-positive controls. Oral coadministration of APAP with AP or AS did not prevent the initial hepatic GSH depletion (15 min-4 hr postdosing). However, hepatic GSH content began to rise in the APAP + AS or AP-treated animals at 4 hr and reached control values within 12 hr postdosing. Urinary mercapturate conjugates were also significantly higher in the APAP + AP or AS-treated animals as compared to APAP alone when measured over a 60-min postdosing period. Plasma sulfobromophthalein (BSP) retention was approximately eight times higher in APAP-treated animals as compared to the APAP + ascorbate ester treatments indicating maintenance of hepatic excretory functions in presence of AP or AS. Prior depletion of hepatic GSH by diethyl maleate (DEM) did not alter hepatoprotective effects of AP or AS in the presence of APAP. Hepatic ascorbate levels also peaked at 4 hours after APAP + AP or AS treatments. The possible role of L-ascorbic acid esters in GSH regeneration following co-administration of a hepatotoxic dose and APAP is discussed.

Early loss of large genomic DNA in vivo with accumulation of Ca 2+ in the nucleus during acetaminophen-induced liver injury

Hepatotoxic doses of acetaminophen cause early impairment of Ca 2+ homeostasis in the liver. This in vivo study considers the nucleus as a possible site of lethal Ca 2+ action by evaluating whether acetaminophen raises Ca 2+ in this compartment, whether DNA becomes altered, and whether DNA changes occur early enough during injury to contribute causally to necrosis. Fed Swiss mice were treated with 600 mg/kg acetaminophen ip and livers and blood samples were collected over time. Total nuclear Ca 2+ accumulation and fragmentation damage to DNA showed modest parallel increases between 2 and 6 hr, followed by > 200% rises at 12 hr mirroring the appearance of frank liver injury (ALT > 10,000 U/liter). However, agarose gel electrophoresis revealed extensive loss of large genomic DNA from 2 hr onward, accompanied by the appearance of periodic DNA fragments. Thus, acetaminophen raised nuclear Ca 2+ concentrations and promoted DNA fragmentation in vivo. The considerable cleavage of DNA seen at late times probably resulted from cell death, whereas loss of large genomic DNA from 2 hr onward appeared at an early enough point in time to be a contributing factor in acetaminophen-induced liver necrosis.

Alkylation of the liver plasma membrane and inhibition of the Ca 2+ ATPase by acetaminophen

Acetaminophen is activated metabolically to yield reactive species that bind covalently to liver cell macromolecules. The extent of covalent binding correlates with the occurrence and severity of hepatic necrosis. We reported previously [J. O. Tsokos-Kuhn, E. L. Todd, J. B. McMillin-Wood and J. R. Mitchell, Molec. Pharmac. 28, 56 (1985)] that active Ca 2+ accumulation of isolated liver plasma membranes is decreased 60–75% after a hepatotoxic dose of acetaminophen in vivo. We now report that the protein of isolated liver plasma membranes was substantially labeled with drug metabolites after administration of [ 3H]acetaminophen. There was no increase in passive membrane permeability that might cause diminished Ca 2+ accumulation. Intravesicular volume and relative purity of the vesicle preparations after acetaminophen were not different from controls. However, (Ca 2+,Mg 2+)-ATPase, a possible biochemical expression of the Ca 2+ pump, was decreased 31% (P < 0.025) after acetaminophen treatment. ATPase activity in both control and treated groups was enhanced by isolating membranes in the presence of 5 mM reduced glutathione (GSH), but the effects of drug treatment were not reversed. A similar effect of GSH on Ca 2+ accumulation was observed previously [J.O. Tsokos-Kuhn, E. L. Todd, J. B. McMillin-Wood and J. R. Mitchell, Molec. Pharmac. 28, 56 (1985)]. These data are consistent with a hypothesis wherein alkylation of membrane proteins by reactive acetaminophen metabolites is a factor in the onset of hepatic necrosis after acetaminophen. They are not consistent with an oxidative stress hypothesis where thiol S-thiolation of membrane components is postulated to produce altered membrane permeability or thiol-reversible alterations in membrane protein structure and enzymatic function.

Acetaminophen hepatotoxicity [formula omitted] is not accompanied by oxidant stress

Hepatotoxic doses of acetaminophen in Fischer 344 rats did not increase biliary efflux of oxidized glutathione. Pretreatment of the animals with bis(2-chloroethyl)-N-nitrosourea inhibited hepatic glutathione reductase by 73 percent but did not potentiate the hepatotoxicity of acetaminophen and did not produce an increase in biliary efflux of oxidized glutathione in response to acetaminophen. Hepatic protein thiol content was not depleted by acetaminophen. A proposed role for oxidant stress mechanisms mediated either by reactive oxygen species or by the direct oxidant action of a reactive metabolite in acetaminophen-induced hepatotoxicity is unsubstantiated and unlikely.