APAP Intoxication Research Articles

Attenuation of Acetaminophen-Induced Hepatotoxicity In Vivo and In Vitro by a 43-kD Protein Isolated from the Herb Cajanus indicus L

ABSTRACTThe aim of this study was to evaluate the hepatoprotective role of a 43-kD protein (Hp-P) isolated from the leaves of Cajanus indicus L. against acetaminophen (APAP)-induced toxicity in mouse liver and in isolated hepatocytes. The hepatotoxicity of APAP and the hepatoprotective activity of Hp-P in vivo were determined by measuring the liver-specific serum marker enzymes alanine amino transferase (ALT) and alkaline phosphatase (ALP) in murine sera and observing the histological changes in the mice liver treated with the protein before and after (2 mg/kg body weight for 5 days) APAP (at a dose of 300 mg/kg body weight for 2 days) administration. The cell viability, LDH leakage, GSH level, and lipid peroxidation were measured in isolated hepatocytes to evaluate the cytotoxic effect of APAP and the protective role of Hp-P in vitro. Experimental results showed that APAP induced hepatotoxicity in vivo as revealed from the changes in serum-specific marker enzyme levels and histology of liver. It also induced cytotoxicity in hepatocytes as observed from the changes in cell viability and LDH leakage. Pretreatment with Hp-P prevented the APAP-induced elevation of ALT and ALP in murine sera. In addition, posttreatment with Hp-P significantly altered most of the changes induced by APAP. Although some natural recovery has been observed in toxin controls, the Hp-P-induced recovering process is more rapid than the natural ones. In histological studies, less centrilobular necrosis was found in the liver treated with Hp-P before and after APAP intoxication compared to the liver treated with APAP alone. Radical scavenging experiment showed that Hp-P scavenges DPPH radicals directly. Studies also showed that APAP-induced reduced cell viability and cellular LDH leakage could be prevented by the combinatorial effect of Hp-P. Besides, treatment of hepatocytes with Hp-P and APAP together maintained the normal GSH level. APAP-induced enhanced lipid peroxidation was also decreased when cells were treated with APAP and Hp-P together. Hp-P alone, on the other hand, did not induce any alterations of the studied parameters. Results of this study have been compared with a known antioxidant, α-tocopherol. Data from both the in vivo (before and after APAP administration) and in vitro studies suggest that Hp-P has potent hepato- and cytoprotective properties against APAP-induced toxicity.

Mild Hypothermia Attenuates Liver Injury and Improves Survival in Mice With Acetaminophen Toxicity

Body temperature may critically affect mechanisms of liver injury in acetaminophen (APAP) hepatotoxicity. In addition, mild hypothermia is used to treat intracranial hypertension in human liver failure without detailed information on its effects on the injured liver itself. Therefore, we investigated the effects of body temperature on the progression of APAP-induced liver injury in mice. Male C57BL6 mice treated with saline or APAP (300 mg/kg intraperitoneally) were maintained at normothermia (35.5-37.5 degrees C) by external warming or were allowed to develop mild hypothermia (32.0-35.0 degrees C) after 2 hours from APAP administration. Mild hypothermia resulted in improved survival after APAP intoxication. Liver damage was reduced, as assessed histologically and by plasma alanine aminotransferase levels. Early effects of hypothermia included a reduction of hepatic congestion and improved recovery of glycogen stores. At later time points (8-12 hours), APAP-treated mice that were maintained at normothermia manifested increased hepatocyte apoptosis, as assessed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling staining and cleavage of poly(adenosine diphosphate-ribose) polymerase. Mild hypothermia did not affect the formation of APAP-protein adducts or the depletion of glutathione, nor did it abrogate hepatocyte DNA synthesis. Mild hypothermia improved survival and attenuated liver injury and apoptosis in APAP-treated mice by reducing hepatic congestion and improving glycogen recovery without affecting hepatic regeneration. Results of the study underscore the need for a strict control of body temperature in animal models of liver failure and suggest that the benefits of mild hypothermia in liver failure may extend beyond those related to reduced cerebral complications.

Acetaminophen 유도 간독성에 대한 백삼과 홍삼 추출물의 간보호 효과

Acetaminophen(APAP) is one of the most extensively used analgesics and antipyreics worldwide. In order to investigate preventive effects of white and red ginseng extracts, male ICR mice pretreated with white or red ginseng extracts(50 or 250 ㎎/㎏/day, for 5 days, orally) before treatment with acetaminophen(800 ㎎/㎏, i.p, single dose). In an attempt to elucidate the possible mechanism of hepatoprotective effect, superoxide dismutase(SOD), catalase(CAT), hydroperoxide, malondialdehyde(MDA) contents were studied. In pretreatment with red ginseng extract(250 ㎎/㎏), the activities of SOD, CAT were generally highest and the hydrogen peroxide content was lowest. The levels of MDA were significantly lower in white and red ginseng extract groups than those in the APAP groups. By treatment with ginseng extract, high content of hydrogen peroxide and increased lipid peroxidation level caused by APAP could be lowered. Also, ginseng extracts were found to increase antioxidative enzyme activity. Finally, the results suggest that the antioxidant effects of (white and red) ginseng extracts prevent oxidative damage by direct antioxidant effects involving SOD, CAT and increasing the ability to synthesize endogenous antioxidants. It was concluded that ginseng can protect against APAP intoxication through its antioxidant properties.

A patient-tailored N-acetylcysteine protocol for acute acetaminophen intoxication

Background: Hepatotoxicity as a result of acetaminophen(APAP) intoxication has become an important problem, but early intervention with N-acetylcysteme (NAC) is effective in preventing hepatic injury. Two NAC regimens are currently approved for acute APAP intoxication: NAC administered orally every 4 hours for 72 hours, and NAC administered intravenously for 20 hours within 8 to 10 hours after ingestion of a potentially hepatotoxic amount of APAP. However, clinical observations suggest that a variable treatment duration may be more appropriate than use of these predetermined, fixed-duration protocols. Objectives: This study investigated the tolerability and efficacy of a patient-tailored NAC protocol for acute APAP intoxication by comparing the incidence of hepatotoxicity in patients receiving this protocol and in historical controls receiving 1 of 2 fixed-duration protocols: oral NAC for 72 hours and intravenous NAC for 20 hours within 8 to 10 hours after ingestion of a potentially hepatotoxic amount of APAP. Methods: This was a retrospective case series study that included all patients admitted through the emergency department (ED) of the National Taiwan University Hospital with a diagnosis of APAP intoxication between October 1997 and October 2002. According to the patient-tailored protocol, which had been used in the ED since 1997, patients with a serum APAP concentration above the limit for possible risk based on a modified Rumack-Matthew nomogram received oral treatment with NAC 140 mg/kg, followed by maintenance doses of 70 mg/kg every 4 hours. NAC treatment was discontinued when the APAP concentration was <10 mg/L and serum aspartate aminotransferase (AST) was <40 IU/L. For the purposes of assessing clinical outcomes, patients were divided into 3 groups based on duration of treatment: the short-course group (≤36 hours), the intermediate-course group (37–72 hours), and the long-course group (≥73 hours). The primary outcome measure was development of hepatotoxicity, defined as a serum AST or alanine aminotransferase concentration >1000 IU/L. Results: Twenty-seven patients were included in thestudy, 17 in the short-course group, 4 in the intermediate-course group, and 6 in the long-course group. The mean (SD) durations of NAC treatment in the respective groups were 22.1 (5.5) hours, 45.0 (8.2) hours, and 97.3 (33.2) hours. All 6 patients (22%) in the long-course group had hepatotoxicity (peak AST range, 1083-9770 IU/L); their treatment duration ranged from 80 to 164 hours. No patients in the short- or intermediate-course group had evidence of hepatotoxicity. One woman in the long-course group in whom initiation of NAC treatment was delayed by 28 hours died of fulminant hepatic failure. The overall incidence of hepatotoxicity was similar to that in the historical controls. Conclusions: In this retrospective case series inpatients who received patient-tailored NAC therapy for acute APAP intoxication, the incidence of hepatotoxicity was low and comparable to that in historical controls who received treatment with 1 of 2 fixed-duration regimens. Use of this protocol may have the potential to shorten hospital stays without increasing the risk to patients. However, the sample size was small, and the findings require confirmation in prospective clinical trials.

Inhibition of Matrix Metalloproteinases Minimizes Hepatic Microvascular Injury in Response to Acetaminophen in Mice

The acetaminophen (APAP)-induced hepatic centrilobular necrosis is preceded by hepatic microcirculatory dysfunction including the infiltration of erythrocytes into the space of Disse. The purpose of this study was to examine the involvement of matrix metalloproteinases (MMPs) in the hepatic microvascular injury elicied by APAP. Male C57Bl/6 mice were pretreated with 2-[(4-biphenylsulfonyl) amino]-3-phenyl-propionic acid, an MMP-2/MMP-9 inhibitor (5 mg/kg, ip) 30 min before oral gavage with 600 mg/kg of APAP. The hepatic microvasculature in anesthetized mice was observed using established in vivo microscopic methods 2 and 6 h after APAP. The levels of mRNAs and activities of MMP-2 and MMP-9 in the liver were increased from 1 h through 6 h after APAP gavage. APAP increased alanine transferase (ALT) levels (41.1-fold) and resulted in centrilobular hemorrhagic necrosis at 6 h. Pretreatment with 2-[(4-biphenylsulfonyl) amino]-3-phenyl-propionic acid attenuated ALT values by 71% as well as the necrosis. APAP decreased the numbers of perfused sinusoids in centrilobular regions by 30% and increased the area occupied by infiltrated erythrocytes into Disse space. 2-[(4-Biphenylsulfonyl) amino]-3-phenyl-propionic acid restored the sinusoidal perfusion to 90% of control levels and minimized extrasinusoidal area occupied by erythrocytes. The present study showed that increased MMPs during APAP intoxication are associated with hepatocellular damage and with hepatic microcirculatory dysfunction including impaired sinusoidal perfusion and infiltration of erythrocytes in Disse space. 2-[(4-Biphenylsulfonyl) amino]-3-phenyl-propionic acid attenuated APAP-induced parenchymal and microvascular injury. These results suggest that MMPs participate in APAP hepatotoxicity mediated by sinusoidal endothelial cell injury, which results in impairment of microcirculation.

Ethanol Binging Enhances Hepatic Microvascular Responses to Acetaminophen in Mice

Chronic alcoholism has been considered to be a risk for acetaminophen (APAP) hepatotoxicity, but little is known about the effect of binge alcohol drinking on APAP-induced liver injury. The present study was conducted to examine the effect of ethanol binging on APAP-induced hepatic microcirculatory dysfunction. Male C57Bl/6 mice received 3 weekly ethanol binges (4 g/kg every 12 h x 5 doses/ week) or water binges. At 12 h after the last gavage, APAP (300 mg/kg) was given by oral gavage. In one group of mice, gadolinium chloride (GdCl3, 10 mg/kg) was intraperitoneally administered 2 and 1 days before the start of each weekly ethanol binge. Ethanol binging enhanced APAP-induced liver injury as indicated by ALT levels. Intravital microscopic study showed that APAP further increased the area occupied by infiltrated erythrocytes into the extrasinusoidal space as well as Kupffer cell phagocytic activity in ethanol-binged mice when compared with water-binged mice, while no significant differences in sinusoidal perfusion and leukocyte adhesion were observed. ALT levels after APAP were exacerbated in ethanol-binged mice treated with GdCl3, but APAP-induced hepatic microcirculatory dysfunction was not changed significantly. These results suggest that ethanol binging increases APAP-induced liver injury by exacerbating infiltration of the Disse space with blood cells. Kupffer cells exert a protective role in the liver against APAP intoxication following ethanol binging.

Role of nitric oxide in hepatic microvascular injury elicited by acetaminophen in mice.

Nitric oxide (NO) is suggested to play a role in liver injury elicited by acetaminophen (APAP). Hepatic microcirculatory dysfunction also is reported to contribute to the development of the injury. As a result, the role of NO in hepatic microcirculatory alterations in response to APAP was examined in mice by in vivo microscopy. A selective inducible NO synthase (iNOS) inhibitor,l-N6-(1-iminoethyl)-lysine (L-NIL), or a nonselective NOS inhibitor, NG-nitro-l-arginine methyl ester (L-NAME), was intraperitoneally administered to animals 10 min before APAP gavage. L-NIL suppressed raised alanine aminotransferase (ALT) values 6 h after APAP, whereas L-NAME increased those 1.7-fold. Increased ALT levels were associated with hepatic expression of iNOS. L-NIL, but not L-NAME, reduced the expression. APAP caused a reduction (20%) in the numbers of perfused sinusoids. L-NIL restored the sinusoidal perfusion, but L-NAME was ineffective. APAP increased the area occupied by infiltrated erythrocytes into the extrasinusoidal space. L-NIL tended to minimize this infiltration, whereas L-NAME further enhanced it. APAP caused an increase (1.5-fold) in Kupffer cell phagocytic activity. This activity in response to APAP was blunted by L-NIL, whereas L-NAME further elevated it. L-NIL suppressed APAP-induced decreases in hepatic glutathione levels. These results suggest that NO derived from iNOS contributes to APAP-induced parenchymal cell injury and hepatic microcirculatory disturbances. L-NIL exerts preventive effects on the liver injury partly by inhibiting APAP bioactivation. In contrast, NO derived from constitutive isoforms of NOS exerts a protective role in liver microcirculation against APAP intoxication and thereby minimizes liver injury.

Acetaminophen and salicylate serum levels in patients with suicidal ingestion or altered mental status

Is universal screening of acetaminophen (APAP) and salicylate (SAL) necessary in patients with a suicidal ingestion or an altered mental status and suspected ingestion? This descriptive, retrospective chart review in an emergency department in a large urban county hospital examined all patients who presented with a history of suicidal ingestion or an altered mental status with a strong suspicion of ingestion from January 1992 through October 1993. APAP and SAL serum levels were measured in 1,820 patients, and charts of patients with APAP serum levels of >1 μg/mL or SAL serum levels of >1 mg/dL were reviewed. The patient's history of ingesting APAP or SAL was recorded, as well as the clinician's interpretation of that level. Sixteen charts were not available. APAP levels of >1 μg/mL were found in 175 (9.6%) patients, 120 (6.5%) of whom were APAP history-positive and 55 (3%) APAP history-negative. None of the APAP history-negative group required therapy with N-acetylcysteine. Eight (0.3%) of the APAP history-negative group had potentially toxic levels of >50 μg/mL. SAL levels of >1 mg/dL were found in 155 (8.5%) patients, 44 (2.5%) of whom were SAL history-positive and 111 (6%) SAL history-negative. Three patients were SAL history-negative but had a significant chronic SAL intoxication. All these patients presented with an altered mental status and had an anion gap of >20 mEq/L. Universal screening found that 0.3% of suicidal ingestions had a potentially toxic APAP intoxication not suggested by history. This incidence of infrequent but potentially life:threatening overdose should prompt clinicians to screen all of their patients with a suspected ingestion. Salicylate screening found that 0.16% of suicidal ingestions had a toxic SAL intoxication not suggested by history, although such intoxication should be suggested by an elevated anion gap and an altered mental status. Since this less severe intoxication is less frequent and usually suggested by commonly obtained laboratory data, universal screening is not indicated, but a more selective approach to screening could be taken.

Histopathology of Acetaminophen-Induced Liver Changes: Role of Interleukin 1α and Tumor Necrosis Factor α

Administration of 500 mg/kg acetaminophen (APAP) to female B6C3F1 mice resulted in well-documented pathophysiological changes in the liver manifested as increased serum concentration of liver enzymes (aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, and serum sorbitol dehydrogenase), centrilobular congestion, and hepatocellular degeneration and necrosis. The role of proinflammatory cytokines, including tumor necrosis factor alpha (TNF-alpha) and interleukin 1 alpha (IL-1 alpha), on the hepatotoxicity of APAP was examined at 4, 8, 12, and 24 hr following APAP administration. Neutralization of TNF-alpha or IL-1 alpha with specific antibodies partially prevented the hepatotoxic effects of APAP at the 4- and 8-hr time points. In addition, prior administration of anti-TNF-alpha antibodies shortened the recovery time following APAP treatment. While IL-1 receptor antagonist (IL-1ra) had only a modest protective effect against APAP-induced liver damage, as determined by serum enzyme release, IL-1ra had no effect on the degree of hepatic congestion or necrosis at any of the time points examined. On the other hand, administration of antibodies against IL-1ra exacerbated APAP-induced liver toxicity. These results suggest that TNF-alpha and IL-1 alpha play an important role in the degree of damage and recovery that the liver undergoes following APAP intoxication.

Role of Proinflammatory Cytokines in Acetaminophen Hepatotoxicity

Acetaminophen (APAP) intoxication has been shown to activate Kupffer cells. Kupffer cell activation is also associated with the release of proinflammatory cytokines which can induce a variety of pathophysiological responses. These studies examined whether proinflammatory cytokines are produced in response to a hepatotoxic dose of APAP, and if so, the role they play in the observed pathological response. Female B6C3F1 mice received 500 mg APAP/kg in the presence and absence of antibodies against tumor necrosis factor-alpha (TNF-alpha), interleukin-1-alpha (IL-1 alpha), and IL-1 receptor antagonist (IL-1ra). Serum TNF-alpha, IL-1 alpha, and liver-associated enzyme levels were measured. In addition, the levels of mRNA transcripts for IL-1 alpha, IL-6, and TNF-alpha from livers of treated mice were examined by reverse transcription-polymerase chain reaction (RT-PCR). Administration of APAP resulted in an immediate reduction in body temperature as well as elevated serum levels of IL-1 alpha and TNF-alpha that reached a peak at 12 and 16 hr, respectively. The reduction in body temperature was partially blocked by injection of antibodies against TNF-alpha or IL-1 alpha. Furthermore, neutralization of TNF-alpha delayed the increase in serum IL-1 alpha and liver enzyme levels. In contrast, pretreatment with IL-1ra antisera exacerbated the effect of APAP on body temperature and increased the release of liver enzymes. These data suggest that TNF-alpha and IL-1 alpha are released in response to APAP intoxication and are responsible for certain pathological manifestations of APAP-induced hepatotoxicity.

Effect of colchicine on acetaminophen-induced liver damage.

The effect of colchicine on liver damage induced by acetaminophen (APAP) intoxication was studied. Wistar male rats pretreated (72 h) with 3-methylcholanthrene (3-MC) (20 mg/kg i.p.) were divided into six groups: animals in group 1 were treated with acetaminophen (APAP) (500 mg/kg p.o.); group 2 consisted of animals that received colchicine (65 micrograms/kg/day p.o.) for 7 days before APAP intoxication; group 3 was treated like group 2, but the dose of colchicine was 300 micrograms/kg/day; animals in groups 4 and 5 received the same doses of colchicine as groups 2 and 3, respectively, but received the vehicle instead of APAP; and rats in group 6 (control) received the equivalent amount of the vehicles. Animals were sacrificed at different times after APAP administration. Reduced glutathione (GSH), lipid peroxidation and glycogen were measured in liver and, gamma-glutamyl transpeptidase (gamma-GTP), and glutamic pyruvic transaminase (GPT) activities were measured in serum. After APAP intoxication, GSH and glycogen decreased very fast (1 h) and remained low for 6 h. Lipid peroxidation increased three times over control 4 h after APAP treatment. Enzyme activities increased at 18 h after intoxication. Pretreatment with 65 micrograms/kg of colchicine failed to prevent liver damage induced by APAP. However, when a dose of 300 micrograms/kg of colchicine was given, levels of lipid peroxidation and serum gamma-GTP activity remained within the control values, while GPT activity and glycogen content were only partially attenuated. It was concluded that colchicine protects against APAP intoxication, probably through its antioxidant properties, possibly acting as a free radical scavenger.

Silymarin protects against paracetamol-induced lipid peroxidation and liver damage.

The effect of silymarin on liver damage induced by acetaminophen (APAP) intoxication was studied. Wistar male rats pretreated (72 h) with 3-methylcholanthrene (3-MC) (20 mg kg-1 body wt. i.p.) were divided into three groups: animals in group 1 were treated with acetaminophen (APAP) (500 mg kg-1 body wt. p.o.), group 2 consisted of animals that received APAP plus silymarin (200 mg kg-1 body wt. p.o.) 24 h before APAP, and rats in group 3 (control) received the equivalent amount of the vehicles. Animals were sacrificed at different times after APAP administration. Reduced glutathione (GSH), lipid peroxidation and glycogen were measured in liver and alkaline phosphatase (AP), gamma-glutamyl transpeptidase (GGTP) and glutamic pyruvic transaminase (GPT) activities were measured in serum. After APAP intoxication, GSH and glycogen decreased very fast (1 h) and remained low for 6 h. Lipid peroxidation increased three times over the control 4 and 6 h after APAP treatment. Enzyme activities increased 18 h after intoxication. In the group receiving APAP plus silymarin, levels of lipid peroxidation and serum enzyme activities remained within the control values at any time studied. The fall in GSH was not prevented by silymarin, but glycogen was restored at 18 h. It was concluded that silymarin can protect against APAP intoxication through its antioxidant properties, possibly acting as a free-radical scavenger.

The Mouse Liver Slice System as an In Vitro Model for Testing the Protective Effect of N-Acetylcysteine Against Hepatotoxic Agents

AbstractSummary: An in vitro liver slice system was used to test the antidotal effect of N-acetylcysteine (NAC) against hepatotoxic agents. Incubation of mouse slices with acetaminophen (APAP) in a concentration of 6.8 mg/ml resulted in lactate dehydrogenase (LDH) leakage from the cells. NAC protected the tissue from APAP-induced hepatocellular damage in a dose-dependent manner. Maximal protection was achieved by 10 mM of the sulfhydryl compound when applied 20 min following APAP intoxication. Longer intervals, concurrent administration, or preincubation of the slices with the SH reagent were less protective against the hepatotoxin. The liver slice system has been used to test NAC antidotal activity against other hepatotoxins known to be metabolized to reactive radicals, such as carbon tetrachloride (CC14), bromobenzene, and furosemide. The hepatocellular damage caused by these agents was slightly reduced by the sulfhydryl agent. These findings may demonstrate the ability of the in vitro liver slice syste...

Acetaminophen hepatotoxicity: Is there a role for prostaglandin synthesis?

The hepatotoxicity of acetaminophen (APAP) overdose depends on metabolic activation to a toxic reactive metabolite via hepatic mixed function oxidase. In vitro studies have indicated that APAP may also be cooxidized by prostaglandin H synthetase. The present experiments were designed to assess the possible contribution of hepatic prostaglandin synthesis to APAP toxicity. Adult fed male mice were overdosed with 400 mg APAP/kg. Liver toxicity was estimated by measurement of serum transaminases. Hypertonic xylitol or sodium chloride (2250 mOsm/l), administered intragastrically to stimulate prostaglandin synthesis, increased APAP toxicity. By contrast, the cyclooxygenase inhibiting drugs aspirin (at 25 mg/kg) and indomethacin (at 10 mg/kg) protected against APAP-induced toxicity. APAP kinetics were not affected by hypertonic xylitol or indomethacin, nor were hepatic glutathione levels in overdosed mice. Imidazole, a nonspecific thromboxane synthetase inhibitor, also protected overdosed mice. This drug prolonged hexobarbital sleeping time and prevented the depletion of hepatic glutathione that followed APAP intoxication. Thus, the data support the conclusion that APAP-induced hepatoxicity may be modulated not only by inhibition of cytochrome P450 mediated oxidation, but also by controlling hepatic cyclooxygenase activity.