Acetaminophen Treatment Research Articles

Acetaminophen and experimental acute myocardial infarction.

Acetaminophen is a widely used analgesic, and recent studies have shown that it has some benefits in the ischemic heart. The purpose of this study was to test the effects of acetaminophen on infarct size, regional myocardial blood flow and the "no-reflow" phenomenon in a rabbit model of coronary artery occlusion (CAO) and reperfusion. Rabbits were assigned to one of four groups: (1) acetaminophen, 75 mg/kg in 75% ethanol, 30 min before CAO, (2) vehicle at 30 min before CAO, (3) acetaminophen at 18 min after CAO (12 min before reperfusion), or (4) vehicle at 18 min of CAO ( n = 10 each group). All rabbits received 30 min of CAO followed by 3 hr reperfusion. The extent of ischemia was similar in all groups comprising 24-27% of the left ventricle. Infarct size (% ischemic zone) was 51 +/- 5, 56 +/- 3 in groups 1 and 2 ( p = ns), 43 +/- 3 and 40 +/- 4 in groups 3 and 4 ( p = ns). Thus acetaminophen did not affect the development of necrosis. Heart rate and blood pressure were unchanged by acetaminophen treatment. Regional myocardial blood flow was similar in all groups during occlusion and at the end of reperfusion. Acetaminophen had no effect on the size of the anatomic zone of no-reflow that developed by 3 hours of reperfusion. We conclude that acetaminophen has a neutral effect in this experimental model of ischemia/reperfusion and appears to be safe in the course of experimental myocardial infarction.

Modulation of acetaminophen-induced hepatotoxicity by the xenobiotic receptor CAR

Zhang J, Huang W, Chua SS, et al (Baylor College of Medicine, Houston, TX) Overdoses of acetaminophen are the leading cause of hospital admission for acute liver failure in the United States. Ingestion of only 2 to 3 times the maximum daily recommended dose of acetaminophen can cause hepatotoxicity, and higher doses result in potentially fatal centrilobular necrosis. Acetaminophen toxicity is increased in both human beings and rodents by pretreatment with various inducers of CYP gene expression, including ethanol and phenobarbital. Because the xenobiotic receptor CAR has recently been shown to mediate the effects of phenobarbital and other phenobarbital-like inducers, the effects of phenobarbital and the potent CAR agonist 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) on acetaminophen toxicity were examined in wild-type and CAR knockout mice. Both strains of mice were treated with inducers or the vehicle control, followed by acetaminophen (250 mg/kg of body weight). The known CAR activators and the high dose of acetaminophen induced expression of 3 acetaminophen-metabolizing enzymes in wild-type mice but not in the CAR null mice. The CAR null mice were resistant to acetaminophen toxicity. Inhibition of CAR activity by administration by the inverse agonist ligand androstanol 1 hour after acetaminophen treatment blocked hepatotoxicity in wild-type but not in CAR null mice. These findings demonstrate that CAR is a central mediator of acetaminophen toxicity in mice and potentially also in human beings. It would appear that CAR is not involved in the toxicity associated with ethanol-dependent induction of CYP2E1 and other targets because CAR activation caused a modest decrease in CYP2E1 mRNA levels. However, activation of either mouse or human CAR by appropriate inducers, including acetaminophen itself, increased the production of APAP-metabolizing enzymes and increased toxicity. Fatal outcomes have been reported to result from the combination of phenobarbital and acetaminophen in human beings. The findings of this study suggest a new strategy for treatment of acetaminophen toxicity based on the use of CAR agonists.

Peroxynitrite formation and sinusoidal endothelial cell injury during acetaminophen-induced hepatotoxicity in mice

IntroductionVascular injury and accumulation of red blood cells in the space of Disse (hemorrhage) is a characteristic feature of acetaminophen hepatotoxicity. However, the mechanism of nonparenchymal cell injury is unclear. Therefore, the objective was to investigate if either Kupffer cells or intracellular events in endothelial cells are responsible for the cell damage.ResultsAcetaminophen treatment (300 mg/kg) caused vascular nitrotyrosine staining within 1 h. Vascular injury (hemorrhage) occurred between 2 and 4 h. This paralleled the time course of parenchymal cell injury as shown by the increase in plasma alanine aminotransferase activities. Inactivation of Kupffer cells by gadolinium chloride (10 mg/kg) had no significant effect on vascular nitrotyrosine staining, hemorrhage or parenchymal cell injury. In contrast, treatment with allopurinol (100 mg/kg), which prevented mitochondrial injury in hepatocytes, strongly attenuated vascular nitrotyrosine staining and injury.ConclusionsOur data do not support the hypothesis that acetaminophen-induced superoxide release leading to vascular peroxynitrite formation and endothelial cell injury is caused by activated Kupffer cells. In contrast, the protective effect of allopurinol treatment suggests that, similar to the mechanism in parenchymal cells, mitochondrial oxidant stress and peroxynitrite formation in sinusoidal endothelial cells may be critical for vascular injury after acetaminophen overdose.

Achieving Normothermia in Patients With Febrile Subarachnoid Hemorrhage: Feasibility and Safety of a Novel Intravascular Cooling Catheter

Fever is common and difficult to control in patients with subarachnoid hemorrhage (SAH). We have previously shown an inverse relationship between fever and outcome in patients with SAH. This was a prospective, single-arm, feasibility trial in which nine patients with SAH underwent temperature management using an intravascular cooling catheter (ICC) to restore and maintain 24 hours of normothermia (36.5 degrees+/-0.2 degrees C). Enrollment occurred after development of a fever of at least 38.3 degrees C within 7 days of SAH that was refractory to acetaminophen treatment. The ICC was placed at the bedside through an introducer sheath via the femoral vein into the inferior vena cava (IVC). Portable X-ray confirmed placement. Normothermia was achieved in seven of the nine patients treated (78%); it was achieved in 100% of the patients with a 14F catheter (n=4) and in 60% of the patients with a 9F catheter (n=5). The two patients not reaching normothermia were not adequately treated for shivering. All other patients reached normothermia irrespective of intubation status. Overall, normothermia was well tolerated and not discontinued because of discomfort or adverse events. Two incidences of deep vein thrombosis were diagnosis by ultrasound that were not associated with clinical sequelae, and IVC filters were placed. No unanticipated adverse events occurred. We have demonstrated that fever can be safely and effectively controlled in patients with SAH for at least 24 hours using an ICC. Future studies are needed to assess the effect of such sustained therapy on outcome in patients with SAH.

Mechanism of Acetaminophen‐Induced Apoptosis in Cultured Cells: Roles of Caspase‐3, DNA Fragmentation Factor, and the Ca2+ and Mg2+ Endonuclease DNAS1L3

We have recently shown that acetaminophen induces many of the apoptotic traits in hepatoma cells and lymphocytes (Boulares et al. (2002d). In an effort to further investigate the mechanism by which non-metabolized acetaminophen induces apoptosis, we have now examined the roles of caspase-3, the DNA fragmentation factor, and the poly(ADP-ribose) polymerase-1-regulated Ca2+ and Mg2+-dependent endonuclease DNAS1L3 in the induction of such death process. This was achieved with the use of MCF-7 cells, a caspase-3-deficient breast adenocarcinoma cell line, thymocytes isolated from DFF45 (the inhibitory and chaperone subunit of the DNA fragmentation factor subunit, DFF40) deficient mice, and HeLa cells, a DNAS1L3-deficient cervical carcinoma cell line. MCF-7 exhibited a marked resistance to acetaminophen treatment. Ectopic expression of human caspase-3 significantly potentiated the cytotoxic effect of acetaminophen and promoted the release of cytochrome c into the cytosol of treated cells suggesting a direct role for caspase-3 in acetaminophen-induced apoptosis. Expression and cleavage of DFF45 were required but not sufficient for acetaminophen-induced internucleosomal DNA fragmentation. DFF45 gene knockout rendered thymocytes resistant against acetaminophen-induced generation of both large and internucleosomal DNA fragments. The treatment of HeLa cells with acetaminophen resulted in internuclesomal DNA fragmentation only after transfection of these cells with a plasmid encoding the DNAS1L3 gene suggesting that this endonuclease is required for acetaminophen-induced internucleosomal DNA fragmentation. DNAS1L3 expression potentiated the cytotoxic effect of acetaminophen in HeLa cells suggesting an active role in the death process induced by this drug. Altogether, these results demonstrate the specific roles of caspase-3, DNA fragmentation factor, and DNAS1L3 in the process of acetaminophen-induced apoptosis in cultured cells.

Modulation of acetaminophen-induced hepatotoxicity by the xenobiotic receptor CAR.

We have identified the xenobiotic receptor CAR (constitutive androstane receptor) as a key regulator of acetaminophen metabolism and hepatotoxicity. Known CAR activators as well as high doses of acetaminophen induced expression of three acetaminophen-metabolizing enzymes in wild-type but not in CAR null mice, and the CAR null mice were resistant to acetaminophen toxicity. Inhibition of CAR activity by administration of the inverse agonist ligand androstanol 1 hour after acetaminophen treatment blocked hepatotoxicity in wild type but not in CAR null mice. These results suggest an innovative therapeutic approach for treating the adverse effects of acetaminophen and potentially other hepatotoxic agents.

Reduced Hepatotoxicity of Acetaminophen in Mice Lacking Inducible Nitric Oxide Synthase: Potential Role of Tumor Necrosis Factor-α and Interleukin-10

Macrophage-derived inflammatory mediators have been implicated in tissue injury induced by a number of hepatotoxicants. In the present studies, we used transgenic mice with a targeted disruption of the gene for inducible nitric oxide synthase (NOS II) to analyze the role of nitric oxide in inflammatory mediator production in the liver and in tissue injury induced by acetaminophen. Treatment of wild-type mice with acetaminophen (300 mg/kg) resulted in centrilobular hepatic necrosis, which was evident within 3 h and reached a maximum at 18 h. This was correlated with NOS II expression and nitrotyrosine staining of the liver, which was most prominent after 6 h. Expression of mRNA for tumor necrosis factor-α (TNF-α), interleukin-10 (IL-10), matrix metalloproteinase-9, and connective tissue growth factor (CTGF) also increased in the liver following acetaminophen treatment of wild-type mice. NOS II knockout mice were found to be less sensitive to the hepatotoxic effects of acetaminophen than wild-type mice. This did not appear to be due to differences in acetaminophen-induced glutathione depletion or adduct formation. In NOS II knockout mice treated with acetaminophen, hepatic expression of TNF-α, as well as CTGF, was significantly increased compared to wild-type mice. In contrast, IL-10 expression was reduced. These data demonstrate that nitric oxide is important in hepatotoxicity induced by acetaminophen. Moreover, some of its effects may be mediated by altering production of pro- and antiinflammatory cytokines and proteins important in tissue repair.

Acetaminophen-Induced Inhibition of Fas Receptor-Mediated Liver Cell Apoptosis: Mitochondrial Dysfunction versus Glutathione Depletion

We reported previously that acetaminophen overdose interrupts the signaling pathway of Fas receptor-mediated apoptosis. The aim of our study was to investigate the mechanism of this effect. Male C3Heb/FeJ mice received a single dose of acetaminophen (300 mg/kg ip) and/or anti-Fas antibody Jo-2 (0.6 mg/kg iv). Some animals were treated with allopurinol (100 mg/kg po) 18 and 1 h before acetaminophen injection. After 90 min of Jo treatment, there was processing of procaspase-3 and a significant increase in liver caspase-3 activity, which is consistent with apoptotic cell death. Treatment with acetaminophen 2.5 h before Jo inhibited the increase in hepatic caspase-3 activity by preventing the processing of the proenzyme. When administered alone, acetaminophen did not induce caspase-3 activation but caused significant liver injury. Acetaminophen treatment alone caused mitochondrial cytochrome c release, depletion of the hepatic ATP content by 55%, and a 10-fold increase in mitochondrial glutathione disulfide levels. Pretreatment with allopurinol prevented the mitochondrial oxidant stress and liver injury due to acetaminophen toxicity but had no effect on Jo-mediated apoptosis. Allopurinol did not affect the initial glutathione depletion after acetaminophen. However, allopurinol restored the sensitivity of hepatocytes to Fas receptor signaling in acetaminophen-treated animals. Histochemical evaluation of DNA fragmentation with the TUNEL assay showed that acetaminophen eliminated Fas receptor-mediated apoptosis in all hepatocytes not just in the damaged cells of the centrilobular area. Our data suggest that acetaminophen-induced mitochondrial dysfunction and not the initial glutathione depletion is responsible for the interruption of Fas receptor-mediated apoptotic signaling in hepatocytes.

NON-STEROIDAL ANTI-INFLAMATORY DRUGS (NSAIDS) AND ACETAMINOPHEN IN THE TREATMENT OF AN ACUTE MUSCLE INJURY

Muscle injuries are common in athletes. Non-steroidal anti-inflammatory drugs (NSAIDs) are frequently used to treat them. It is not known whether the anti-inflammatory effects of these drugs are important or whether their effectiveness is a result of their central analgesic effect. PURPOSE: evaluate and compare the effects of non-steroidal anti-inflammatory medication (NSAID), an analgesic (acetaminophen) and placebo in an experimental, acute muscle contusion model. METHODS: A standardized, unilateral, non-penetrating injury was created to the tibialis anterior muscle of 96 adult, male mice. Four treatment groups were used: Group 1 placebo treatment, Group 2 rofecoxib, an NSAID, treatment following the injury, Group 3 rofecoxib treatment starting 24 hrs prior to the injury, Group 4 acetaminophen treatment following the injury. The muscle and the contralateral normal muscle were evaluated at 2, 5 and 7 days post-injury with grading of gait, wet weight and histology. RESULTS: Group 1 had significant more gait disturbances at day 2 than all other groups (P < 0.05). No differences were found at day 5 and 7. Wet weights showed an increase at day 2 in Group 1 (P < 0.01). Again no differences were at day 5 and 7. Histology revealed similar inflammatory changes at day 2 in all groups with regeneration of muscle fibers at day 5 and 7. CONCLUSION: The results of in this muscle injury study indicate that rofecoxib as an NSAID and acetaminophen as a non-NSAID analgesic have similar effects. The lack of differences in wet weights and histology suggest that the anti-inflammatory effects of rofecoxib are not an important feature of its action. Similarly, administration of the NSAID prior to the injury does not appear to enhance its effectiveness.

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.

Retinol potentiates acetaminophen-induced hepatotoxicity in the mouse: mechanistic studies.

This study was designed to elucidate the mechanism of retinol's potentiation of acetaminophen-induced hepatotoxicity. To accomplish this, the major bioactivation and detoxification pathways for acetaminophen were investigated following retinol (75 mg/kg/day, 4 days), acetaminophen (400 mg/kg), and retinol + acetaminophen treatment. Hepatic microsomes were used to determine the catalytic activity and polypeptide levels of cytochrome P450 enzymes involved in the murine metabolism of acetaminophen. Results showed that the catalytic activity and polypeptide levels of CYP1A2, CYP2E1, and CYP3A were unchanged in the treatment groups compared to vehicle and untreated controls. In combination, retinol + acetaminophen caused a significantly greater depletion of GSH compared to corn oil + acetaminophen (0.36 +/- 0.11 vs 0.89 +/- 0.19 micromol/g, respectively, p < 0.05). This greater GSH depletion correlated with a higher degree of hepatic injury in the retinol + acetaminophen-treated animals but is probably not the cause of the potentiated injury since the results showed that retinol treatment itself did not alter hepatic glutathione (3.34 +/- 0.43 vs 3.44 +/- 0.46 micromol/g for retinol vs vehicle, respectively). However, hepatic UDPGA stores were decreased in the retinol-treated group compared to untreated and corn oil controls (54.6 +/- 10.6 vs 200.6 +/- 17.6 nmol/g for retinol and untreated control, respectively, p < 0.001). This demonstrates that there is significantly less hepatic UDPGA available for conjugation following retinol administration. The results suggest that decreased hepatic UDPGA is likely the cause of retinol's potentiation of acetaminophen-induced hepatic injury.

Role of taurine in preventing acetaminophen-induced hepatic injury in the rat.

Acetaminophen overdose causes acute liver injury in both humans and animals. This study was designed to investigate the potential role of the conditionally essential amino acid taurine in preventing acetaminophen-induced hepatotoxicity. Male Sprague-Dawley rats were administered acetaminophen (800 mg/kg) intraperitoneally. Taurine (200 mg/kg) was given 12 h before, at the time of, and 1 or 2 h after acetaminophen injection. Acetaminophen treatment increased the plasma levels of aspartate transaminase, alanine aminotransferase, and alkaline phosphatase and caused hepatic DNA fragmentation and hepatocyte necrosis. Taurine administered before, simultaneously with, or 1 h after acetaminophen resulted in significant improvement in hepatic injury as represented by decrease of hepatocellular enzyme release and attenuation of hepatocyte apoptosis and necrosis, and this correlated with taurine-mediated attenuation of hepatic lipid peroxidation. These results indicate that taurine possesses prophylactic and therapeutic effects in acetaminophen-induced hepatic injury.

Acetaminophen elicits anti-estrogenic but not estrogenic responses in the immature mouse

Acetaminophen is a widely used analgesic, which has exhibited evidence of estrogenic activity in estrogen-receptor positive human breast cancer cells. However, there is limited in vivo experimentation regarding the estrogenicity of acetaminophen. Therefore, the present study examined the in vivo estrogenic potential of acetaminophen using the immature female mouse model. Specifically, C57Bl/6 female mice were treated with acetaminophen (100, 200 and 250 mg/kg per day, i.p.) for 3 consecutive days. Positive controls received estradiol (10 μg/kg per day, i.p.) for 3 consecutive days. Markers of estrogenic activity examined were uterine weight, uterine peroxidase activity and progesterone receptor (PR) up-regulation. The results demonstrated that, at all three doses, acetaminophen did not significantly increase uterine weight, uterine peroxidase activity or PR levels. However, the co-administration of E 2 and acetaminophen (200 mg/kg per day, 3 days, i.p.) resulted in a decrease in the E 2-induced upregulation of uterine and hepatic nuclear PR (uterine PR values of 39.7±6.6 and 23.7±3.4 fmol/mg for E 2+vehicle and E 2+acetaminophen, respectively, P<0.05). Additionally, the estradiol-induced increase in uterine peroxidase was decreased 75% by acetaminophen (200 mg/kg per day, 3 days, i.p.). Therefore, in the immature mouse model acetaminophen treatment did not elicit estrogenic activity. However, acetaminophen had a limited ability to antagonize the effects of E 2.

Acetaminophen hepatotoxicity precipitated by short-term treatment of rats with ethanol and isopentanol: Protection by triacetyloleandomycin

Ethanol and isopentanol are the predominant alcohols in alcoholic beverages. We have reported previously that pretreatment of rats with a liquid diet containing 6.3% ethanol plus 0.5% isopentanol for 7 days results in a synergistic increase in acetaminophen hepatotoxicity, compared with rats treated with either alcohol alone. Here, we investigated the role of CYP3A in acetaminophen hepatotoxicity associated with the combined alcohol treatment. Triacetyloleandomycin, a specific inhibitor of CYP3A, protected rats pretreated with ethanol along with isopentanol from acetaminophen hepatotoxicity. At both 0.25 and 0.5 g acetaminophen/kg, triacetyloleandomycin partially prevented elevations in serum levels of alanine aminotransferase. At 0.25 g acetaminophen/kg, triacetyloleandomycin completely protected 6 of 8 rats from histologically observed liver damage, and partially protected the remaining 2 rats. At 0.5 g acetaminophen/kg, triacetyloleandomycin decreased histologically observed liver damage in 7 of 15 rats. In rats pretreated with ethanol plus isopentanol, CYP3A, measured immunohistochemically, was decreased by acetaminophen treatment. This effect was prevented by triacetyloleandomycin. These results suggest that CYP3A has a major role in acetaminophen hepatotoxicity in animals administered the combined alcohol treatment. We also found that exposure to ethanol along with 0.1% isopentanol for only 3 days resulted in maximal increases in acetaminophen hepatotoxicity by the combined alcohol treatment, suggesting that short-term consumption of alcoholic beverages rich in isopentanol may be a risk for developing liver damage from acetaminophen.

Impairment of TNF-α expression and secretion in primary rat liver cell cultures by acetaminophen treatment

Tumor necrosis factor-α (TNF-α) is assumed to act as a mediator in toxic liver injury, aggravating the primary damage to the parenchymal liver cell, but also stimulating liver regeneration. Reports on the effect of acetaminophen in vivo on TNF-α transcript concentrations and serum TNF-α concentrations, under different experimental, or clinical conditions have yielded controversial results. We used primary rat hepatocyte and Kupffer cell cultures to test the direct action of subtoxic and toxic concentrations of acetaminophen on TNF-α expression and release. We observed a dose-dependent decrease of TNF-α mRNA in the hepatocytes, and of TNF-α release into the medium of hepatocyte cultures. The data also indicate an impairment of TNF-α release in Kupffer cell cultures after treatment with nontoxic, as well as with toxic, acetaminophen concentrations. The results suggest that inhibition of TNF-α expression and release in the liver is a consequence of acetaminophen exposure. It is at present unknown how this effect modulates the course of acetaminophen intoxication.

Acute Effects of Acetaminophen on Renal Function and Urinary Excretion of Some Proteins and Enzymes in Patients with Kidney Disease

The acute effects of acetaminophen, a commonly used as analgesic drug, upon the urinary excretion of some proteins and enzymes as markers of kidney damage, was investigated. Patients with chronic glomerulo-nephritis (GN) and Balkan endemic nephropathy (BEN), having kidney vulnerable to toxic drugs, were enrolled in the study. Timed urine specimens were collected: before drug administration, and in 3-hour periods for 24 hours after an oral dose of 2 g of acetaminophen.Urinary excretion of albumin before acetaminophen treatment was significantly higher in patients with GN and BEN than in healthy adults, however, β2-miicroglobulin excretion was increased in BEN patients only. Urinary excretion of creatinine markeldly increased immediately after acetaminophen ingestion. Urinary excretion of total protein and albumin was not changed after acetaminophen administration. However, acetaminophen treatment produced a significant increase in β2-miicroglobulin excretion in patient with BEN and GN, and in clinically healthy members of nephropathic families.Excretion of aminopeptidase N (APN) activity before acetaminophen treatment was significantly higher in patients with GN, however, NAGA excretion was higher in both GN and BEN patients than in healthy controls. After acetaminophen administration urinary excretion of APN, di-peptidylpeptidase IV (DPP IV), γ-glutamyltranspeptidase (GGT) and N-acetyl-β-D-glucosaminidase (NAGA) did not increase significantly in any group studied.This study has shown that urinary excretion of APN, DPP IV NAGA and GGT, as markers of kidney brush border and lysosomal damage, did not change after 2 g of acetaminophen taken orally. β2-miicroglobulin was a marker of acute acetaminophen nephrotoxicity in kidney disease patients and in clinically healthy adults from nephropathic families.

A Protective Role for Metallothionein in Acetaminophen-caused Liver Toxicity via Oxidative Stress(PROCEEDINGS OF 24TH SYMPOSIUM ON TOXICOLOGY AND ENVIRONMENTAL HEALTH)

To investigate the possible involvement of metallothionein (MT) in acetaminophen (AC)-induced liver damage, MT-I and MT-II gene knock-out transgenic mice (MT-null mice) and wild-type control mice were i.p. treated with AC at a single dose of 250mg/kg and compared. At 24h after AC treatment severe liver damage characterized by necrosis of hepatocytes and increased serum GPT activity was found in MT-null mice while a limited degree of change such as a slight increase in serum GPT activity without necrosis was observed in wild-type mouse liver. MT-null hepatocytes also showed elevated PCNA (proliferating cell nuclear antigen)-positive activity around the necrosis area. AC administration resulted in an increase in lipid peroxidation in MT-null mice, but not in the wild-type mice. The liver in the wild-type mice showed an increased MT amount after AC treatment. These results indicate that MT acts as an endogenous defensive factor against AC-induced hepatotoxicity via oxidative stress.

Induction of an ATPase inhibitor protein by propylthiouracil and protection against paracetamol (acetaminophen) hepatotoxicity in the rat.

1. The purpose of the present study was to test the following hypothesis: propylthiouracil (PTU) treatments of rats induces an increase in the concentration and activity of the mitochondrial ATPase (m-ATPase) inhibitor protein (IF1). The PTU-induced elevated baseline levels of this inhibitor protein inactivated m-ATPase, and prevented hepatotoxicity by a toxic dose of acetaminophen (AAP) (paracetamol), by maintaining hepatic adenosine 5'-triphosphate (ATP) levels. 2. Male Wistar rats were either gavaged with a toxic dose of AAP alone, or after pretreatment with PTU for periods of 3 and 12 days. 3. Twenty four hours after acetaminophen treatment alone, toxicity was manifested by: an approximately 10 fold increase in serum transaminase levels (serum glutamic oxaloacetic transaminase and serum glutamic pyruvic transaminase); depletion of hepatic reduced glutathione (GSH) and ATP levels; loss of inhibitor protein activity, and extensive pericentral necrosis of the hepatocytes. Propylthiouracil pretreatment for 12 days enhanced the concentration of the following metabolites in the liver: ATP (1.5 fold), ATPase inhibitor protein (IF1) (4.5 fold), and reduced glutathione (1.3 fold), while the activity of the inhibitor protein increased 2 fold. When the PTU treated rats were challenged with AAP, transaminases were not elevated, and only sporadic areas of necrosis were detected by histological examination of the liver tissue. In contrast to the 12 day treatment with PTU the 3 day treatment had no protection against AAP. No histological evidence of protection was manifested and the transaminases were not different from AAP treated controls. Most of the protective metabolites were depleted. 4. Our findings suggest that PTU-induced increased concentration of inhibitor protein and GSH, are contributing factors in the prevention of hepatotoxicity by maintaining hepatic m-ATP levels and reducing the harmful effect of the toxic metabolite of AAP.

Influence of acetaminophen treatment and hydrogen peroxide treatment on the release of a CINC-related protein and TNF-α from rat hepatocyte cultures

Western blot analysis of conditioned media from hepatocytes exposed to H2O2 revealed that a 28 kDa protein was released dose-dependently in response to 1–10 mM H2O2. The 28 kDa protein was present in freshly isolated hepatocytes and exhibited cross-reactivity towards an antibody against CINC/gro. The intracellular amount of the protein decreased in parallel to the H2O2-induced release into the medium. The CINC-related protein was absent in media harvested after 1 h of treatment. The delivery of CINC-related protein correlated with the extent of cell damage as judged from lactate dehydrogenase leakage. Likewise, exposure of hepatocytes to 10–50 mM acetaminophen resulted in a dose-dependent release of the CINC-related protein after 24 h of culture. In contrast, monomeric CINC (molecular weight approximately 6.5 kDa) but not the 28 kDa CINC-related protein was released by lipopolysaccharide (LPS)-stimulated Kupffer cells. The amount of monomeric CINC liberated by Kupffer cells was diminished upon acetaminophen-treatment. Also, the release of tumor necrosis factor-α by hepatocytes was reduced after exposure to high acetaminophen doses (40–50 mM). In contrast to this finding, TNF-α release from hepatocyte cultures was not affected after H2O2 treatment. These data suggest that damaged hepatocytes release proinflammatory cytokines which may aggravate liver injury through activation of neutrophils and monocytes. The results indicate that the appearance of the CINC-related protein is due to impairment of plasma membrane integrity as the consequence of massive cell damage. In addition, APAP inhibited the release of monomeric CINC from LPS-activated Kupffer cells and of TNF-α from hepatocytes even at concentrations that were not sufficient to affect cell viability.

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.