Different effects of selective β1‐adrenoceptor antagonists,nebivolol or atenolol in acetaminophen‐induced hepatotoxicity of rats

Acetaminophen (APAP) overdose is a common cause of acute liver failure, and beta‐blockers are commonly used drugs in clinical practice. This study aimed to evaluate the effect of two different beta‐blocker agents as nebivolol and atenolol against APAP‐induced hepatotoxicity. Male Wistar rats were treated with APAP (2 g/kg/day, p.o.) to induce hepatotoxicity. Our results showed that nebivolol and atenolol reduced heart rate and blood pressure. Nebivolol (5 mg/kg/day, p.o.) for 14 days has a hepatoprotective effect shown by significant decrease in hepatic injury parameters (serum AST and ALT) with significant suppression of hepatic malondialdehyde (MDA) and nitric oxide (NO) which were elevated with APAP administration. Also, nebivolol increased reduced glutathione (GSH) which was reduced with APAP administration. Moreover, immunohistochemical examination revealed that nebivolol treatment markedly reduced inducible nitric oxide synthase (iNOS) expression, while expression of endothelial nitric oxide synthase (eNOS) was markedly enhanced, as compared to APAP group. The protective effects of nebivolol were also verified histopathologically. On the other hand, as compared to APAP group, oral administration of atenolol (50 mg/kg) increased hepatic injury parameters but did not change hepatic NO, MDA, and GSH. In conclusion, this study revealed that nebivolol not atenolol is protective against APAP‐induced hepatotoxicity possibly, in part, through its antioxidant activity, inhibition of iNOS expression, and induction of eNOS expression.     

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-acetylcysteine (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 (/=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 the study, 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.     

Influence of feeding schedule on 24-h rhythm of hepatotoxicity induced by acetaminophen in mice

The influence of feeding schedule on the chronopharmacological aspects of acetaminophen (APAP) was examined in mice housed under 12-h light/dark cycle (lights on from 7:00 AM to 7:00 PM) with food and water ad libitum feeding (ALF) or under repeated time-restricted feeding (feeding time between 9:00 AM and 5:00 PM) for 2 weeks before the experiment. For the ALF group, there was a significant 24-h rhythm of mortality after APAP (600 mg/kg i.p.) injection. Peak mortality was observed after APAP injection at 9:00 PM and 1:00 AM, and nadir mortality was observed after drug injection at 9:00 AM. Hepatotoxicity after APAP (300 mg/kg i.p.) injection at 9:00 PM was significantly more severe than that after drug injection at 9:00 AM. Immunohistochemical staining using anti-APAP antibody 2 h after APAP injection was detected in centrilobular hepatocytes after drug injection at 9:00 PM but not after drug injection at 9:00 AM. CYP2E1 activity and hepatic glutathione (GSH) levels in untreated mice showed significant 24-h rhythms associated with APAP toxicity rhythm. The reduction in hepatic GSH levels after APAP injection at 9:00 PM was greater than that after drug injection at 9:00 AM. On the other hand, manipulation of the feeding schedule modified APAP hepatotoxicity rhythmicity, CYP2E1 activity, and GSH levels in the liver. Manipulation of the feeding schedule and choosing the most appropriate time of the day for drug administration may help to achieve rational chronopharmacology of some drugs including APAP in specific experimental and clinical situations.     

Comparison of the effects of sodium sulfate and N-acetylcysteine on the hepatotoxicity of acetaminophen in mice

N-acetylcysteine (NAC) has been proposed to decrease the toxicity of acetaminophen (AA) via two mechanisms: by increasing cysteine availability for hepatic glutathione biosynthesis and by increasing inorganic sulfate levels, which would increase AA sulfation and elimination. Because administration of sodium sulfate also reportedly decreases AA-induced toxicity, we have investigated the role of inorganic sulfate in the antidotal properties of NAC. Simultaneous administration of NAC (4 mmol/kg) and AA (2.5 and 4 mmol/kg) to male mice prevented AA-induced lethality and hepatotoxicity whereas sodium sulfate (4 mmol/kg) did not. Neither NAC nor sodium sulfate produced significant changes in the half-life (44 min) or clearance (9.0 ml/min/kg) of AA (4.0 mmol/kg) from blood nor were the amounts of AA-sulfate, AA-cysteine or AA-mercapturate excreted in urine altered. Injection of either sodium sulfate or NAC increased serum sulfate concentration and prevented the depletion in serum sulfate produced by AA. Hepatic adenosine 3'-phosphate 5'-phosphosulfate concentrations were decreased 15 and 30 min after AA and injection of either sodium sulfate or NAC lessened this effect. The concentration of glutathione in liver was decreased markedly after AA. NAC attenuated this effect but sodium sulfate did not. Sodium sulfate did not decrease covalent binding of tritium derived from [3H]AA to liver protein whereas NAC decreased binding by 25%. These findings show that administration of sodium sulfate increases serum sulfate concentration and hepatic adenosine 3'-phosphate 5'-phosphosulfate levels but does not protect against acetaminophen-induced hepatotoxicity in mice.(ABSTRACT TRUNCATED AT 250 WORDS)     

N‐acetylcysteine prevents glutathione decrease and does not interfere with paracetamol antinociceptive effect at therapeutic dosage: a randomized double‐blind controlled trial in healthy subjects

Paracetamol (APAP) may lead to hepatic changes even at therapeutic dosages. Glutathione (GSH) plays a pivotal role in APAP metabolism as it allows the detoxification of a toxic metabolite. N‐Acetylcysteine (NAC) is APAP antidote, is also largely used as a mucoactive drug and is often associated with APAP. This study aims at evaluating if 1‐ NAC modifies APAP pain efficacy and 2‐ NAC prevents glutathione depletion with APAP at therapeutic doses. This double‐blind randomized controlled study (NCT02206178) was carried out in 24 healthy volunteers. APAP was given for 4 days (1 g ×4 daily) with NAC or with placebo. Thermal pain tests, whole blood GSH, and hepatic enzymes (ASAT, ALAT) were measured before (D0) and after (D4) oral APAP‐NAC or APAP‐placebo intake. anova for repeated measures adapted to cross‐overdesign was performed and a two‐tailed type I error was fixed at 5%. The primary endpoint was the area under the curve (0–240 min) of pain intensity (Numerical Scale) after thermal pain stimulation using Pathway‐Medoc^®. APAP antinociceptive effect was similar in both groups. GSH was maintained to its baseline value in the APAP/NAC group but diminished in the APAP/placebo group (P = 0.033). This study shows for the first time that APAP antinociceptive effectiveness is not influenced by NAC. It also shows that the effect of APAP at therapeutic dosage on GSH may be counteracted by NAC. These issues are particularly important for patients as APAP is often prescribed for years as a first‐line pain treatment and further trials in patients are now warranted.     

Potentiation of the hepatotoxic effect of acetaminophen by prior administration of salicylate

The effect of sodium salicylate (SS) pretreatment on acetaminophen (APAP) metabolism and hepatotoxicity in mice was studied. Mice were given a single oral dose of SS (100 mg/kg) 1 hr before graded doses of APAP (150-500 mg/kg). Liver histology, serum hepatic enzymes (serum glutamic-oxaloacetic transaminase, serum glutamic-pyruvic transaminase and isocitric dehydrogenase) and APAP metabolites in urine were examined 24 hr after APAP treatment. Free plasma APAP and liver glutathione were determined over 24 hr after treatment with 400 mg/kg of APAP alone or after SS pretreatment. At 500 mg of APAP per kg, mortality rate was 38% in SS + APAP group; no mortality was seen among animals treated with APAP alone. Centrilobular hepatic hemorrhagic necrosis and/or vacuolation were observed in both treatments. Mitosis of hepatocytes was increased in all APAP-treated mice. Incidence of hepatic necrosis and mean lesion grades at 300- and 500-mg/kg doses increased in mice pretreated with SS. Mice that received SS + APAP had significantly higher levels of serum glutamic-oxaloacetic transaminase, serum glutamic-pyruvic transaminase and isocitric dehydrogenase at all doses compared to mice treated with APAP alone. APAP glucuronide and sulfate conjugates decreased and APAP mercapturate conjugate increased in urine of mice receiving SS + APAP treatment. Free plasma APAP was significantly higher 2 hr after APAP treatment in SS + APAP-treated mice as compared to mice that received APAP alone. Hepatic glutathione levels were similarly decreased over 24 hr in both groups. These data demonstrate that SS pretreatment alters APAP biotransformation profile and potentiates the hepatotoxic effect of APAP in mice.     

Hepatotoxicity Despite Early Administration of Intravenous N-Acetylcysteine for Acute Acetaminophen Overdose

Objectives:  The objective was to evaluate the effectiveness of intravenous N -acetylcysteine (IV NAC; 300 mg/kg over 21 hours) in early acute acetaminophen (APAP) overdose patients.
Methods:  This observational case series included patients hospitalized between 2004 and 2007 for acute APAP overdoses and who were reported to a regional poison center. Inclusion criteria were plasma APAP concentrations on or above the treatment line on the Rumack-Matthew nomogram, administration of IV NAC within 8 hours of ingestion, and follow-up to known outcome. The hospital chart of each patient who received IV NAC for longer than the standard 21 hours was reviewed. Hepatotoxicity was defined as hepatic aminotransferase levels greater than 1,000 IU/L.
Results:  Seventy-seven patients met inclusion criteria and received at least 21 hours of IV NAC for an acute APAP overdose. Seven patients received antidotal therapy for greater than 21 hours. These patients tended to have ingested combination preparations, have very high initial plasma APAP concentrations, and had persistently elevated plasma concentrations during their hospital stay. Hepatotoxicity occurred in 4 patients (5.2%, 95% confidence interval [CI] = 0.2% to 10.1%), including 1 death and 1 liver transplantation.
Conclusions:  Hepatotoxicity developed in 5.2% of cases, suggesting that the 21-hour IV NAC regimen is suboptimal in some patients. In addition to high initial plasma APAP concentrations, APAP product formulation and persistently elevated plasma APAP concentrations were identified as factors possibly associated with developing hepatotoxicity. The authors propose a tailored approach to the discontinuation of IV NAC and point out the need for reevaluation of optimal doses and duration of therapy.     

Refining the level for anticipated hepatotoxicity in acetaminophen poisoning

Treatment of an acetaminophen overdose with N-acetyl cysteine usually is based on the position of the 4-h acetaminophen (APAP) level on the Rumack-Matthew nomogram; however, there is disagreement on the level at which clinically relevant hepatotoxicity occurs. A retrospective review of all acute adult formulation APAP exposures reported to our poison center between 1986 and 1993 was performed and cases corresponding to the “possible risk or toxicity” range on the nomogram were identified. Our current poison center protocol for APAP poisoning does not recommend treatment with N-acetylcysteine (NAC) in low-risk patients if the 4-h serum APAP level or the extrapolated equivalent falls within the possible toxicity range on the nomogram. Seventeen cases met the inclusion criteria for the study and received no NAC; six additional patients met inclusion criteria but received one or two doses of NAC before therapy was discontinued. No patients in either group demonstrated clinical evidence of hepatotoxicity. This pilot study suggests that patients with no risk factors and APAP levels in the “possible risk” range may not require NAC therapy.     

Effects of reduced glutathione and n-acetylcysteine on lidocaine metabolism in cimetidine treated rats

Summary— The aim of this work was to evaluate the effects of exogenous glutathione (GSH) and N-acetylcysteine (NAC) on the formation of monoethylglycinexylidide (MEGX) from lidocaine in rats with and without the administration of cimetidine. GSH and NAC were administered intraperitoneally (ip) (1 mmol/kg) 1 hour before treatment with cimetidine (0.5 mmol/kg) or saline, and 1 hr later all rats were injected ip with lidocaine (1 mg/kg). Blood samples were drawn 30 min after the lidocaine injection. MEGX and lidocaine serum concentrations were determined by means of fluorescence polarization immuno-assay using the TDX system. Cimetidine produced a decrease in MEGX levels (from 210 ± 18 to 164 ± 13 ng/mL) and a parallel increase in lidocaine levels (from 73 ± 22 to 172 ± 47 ng/mL), consistent with cytochrome P-450 3A inhibition. Both GSH and NAC produced a significant decrease in MEGX levels (151 ± 16 and 139 ± 14 ng/mL, respectively), but no significant increase in lidocaine levels were found. As compared to the cimetidine group, pre-treatment using either GSH or NAC with Cimetidine produced a marked decrease in lidocaine levels (37 ± 27 and 63 ± 28 ng/mL, respectively) and no modification of MEGX levels (155 ± 12 and 165 ± 22 ng/mL, respectively). These results suggest that GSH and NAC might accelerate the lidocaine metabolism while counteracting the inhibitory effect of Cimetidine.     

Acetaminophen-induced hepatotoxicity in mice occurs with inhibition of activity and nitration of mitochondrial manganese superoxide dismutase

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

A novel mechanism for the enhancement of acetaminophen hepatotoxicity by phenobarbital

Pretreatment of mice with multiple doses of phenobarbital (PB) potentiates N-acetyl-para-aminophenol (APAP) hepatotoxicity through induction of cytochrome P-450, thus increasing the formation of APAP-reactive metabolites. The objective of this report is to investigate the effect of a single oral dose of PB on APAP hepatotoxicity in mice. PB was administered (150 mg/kg) 1 hr before oral administration of APAP (400 mg/kg). Blood, liver and urine were collected from mice at 2, 4, 8, 12 and 24 hr after APAP treatment. Mortality rate and incidence of gross hepatic lesions were significantly higher in mice pretreated with PB than in mice treated with APAP alone. At 8, 12 and 24 hr post-APAP treatment, serum glutamic oxalacetic transaminase activity was significantly higher in mice receiving the combination treatment. Hepatic glutathione levels were significantly lower at 1 and 2 hr in mice pretreated with PB. Urinary excretion of APAP mercapturate, APAP sulfate and free APAP increased, whereas APAP glucuronide decreased, in mice pretreated with PB compared with mice treated with APAP alone. Covalent binding of [3H]APAP to hepatic microsomes was markedly increased after PB pretreatment. PB pretreatment was found to deplete uridine diphosphate glucuronic acid in livers of mice at 1 and 2 hr post-APAP treatment. These results indicate that the biochemical mechanism by which a single dose of PB enhances APAP hepatotoxicity does not involve cytochrome P-450 induction; interference with APAP glucuronidation may occur.     

Effect of melatonin on cadmium-induced hepatotoxicity in male Sprague-Dawley rats.

Effect of melatonin on toxicity of cadmium (Cd) was studied in male SD rats co-administered daily Cd (1 mg/kg b.w., s.c.) with melatonin (10 mg/kg b.w., i.p.) for 15 days. Cd alone injection decreased GSH concentrations in the liver and RBC by 35% and 43% compared with those in saline-treatment group, but not in the kidney and whole brain. The activity of GSSG-reductase was significantly decreased in the liver of Cd alone injected rats, while melatonin given in combination with Cd failed to prevent the Cd-induced decreased activity of hepatic GSSG-reductase. However, the hepatic GSH concentration decreased by Cd alone was restored by melatonin treatment, and the melatonin also ameliorated Cd-induced histopathological changes in the liver. Therefore, data indicate that melatonin restores the reduction of hepatic GSH level induced with Cd regardless of GSSG-reductase activity, and suggests that melatonin may ameliorate Cd-induced hepatotoxicity.     

四氯化碳和醋氨酚急性肝损伤犬模型的比较

摘 要 目的：研究比较四氯化碳（CCl4）和醋氨酚（APAP）所致急性肝损伤犬模型的异同，为药物性肝炎的相关研究提供动物模型选择的实验依据。方法：24只健康杂种犬随机分为CCl4组和APAP 组（n=12）。CCl4组一次性腹腔注射50.0%（W/W）CCl4花生油溶液，0.9ml/kg体重。APAP组三次多点皮下注射600ｇ/Ｌ APAP溶液，首次剂量为750mg/kg体重，于首次注射后9ｈ和24ｈ分别再次注射，剂量为200mg/kg体重。分别观察各犬的全身情况、记录生存时间，测定血液生化指标，包括丙氨酸氨基转移酶（ALT）、丙氨酸氨基转移酶与天门冬氨酸氨基转移酶比值（AST/ALT）、血清总胆红素（TBIL）、凝血酶原时间（PT）、血氨（AMMO）、尿素氮（BUN），犬死亡时或存活15d时取肝脏组织行病理检查。结果：二组生存率比较差异无显著性意义（P>0.05）。二组血液生化指标3天内达高峰；CCl4对肝功能的影响比APAP明显（P<0.05），而APAP肾毒性较CCl4大（P<0.05）；肝炎表现CCl4组较重。肝脏病理改变CCl4较显著。肝外表现CCl4注射后迅速出现消化道和神经症状改变，其精神、饮食变化较APAP模型犬显著。结论：CCl4对犬的生存状态影响和肝脏的损害程度均高于APAP，而APAP的肾毒性较CCl4显著。     

The protective effect of sesamol against mitochondrial oxidative stress and hepatic injury in acetaminophen-overdosed rats

An acetaminophen (APAP) overdose induces oxidative stress and acute hepatic injury or even death. We investigated the prophylactic effect of sesamol (SM) on mitochondrial oxidative stress, hydroxyl-radical-generated lipid peroxidation, and hepatic injury in APAP-overdosed rats. Six male Wistar rats (APAP group) were given only oral APAP (1,000 mg/kg) to induce mitochondrial oxidative-stress-associated hepatic injury, and another six (ASM group) were given the same dose of oral APAP, and then, immediately afterward, were injected with SM (10 mg/kg, i.p.), to assess its prophylactic effects. In the APAP group, APAP had significantly increased the levels of 1) serum aspartate transaminase and alanine transaminase, 2) centrilobular necrosis, 3) ferrous ions, 4) hydrogen peroxide, 5) hydroxyl radicals, and 6) lipid peroxidation, and decreased 7) mitochondrial aconitase activity in the rats' liver tissue 24 h later. In the ASM group, SM had prevented significant rises in the levels of 1) to 6) and a significant decrease (7). Therefore, we hypothesize that the protective effect of SM in APAP-overdosed rats is associated with maintaining the mitochondrial aconitase activity, ferrous ions (Fe2+), and hydrogen peroxide levels and inhibiting hydroxyl-radical-associated lipid peroxidation and hepatic injury.     

Acetaminophen psi parameter: A useful tool to quantify hepatotoxicity risk in acute acetaminophen overdose

Context. The risk of hepatotoxicity secondary to acute acetaminophen overdose is related to serum acetaminophen concentration and lag time from ingestion to N-acetylcysteine (NAC) therapy. Psi (Greek letter ψ) is a toxicokinetic parameter that takes the acetaminophen level at 4 h post-ingestion ([APAP]_{4 h}) and the time-to-initiation of NAC (tNAC) into account and was found to be significantly predictive of hepatotoxicity in Canadian patients with acetaminophen overdose treated with intravenous NAC. Objective. We report the relationship of psi and hepatotoxicity in a Thai population with acute acetaminophen overdose. Methods. This is a retrospective study of patients with acute paracetamol overdose during January 2004 to June 2009 at Siriraj Hospital. Patients were treated with the standard 21-h intravenous NAC regimen. Univariate analyses were performed with logistic regression to assess the relationships of psi, [APAP]_{4 h}, and tNAC, and hepatotoxicity. Results. A total of 127 patients were enrolled. The median (interquartile range; IQR) of [APAP]_{4 h} was 267.8 (196.0–380.0) mg/L. The median (IQR) of tNAC was 8.5 (6.2–12.0) h. Thirteen patients (10.2%) developed hepatotoxicity. Univariate analysis revealed [APAP]_{4 h}, tNAC, and psi as statistically significant predictors of hepatotoxicity. Discussion and conclusion. The psi parameter is a reliable prognostic tool to predict hepatotoxicity secondary to acute acetaminophen overdose treated with intravenous NAC. Our evidence shows that psi may be a more superior tool than either acetaminophen level or time-to-initiation of NAC at predicting hepatotoxicity.     

Acetaminophen-induced hepatotoxicity and protein nitration in neuronal nitric-oxide synthase knockout mice

In overdose acetaminophen (APAP) is hepatotoxic. Toxicity occurs by metabolism to N-acetyl-p-benzoquinone imine, which depletes GSH and covalently binds to proteins followed by protein nitration. Nitration can occur via the strong oxidant and nitrating agent peroxynitrite, formed from superoxide and nitric oxide (NO). In hepatocyte suspensions we reported that an inhibitor of neuronal nitric-oxide synthase (nNOS; NOS1), which has been reported to be in mitochondria, inhibited toxicity and protein nitration. We recently showed that manganese superoxide dismutase (MnSOD; SOD2) was nitrated and inactivated in APAP-treated mice. To understand the role of nNOS in APAP toxicity and MnSOD nitration, nNOS knockout (KO) and wild-type (WT) mice were administered APAP (300 mg/kg). In WT mice serum alanine aminotransferase (ALT) significantly increased at 6 and 8 h, and serum aspartate aminotransferase (AST) significantly increased at 4, 6 and 8 h; however, in KO mice neither ALT nor AST significantly increased until 8 h. There were no significant differences in hepatic GSH depletion, APAP protein binding, hydroxynonenal covalent binding, or histopathological assessment of toxicity. The activity of hepatic MnSOD was significantly lower at 1 to 2 h in WT mice and subsequently increased at 8 h. MnSOD activity was not altered at 0 to 6 h in KO mice but was significantly decreased at 8 h. There were significant increases in MnSOD nitration at 1 to 8 h in WT mice and 6 to 8 h in KO mice. Significantly more nitration occurred at 1 to 6 h in WT than in KO mice. MnSOD was the only observed nitrated protein after APAP treatment. These data indicate a role for nNOS with inactivation of MnSOD and ALT release during APAP toxicity.     

Predicting acute acetaminophen hepatotoxicity with acetaminophen-aminotransferase multiplication product and the Psi parameter

Context. Prediction of potential hepatotoxicity is important for individualizing therapy with N-acetylcysteine (NAC) in patients with acute acetaminophen overdose. Acetaminophen-aminotransferase multiplication product (APAP × AT) and the Psi Parameter (Psi) have been reported to be the predictors of acetaminophen hepatotoxicity. Objective. To determine the validity of APAP × AT and Psi in predicting hepatotoxicity secondary to acute acetaminophen overdose. Materials and methods. We retrospectively reviewed acute acetaminophen overdose cases who were treated with NAC at Siriraj Hospital, Thailand during January 2004–June 2012. The patients’ ages were 12 years or more. Initial acetaminophen concentration (mg/L) and aminotransferase (IU/L) were multiplied to obtain APAP × AT. Psi were derived from initial acetaminophen concentrations (mg/L) and lag time (hours) to NAC therapy. The cut-off values for APAP × AT and Psi were 1500 mg?IU/L² and 5 mM?h, respectively. Hepatotoxicity (defined as aspartate or alanine aminotransferase (ALT) greater than 1000 IU/L) was the outcome of interest. Results. A total of 255 patients were included, 32 of whom developed hepatotoxicity. APAP × AT had sensitivity, specificity, and negative likelihood ratio of 90.6%, 62.8%, and 0.2, respectively. The sensitivity of Psi, specificity, and negative likelihood ratio were 96.9%, 91.5%, and 0.0, respectively. The areas under the curve of the receiver operating characteristic (ROC) curve for APAP × AT and Psi were 0.82 and 0.96, respectively, with a statistically significant difference between the two methods (p = 0.002). APAP × AT showed higher specificity (92.5%) in patients who presented 8–24 h after the overdose. Discussion and conclusion. Psi and APAP × AT are valid clinical tools in predicting hepatotoxicity secondary to acute acetaminophen overdose in adults. APAP × AT is useful in predicting a low likelihood of hepatotoxicity after standard NAC therapy among late-presenting patients.     

Inhibition by ranitidine of acetaminophen conjugation and its possible role in ranitidine potentiation of acetaminophen-induced hepatotoxicity

Pretreatment with ranitidine (RA) potentiates the hepatotoxicity of acetaminophen (APAP) in male Fischer 344 rats. The present studies were undertaken to investigate the role of APAP metabolism in this potentiation. Administration of RA (50 mg/kg p.o.) to male Fischer 344 rats 30 min before [3H]APAP (750 mg/kg p.o.) increased the plasma concentrations of acetaminophen at 2 hr (193%) and 4 hr (277%) after APAP. Covalent binding of [3H]APAP-related material to hepatic macromolecules in RA-pretreated animals was similar to APAP alone values up to 12 hr after treatment; however, 24 hr after APAP, binding in the RA-pretreated animals was twice that observed in animals given [3H]APAP alone. Urinary excretion (0-24 hr) of APAP and APAP glucuronide were reduced in ranitidine-pretreated animals to 64 and 66% of control, respectively, indicating that in vivo RA altered APAP conjugation with glucuronic acid. APAP uridine diphosphoglucuronyltransferase activity in rat hepatic microsomes was competitively inhibited by RA (0.1-2 mM). The Ki apparent for RA inhibition of APAP uridine diphosphoglucuronyltransferase was 0.04 mM. In contrast, neither APAP nor 4-nitrophenol sulfotransferase activity in rat hepatic cytosol was inhibited by RA at concentrations up to 5 mM. Together, these results support the suggestion that RA-mediated alterations of APAP conjugation may explain the potentiation of APAP-induced hepatotoxicity by RA in rats.