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Context: Haemodialysis is sometimes used for patients with massive acetaminophen overdose when signs of "mitochondrial paralysis" (lactic acidosis, altered mental status, hypothermia and hyperglycaemia) are present. The role of haemodialysis is debated, in part because the evidence base is weak and the endogenous clearance of acetaminophen is high. There is also concern because the antidote acetylcysteine is also dialyzable. We prospectively measured serum acetylcysteine concentrations during haemodialysis in three such cases., Case Details: Three adults each presented comatose and acidemic 10 to ~18 h after ingesting > 1000mg/kg of acetaminophen. Two were hypothermic and hyperglycaemic. Serum lactate concentrations ranged from 7 mM to 12.5 mM. All three were intubated, and initial acetaminophen concentrations were as high as 5980 μM (900 μg/mL). An intravenous loading dose of 150 mg/kg acetylcysteine was initiated between 10.8 and ~18 h post ingestion, and additional doses were empirically administered during haemodialysis to compensate for possible antidote removal. A single run of 3-4 h of haemodialysis removed 10-20 g of acetaminophen (48-80% of remaining body burden), reduced serum acetaminophen concentrations by 56-84% (total clearance 3.4-7.8 mL/kg/min), accelerated native acetaminophen clearance (mean elimination half-life 580 min pre-dialysis, 120 min during and 340 min post-dialysis) and corrected acidemia. Extraction ratios of acetylcysteine across the dialysis circuit ranged from 73% to 87% (dialysance 3.0 to 5.3 mL/kg/min). All three patients recovered fully, and none developed coagulopathy or other signs of liver failure., Discussion: When massive acetaminophen ingestion is accompanied by coma and lactic acidosis, emergency haemodialysis can result in rapid biochemical improvement. As expected, haemodialysis more than doubles the clearance of both acetaminophen and acetylcysteine. Because acetylcysteine dosing is largely empirical, we recommend doubling the dose during haemodialysis, with an additional half-load when dialysis exceeds 6 h.

The kappa-opioid receptor (KOR) is the primary target for the endogenous opioid peptide dynorphin (DYN), and KORs reside within brain circuitry underlying the complex integration of information related to different behavioral domains such as motivation, negative affect, and decision-making. Alterations in extended amygdala DYNs and KOR function following chronic alcohol exposure have been shown to mediate escalated alcohol self-administration during acute withdrawal. In addition to excessive alcohol consumption and increased negative affect, other symptoms of alcohol dependence include compromised impulse control. Given that DYN and KOR expressions are dysregulated within prefrontal brain circuitry associated with decision-making and impulse control in alcohol-dependent humans and rodents, and have been shown to modify multiple neurotransmitter systems associated with impulse-control disorders, we hypothesized that KOR activation could contribute to impulsive phenotypes. To test this hypothesis, separate cohorts of male Wistar rats were trained in one of the two animal models of impulsivity: delay-discounting (DD) or stop-signal reaction time (SSRT) tasks, and once stable responding was observed, received intracerebroventricular (ICV) infusions of the KOR agonist U50,488 (0-50 μg) according to a within-subject dosing regimen. The results demonstrated a dissociable effect of U50,488 on impulsive phenotypes related to intolerance to delay or response inhibition, with selective effects in the SSRT. Furthermore, the pro-impulsive effects of KOR activation were rescued by pretreatment with the KOR antagonist nor-binaltorphimine (nor-BNI). Therefore, KOR activation was shown to induce an impulsive phenotype that was nor-BNI-sensitive. Dysregulation of impulsive behavior by increased DYN/KOR activity could serve to increase vulnerability for the initiation, or perpetuate existing patterns of excessive alcohol abuse and can enhance the probability of relapse in dependent individuals. Furthermore, KOR-mediated impulsivity has implications for numerous neuropsychiatric disorders.

Nitric oxide modulates pain development. However, there is no evidence on the effect of nitroxyl (HNO/NO⁻) in nociception. Therefore, we addressed whether nitroxyl inhibits inflammatory hyperalgesia and its mechanism using the nitroxyl donor Angeli's salt (AS; Na₂N₂O₃). Mechanical hyperalgesia was evaluated using a modified Randall and Selitto method in rats, cytokine production by ELISA and nitroxyl was determined by confocal microscopy in DAF (a cell permeable reagent that is converted into a fluorescent molecule by nitrogen oxides)-treated dorsal root ganglia neurons in culture. Local pre-treatment with AS (17-450 μg/paw, 30 min) inhibited the carrageenin-induced mechanical hyperalgesia in a dose- and time-dependent manner with maximum inhibition of 97%. AS also inhibited carrageenin-induced cytokine production. AS inhibited the hyperalgesia induced by other inflammatory stimuli including lipopolysaccharide, tumor necrosis factor-α, interleukin-1β and prostaglandin E2. Furthermore, the analgesic effect of AS was prevented by treatment with ODQ (a soluble guanylate cyclase inhibitor), KT5823 (a protein kinase G [PKG] inhibitor) or glybenclamide (an ATP-sensitive K⁺ channel blocker), but not with naloxone (an opioid receptor antagonist). AS induced concentration-dependent increase in fluorescence intensity of DAF-treated neurons in a l-cysteine (nitroxyl scavenger) sensitive manner. l-cysteine did not affect the NO⁺ donor S-Nitroso-N-acetyl-DL- penicillamine (SNAP)-induced anti-hyperalgesia or fluorescence of DAF-treated neurons. This is the first study to demonstrate that nitroxyl inhibits inflammatory hyperalgesia by reducing cytokine production and activating the cGMP/PKG/ATP-sensitive K⁺ channel signaling pathway in vivo., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The compound (±)-trans-4-hydroxy-6-propyl-1-oxocyclohexan-2-one [(±)-δ-lactone] was isolated from the plant Vitex cymosa Bertero, and determined to be the active principle. The present study aimed to evaluate the antinociceptive effect of (±)-δ-lactone and to elucidate its mechanism of action. Mice were subjected to in-vivo models of acute pain (acetic acid-induced abdominal writhing, formalin and hot-plate tests) and the open-field test. (±)-δ-Lactone, administered orally (6-900 µmol/kg), exerted a dose-dependent antinociceptive effect in the acetic acid-induced abdominal writhing, formalin and hot-plate tests. (±)-δ-Lactone administered by the intrathecal (i.t.) and subplantar (s.p.) routes (10-600 nmol) exerted concentration-dependent antinociceptive effects in the formalin test, showing its spinal and peripheral activity, respectively. In the hot-plate test, (±)-δ-lactone was also active when administered i.t., confirming its spinal effect. The previous intraperitoneal (i.p.) application of naloxone, yohimbine, mecamylamine or glibenclamide did not alter the effect produced by the i.t. administration of (±)-δ-lactone, whereas the previous application of atropine and L-arginine significantly reduced its effects in the formalin and hot-plate tests. The previous i.p. application of L-NAME enhanced the antinociceptive effect of the i.t. administration of (±)-δ-lactone in the formalin and hot-plate tests. The previous i.p. application of L-NAME and L-arginine increased and decreased, respectively, the activity of (±)-δ-lactone administered by s.p. administration. These results indicate that (±)-δ-lactone has significant spinal and peripheral antinociceptive activity, and that its effects are at least partially mediated by a reduced nitric oxide production/release, most likely through mechanisms involving the cholinergic system.

Acetaminophen (APAP)-induced liver injury is the leading cause of acute liver failure in many countries. This study determined the extent of liver protein sulfhydryl depletion not only in whole liver homogenate but also in the zonal pattern of sulfhydryl depletion within the liver lobule. A single oral gavage dose of 150 or 300 mg/kg APAP in B6C3F1 mice produced increased serum alanine aminotransferase levels, liver necrosis, and glutathione depletion in a dose-dependent manner. Free protein sulfhydryls were measured in liver protein homogenates by labeling with maleimide linked to a near infrared fluorescent dye followed by SDS-polyacrylamide gel electrophoresis. Global protein sulfhydryl levels were decreased significantly (48.4%) starting at 1 hour after the APAP dose and maintained at this reduced level through 24 hours. To visualize the specific hepatocytes that had reduced protein sulfhydryl levels, frozen liver sections were labeled with maleimide linked to horseradish peroxidase. The centrilobular areas exhibited dramatic decreases in free protein sulfhydryls while the periportal regions were essentially spared. These protein sulfhydryl-depleted regions correlated with areas exhibiting histopathologic injury and APAP binding to protein. The majority of protein sulfhydryl depletion was due to reversible oxidation since the global- and lobule-specific effects were essentially reversed when the samples were reduced with tris(2-carboxyethy)phosphine before maleimide labeling. These temporal and zonal pattern changes in protein sulfhydryl oxidation shed new light on the importance that changes in protein redox status might play in the pathogenesis of APAP hepatotoxicity.

Background: Treatment of single-time-point ingestion acute paracetamol (acetaminophen) poisoning with N-acetylcysteine (NAC) is guided by plotting a timed plasma paracetamol concentration on established nomograms. Guidelines in the UK differ from those in the U.S. and Australasia by having two treatment lines on the nomogram. Patients deemed to be at 'normal' risk of hepatotoxicity are treated using the treatment line starting at 200 mg/L at 4 h post-ingestion; those at higher risk are treated using the 'high risk' treatment line starting at 100 mg/L at 4 h post-ingestion., Aim: To examine the effect on treatment numbers if UK guidelines were to adopt a single treatment line nomogram or lower, risk-stratified treatment lines., Methods: We undertook a retrospective analysis of a series of acute single-time-point paracetamol poisonings presenting to our inner city emergency department. Treatment numbers and effect on treatment costs were modelled for three alternative scenarios: a 150 line-a combined single treatment line starting at a 4 h concentration of 150 mg/L, a 100 line-a combined single treatment line starting at a 4 h concentration of 100 mg/L, and a 150/75 line-a double treatment line at the lower concentrations of 150 mg/L for normal risk and 75 mg/L for high risk patients., Results: A total of 1,214 cases were identified. Under current UK guidance, 133 (11.0%) high risk cases and 98 (8.1%) normal risk cases needed treatment (total 231, 19.0%). A 150 line would result in 87 (7.2%) high risk cases and 155 (12.8%) normal risk cases needing treatment (total 242, 19.9%). A 100 line would result in 133 (11.0%) high risk and 251 (20.7%) normal risk cases needing treatment (total 384, 31.6%). A 150/75 line would result in 153 (12.6%) high risk and 155 (12.8%) normal risk cases needing treatment (total 308, 25.4%)., Conclusions: Both a 100 line and a 150/75 line would result in a large increase in the number of patients being treated and an associated increase in the costs of treatment. A single 150 mg/L treatment line would simplify treatment algorithms and lead to a similar number of patients being treated with NAC overall. A potential concern however is whether any of the high risk cases that would no longer be treated might develop significant hepatotoxicity. After consideration of the evidence for dual treatment lines, we feel that these risks are small and that it is worth reconsidering a change of treatment recommendations to a single 150 line.

During a migraine attack capsaicin-sensitive trigeminal sensory nerves release calcitonin gene-related peptide (CGRP), producing cranial vasodilatation and central nociception; hence, trigeminal inhibition may prevent this vasodilatation and abort migraine headache. This study investigated the role of spinal α₂-adrenoceptors and their subtypes (i.e. α(2A), α(2B) and/or α(2C)-adrenoceptors) in the inhibition of the canine external carotid vasodilator responses to capsaicin. Anaesthetized vagosympathectomized dogs were prepared to measure arterial blood pressure, heart rate and external carotid conductance. The thyroid artery was cannulated for one-min intracarotid infusions of capsaicin, α-CGRP and acetylcholine. A cannula was inserted intrathecally for spinal (C₁-C₃) administration of 2-amino-6-ethyl-4,5,7,8-tetrahydro-6H-oxazolo-[5,4-d]-azepin-dihydrochloride (B-HT 933; a selective α₂-adrenoceptor agonist) and/or the α₂-adrenoceptor antagonists rauwolscine (α(2A/2B/2C)), 2-[(4,5-dihydro-1H-imidazol-2-yl)methyl]-2,3-dihydro-1-methyl-1H-isoindole maleate (BRL44408; α(2A)), imiloxan (α(2B)) or acridin-9-yl-[4-(4-methylpiperazin-1-yl)-phenyl]amine (JP-1302; α(2C)). Infusions of capsaicin, α-CGRP and acetylcholine dose-dependently increased the external carotid conductance. Intrathecal B-HT 933 (1000 and 3100 μg) inhibited the vasodilator responses to capsaicin, but not those to α-CGRP or acetylcholine. This inhibition, abolished by rauwolscine (310 μg), was: (i) unaffected by 3,100 μg imiloxan; (ii) partially blocked by 310 μg of BRL44408 or 100 μg of JP-1302; and (iii) abolished by 1,000 μg of BRL44408 or 310 μg of JP-1302. Thus, intrathecal B-HT 933 inhibited the external carotid vasodilator responses to capsaicin. This response, mediated by spinal α₂-adrenoceptors unrelated to the α(2B)-adrenoceptor subtype, resembles the pharmacological profile of α(2C)-adrenoceptors and, to a lesser extent, α(2A)-adrenoceptors., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Acetaminophen (APAP) is a widely used analgesic and antipyretic drug, but at high dose it leads to undesirable side effects, such as hepatotoxicity and nephrotoxicity. The present study demonstrates the comparative hepatoprotective and nephroprotective activity of hesperidin (HD), a naturally occurring bioflavonoid against APAP induced toxicity. APAP induces hepatotoxicity and nephrotoxicity as was evident by abnormal deviation in the levels of antioxidant enzymes. Moreover, APAP induced renal damage by inducing apoptotic death and inflammation in renal tubular cells, manifested by an increase in the expression of caspase-3, caspase-9, NFkB, iNOS, Kim-1 and decrease in Bcl-2 expression. These results were further supported by the histopathological examination of kidney. All these features of APAP toxicity were reversed by the co-administration of HD. Therefore, our study favors the view that HD may be a useful modulator in alleviating APAP induced oxidative stress and toxicity., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Rosa damascena (RD) is a widely cultivated ornamental plant. It acts as an astringent, aperients, carminative, and refrigerant and is used in respiratory disorders, tonsillitis, eye disorders, migraines, gynecological disorders, and menopausal symptoms. The aim of this study is to investigate the hepatoprotective activity of the aqueous extract of RD flowers at different oral dose levels (250, 500, and 1000 mg/kg body weight) on acetaminophen (2 g/kg oral N-acetyl-p-aminophenol [APAP])-induced toxicity in rats. APAP administration altered various biochemical parameters, including serum transaminases, serum alkaline phosphatase, lactate dehydrogenase, albumin, bilirubin, urea and creatinine, hepatic lipid peroxidation, and reduced glutathione levels. Adenosine triphosphatase and glucose-6-phosphatase activity in the liver was decreased significantly in animals treated with APAP. These values are retrieved significantly by treatment with RD extract at all 3 doses in dose-dependant manner. Apart from these, histopathological changes also reveal the protective nature of the RD extract against acetaminophen-induced necrotic damage of hepatic tissues. In conclusion, these data suggest that the aqueous extract of RD may prevent hepatic damage from APAP-induced toxicity in rats and is likely to be mediated through its antioxidant activities.

The present investigation was planned to evaluate the nephroprotective activity of jaggery against acetaminophen (APAP)-induced renal damage in rats. The protective activity of jaggery at different doses (250, 500, and 750 mg/kg, orally) was evaluated against oxidative damage induced by APAP administration (2 g/kg, once orally in acute exposure; 20 mg/kg, orally for 21 days in subchronic exposure) in rats. APAP administration significantly increased the levels of serum urea, creatinine, and renal lipid peroxidation (LPO), whereas substantial decreases were observed in levels of glutathione (GSH), adenosine triphosphatase (ATPase), superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), and glutathione peroxidase (GPx) enzymatic activities after APAP administration. Administration of jaggery significantly moved the studied parameters toward normal levels and also reversed the histopathologic alterations. Thus, jaggery can be used to reduce renal damage and may serve as an alternative medicine in the treatment of renal etiologies.

Background: Acetaminophen is an analgesic drug that is used safely in therapeutic doses. At high doses, it causes hepatotoxicity, resulting in hepatic necrosis. Some medications and methods are available for treatment of acetaminophen overdose. However, results are inconsistent, and sufficient outcomes cannot always be obtained., Objective: The mechanism of action of acetaminophen has not been fully understood. It has been suggested that it exerts its effects on GABA receptors. Flumazenil has been experimentally proven to produce an antagonism on acetaminophen's analgesic effect.The purpose of this study was to determine whether flumazenil antagonized the toxic effects of acetaminophen overdose in rats., Methods: A total of 49 Wistar albino rats weighing between 250 - 350 g were used in the study. Nine rats were examined for a preliminary study, and the other rats were randomly divided into five groups with eight subjects in each., Control Group: Saline; Acetaminophen group: 3 g/kg acetaminophen; Experimental Group F1: 3 g/kg acetaminophen + 0.1 mg/kg flumazenil; Experimental group F2: 3 g/kg acetaminophen + 1 mg/kg flumazenil; Experimental group F3: 3 g/kg acetaminophen + 10 mg/kg flumazenil. Acetaminophen was administered in a 3 ml saline solution by way of gastric catheter. Flumazenil was administered by way of intraperitoneal injections. Serum levels of acetaminophen, AST, ALT, LDH, ALP and bilirubin were recorded over a 24-hour period., Results: Serum acetaminophen levels were similar between the groups. The AST, ALT, ALP, LDH, total bilirubin and direct bilirubin levels of Group A were significantly higher compared with the Group C, Group F1, Group F2 and Group F3. There was not a statistically significant difference in the AST, ALT, ALP, LDH, total bilirubin or direct bilirubin levels of the flumazenil-administered groups., Conclusion: Flumazenil's prevention of the acetaminophen-induced increase in liver enzymes is promising. There is some indication that flumazenil could be used in treatment of acetaminophen intoxication (Tab. 2, Ref. 25).

This study was carried out to compare the hepatoprotective effect of methionine on paracetamol treated rats at both the peaks of toxicity and absorption. Female Wistar rats were divided into 17 groups consisting of eight rats per group and treated with different doses of paracetamol/methionine (5:1). Each control rat received 5 ml of physiologic saline. The study was terminated at two different end points -the 4th and 16th hours. Results show that rats administered with toxic doses (1000 mg/kg, 3000 mg/kg, 5000 mg/kg BW) of paracetamol exhibited significant increases in the levels of ALT, AST, γ- GT compared with controls. These increases were much higher at the 16th than 4th hour but serum total protein, albumin and globulin were significantly decreased by the end of the 16th hour. Histology results of rats in the 3000 and 5000 mg/kg (by the end of the 16th hour) confirmed hepatic damage, light microscopic evaluation of liver showed remarkable centrilobular necrosis. Moreover, the presence of mononuclear cells in liver section of rats intoxicated with APAP (5000 mg/kg) suggests a possible involvement of inflammatory process which resulted in regurgitation of bilirubin leading to its elevated level as well as increase activity of ALP. The hepatoprotective effect of methionine, on the other hand, was demonstrated in these rats at the 4th and 16th hours, and both results were comparable and therefore not significantly different but elevation in GGT level still persisted. In conclusion, data obtained from this study suggest that these agents may be capable of inducing GGT, although further study is required to establish a possible relationship between methionine and this enzyme in some other animal species.

Background: Acetaminophen is one of the most commonly encountered medications in self-poisoning, with a high rate of morbidity. The prevalence and characteristics of acetaminophen intoxication associated with long hospital stay in patients are not well defined., Objectives: This study aims to identify the clinical and demographic factors associated with the length of in-hospital stay (LOS), and to evaluate the effect of early treatment of acetaminophen overdose patients (≤8 hours) by intravenous N-acetylcysteine (IV-NAC) on hospital stay., Methods: This is a retrospective cohort study of hospital admissions for acetaminophen overdose conducted over a period of 5 years from 1 January 2004 to 31 December 2008. Patients were divided into two groups: LS group patients had a long hospital stay (> median hours stay in hospital) and SS group patients had a short hospital stay (≤ median hours stay in hospital). Variables were abstracted from medical records for comparison between the two groups. A total of 20 variables were identified for comparison. Parametric and non-parametric tests were used to test differences between groups depending on the normality of the data. SPSS 15 was used for data analysis., Results: Of the 305 patients, 11 factors were identified in the univariate analysis as associated with LS. Three independent factors were found to be significant predictors of LS in the multivariate analysis. The factors associated with LS were seen among patients with a history of abdominal pain after ingestion of acetaminophen (p = 0.04), who were on IV-NAC administration (p < 0.001) and had an acutely depressed mood (p = 0.003). Late time to NAC infusion of more than 8 hours was associated with LS rather than SS (96 patients [57%] and 6 [24%], respectively; p = 0.003)., Conclusion: Patients with long hospital stay have different clinical characteristics compared to patients with short hospital stay. We identified time to IV-NAC administration is a potentially modifiable factor that may lead to prolonged hospital stay. When risk assessment indicates that NAC is required, it is highly recommended that NAC be started in the first hours of admission to reduce the LOS.

Mosapride citrate (mosapride), a prokinetic agent with 5-HT(4)-receptor agonistic activity, is known to enhance gastric emptying and alleviate symptoms in patients with functional dyspepsia (FD). As hyperalgesia and delayed gastric emptying play an important role in the pathogenesis of FD, we used in this study balloon gastric distension to enable abdominal muscle contractions and characterized the visceromotor response (VMR) to such distension in conscious rats. We also investigated the effects of mosapride on gastric distension-induced VMR in the same model. Mosapride (3-10 mg/kg, p.o.) dose-dependently inhibited gastric distension-induced VMR in rats. However, itopride even at 100 mg/kg failed to inhibit gastric distension-induced VMR in rats. Additionally, a major metabolite M1 of mosapride, which possesses 5-HT(3)-receptor antagonistic activity, inhibited gastric distension-induced VMR. The inhibitory effect of mosapride on gastric distension-induced visceral pain was partially, but significantly inhibited by SB-207266, a selective 5-HT(4)-receptor antagonist. This study shows that mosapride inhibits gastric distension-induced VMR in conscious rats. The inhibitory effect of mosapride is mediated via activation of 5-HT(4) receptors and blockage of 5-HT(3) receptors by a mosapride metabolite. This finding indicates that mosapride may be useful in alleviating FD-associated gastrointestinal symptoms via increase in pain threshold.

Background: The aim of this study was to assess any interaction between ondansetron and paracetamol on a model of post-fracture pain in mice., Methods: In protocol A, after fracture of the tibia, mice were assigned to four groups: paracetamol 30 mg kg⁻¹, paracetamol 50 mg kg⁻¹, paracetamol 100 mg kg⁻¹, or a saline vehicle i.p. In protocol B, after fracture of the tibia, mice were randomized to receive either paracetamol (100 mg kg⁻¹) plus saline (vehicle), paracetamol (100 mg kg⁻¹) plus ondansetron (1 mg kg⁻¹), paracetamol (100 mg kg⁻¹) plus ondansetron (2 mg kg⁻¹), saline plus ondansetron (2 mg kg⁻¹), or saline plus saline i.p. Three tests were used to assess pain behaviour: von Frey filament application, hot-plate test, and a subjective pain scale. Rescue analgesia with morphine was administered as necessary., Results: In protocol A, paracetamol (100 mg kg⁻¹)-treated animals had less mechanical nociception, thermal nociception, and a lower subjective pain scale rating, when compared with those receiving paracetamol at 30 or 50 mg kg⁻¹ or saline [ED₅₀ paracetamol=46.3 (6.34) mg kg⁻¹]. No difference was found between paracetamol (30 mg kg⁻¹) and saline-treated animals. In protocol B, the mechanical withdrawal threshold, the thermal withdrawal latency, and the subjective pain scale were lower after injection of paracetamol (100 mg kg⁻¹)+saline, paracetamol (100 mg kg⁻¹)+ondansetron (1 mg kg⁻¹), and paracetamol (100 mg kg⁻¹)+ondansetron (2 mg kg⁻¹), whereas in mice receiving saline+ondansetron (2 mg kg⁻¹) or saline+saline, there was no difference., Conclusion: We found that paracetamol 100 mg kg⁻¹ blocked the development of hyperalgesia and allodynia after fracture pain and ondansetron did not modify the antinociceptive effect of paracetamol in this model.

This study was undertaken to evaluate the protective effect of thymoquinone (TQ) against acetaminophen-induced hepatotoxicity. Mice were given TQ orally at three different doses (0.5, 1 and 2mg/kg/day) for 5 days before a single hepatotoxic dose of acetaminophen (500 mg/kg i.p.). TQ supplementation dramatically reduced acetaminophen-induced hepatotoxicity, in a dose-dependent manner, as evidenced by decreased serum alanine aminotransferase (ALT) activities. Acetaminophen (500 mg/kg i.p.) resulted in a significant increase in serum ALT and total nitrate/nitrite, hepatic lipid peroxides and a significant decrease in hepatic reduced glutathione (GSH) and ATP in a time-dependent manner. Interestingly, supplementation of TQ (2mg/kg/day) for 5 days before acetaminophen administration resulted in reversal of acetaminophen-induced increase in ALT, total nitrate/nitrite, lipid peroxide and a decrease in GSH and ATP. Moreover, TQ did not affect acetaminophen-induced early decrease in hepatic GSH indicating lack of the effect on the metabolic activation of acetaminophen. In conclusion, TQ is effective in protecting mice against acetaminophen-induced hepatotoxicity possibly via increased resistance to oxidative and nitrosative stress as well as its ability to improve the mitochondrial energy production., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

Extracts of Boerhaavia diffusa leaves were evaluated for antioxidant and hepatoprotective properties in the acetaminophen-induced liver damage model. Antioxidative evaluation of ethanolic extract gave total phenolic content, total flavonoid content, vitamin C content and vitamin E content and the levels of selenium and zinc as 6.6+/-0.2mg/g tannic acid equivalent, 0.092+/-0.003 mg/g quercetin equivalent, 0.21+/-0.03 mg/g, 0.054+/-0.002 mg/g, 0.52+/-0.05 ppm and 9.28+/-0.16 ppm, respectively. The DPPH scavenging capacity and the reductive potential were 78.32+/-2.41% and 0.65+/-0.02 mg/g ascorbic acid, respectively. Pretreatment with aqueous and ethanolic extracts decreased the activities of alkaline phosphatase, lactate dehydrogenase, alanine aminotransferase, aspartate aminotransferase, and the level of bilirubin in the serum that were elevated by acetaminophen. The two extracts also ameliorated the elevation in the activities of the enzymes in the liver. Acetaminophen intoxication led to reduction in serum and liver albumin levels which were not significantly increased by pretreatment with the extracts. The extracts also protected against acetaminophen induced lipid peroxidation. These results indicated that leaf extracts from B. diffusa possess hepatoprotective property against acetaminophen-induced liver damage which may be mediated through augmentation of antioxidant defenses., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

Previously, the gastric pentadecapeptide BPC 157, (PL 14736, Pliva) has been shown to have several beneficial effects, it exert gastroprotective, anti-inflammatory actions, stimulates would healing and has therapeutic value in inflammatory bowel disease. The present study aimed to study the effect of naloxone and BPC 157 on morphine-induced antinociceptive action in hot plate test in the mouse. It was found that naloxone and BPC 157 counteracted the morphine (16 mg/kg s.c.) - analgesia. Naloxone (10 mg/kg s.c.) immediately antagonised the analgesic action and the reaction time returned to the basic values, the development of BPC 157-induced action (10 pg/kg, 10 ng/kg, 10 microg/kg i.p.) required 30 minutes. When haloperidol, a central dopamine-antagonist (1 mg/kg i.p.), enhanced morphine-analgesia, BPC 157 counteracted this enhancement and naloxone reestablished the basic values of pain reaction. BPC 157, naloxone, and haloperidol per se failed to exert analgesic action. In summary, interaction between dopamine-opioid systems was demonstrated in analgesia, BPC 157 counteracted the haloperidol-induced enhancement of the antinociceptive action of morphine, indicating that BPC acts mainly through the central dopaminergic system.

Acetaminophen (APAP) can cause life-threatening renal damages and there is no specific treatment for APAP-induced renal damage. The aim of this study was to investigate the protective effects of curcumin (CMN) on APAP-induced nephrotoxicity. Nephrotoxicity was induced in male Wistar Albino rats by the administration of a single dose of 1000 mg/kg APAP intraperitoneally (i.p.). Some of these rats also received i.p. CMN (200mg/kg) at 30 min after the administration of APAP. Twenty-four hours after the administration of APAP, all the rats were sacrificed with a high dose of ketamine. Urea and creatinine levels were measured in the blood, and the levels of malondialdehyde (MDA) and glutathione (GSH), and antioxidant enzyme activity were determined in the renal tissue. Histopathological changes were studied. APAP administration caused elevated levels of renal MDA, and marked depletion of GSH levels and antioxidant enzyme activity, and deteriorated the renal functions as assessed by the increased plasma urea and creatinine levels as compared to control rats. CMN markedly reduced the elevated MDA levels, significantly increased the antioxidant enzyme activity and normalized the altered renal morphology in rats treated with APAP. CMN might be a potential candidate agent against APAP-induced nephrotoxicity, but further studies are required to identify this issue before clinical application becomes possible.

Certain disease conditions can modify drug-induced toxicities, which, in turn, may cause a medication-related health crisis. Therefore, preclinical investigations into the alterations in drug-induced toxicities using appropriate disease animal models are very important. This paper reviews the reported data related to the effects of diabetes and hypertriglyceridemia, common lifestyle-related diseases in a modern society, on acetaminophen (APAP)-induced hepatotoxicity and nephrotoxicity in rats and mice. It has generally been reported that diabetes protects rats and mice from APAP-induced hepatotoxicity and there are several reports that help to speculate on the effects of diabetes on APAP-induced nephrotoxicity. In fructose-induced hypertriglyceridemic rats, hepatotoxicity of APAP becomes apparently less severe, whereas nephrotoxicity of APAP becomes significantly more severe. The mechanisms of alteration of APAP-induced hepatorenal toxicity under diabetic and hypertriglyceridemic conditions are also discussed in this paper.

Oxidative stress is a major cause of drug induced hepatic diseases. The present study aims to investigate the antioxidative signaling mechanism of a protein isolated from the herb, Cajanus indicus against acetaminophen induced necrotic cell death. We found that incubation of hepatocytes with the protein prevented acetaminophen-induced loss in cell viability, reduction in glutathione level and enhancement of reactive oxygen species generation. Treatment of mice with the protein before administration of acetaminophen also reduced serum nitrite and TNF-alpha formation. Moreover, it counteracted acetaminophen-induced loss in mitochondrial membrane potential, loss in adenosine tri phosphate and rise in intracellular calcium. Investigating the cell signaling pathways, we found that the protein exerts its protective action via the activation of NF-kappaB and Akt and deactivation of STAT-1. Surprisingly, no role of ERK1/2 or STAT-3 was found in the protein-mediated protection of hepatocytes during acetaminophen exposure. Finally, we found that acetaminophen introduces necrosis as the primary phenomena of cell death and protein treatment decreased the necrotic process as evident from the DNA fragmentation and flow-cytometry studies. In addition, administration of the protein to mice before acetaminophen application showed fewer number of TUNEL positive cells. Combining, data suggest that the protein possesses cytoprotective activity against acetaminophen-induced oxidative cellular damage and prevents hepatocytes from necrotic death.

Tomato products containing lycopene are believed to be associated with decreased risk of chronic diseases including cancer, and its effects are suggested to be due to antioxidant effect of lycopene. The aim of this research was to study the effects of tomato extract on acetaminophen (APAP), amiodarone (ADN) and cyclosporine A (CsA)-induced liver, lung and kidney toxicity, respectively. Previous studies have shown that free radical reactions may play important roles in toxicity of these drugs. Rats received a single dose of APAP (750mg/kg, i.p.) before treatment with tomato extract (5mg/kg, oral) for seven consecutive days, ADN (100mg/kg, i.p.) plus tomato extract (5mg/kg, oral) for 10 consecutive days, or CsA (250mg/kg, i.p.) plus tomato extract (5mg/kg, oral) for 14 consecutive days. At the end of each treatment, the animals were sacrificed and the related organ tissues were collected for biochemical and histopathological examinations. Simultaneous treatment of tomato extract ameliorated tissue damage, biochemical indices, and oxidative stress parameters against APAP-induced acute hepatotoxicity, but had less beneficial effects on ADN-induced lung toxicity and little effect against CsA-induced nephrotoxicity. Therefore, tomato products may be beneficial for the prevention and therapy of toxicity induced by ADN and APAP.

This study was conducted to examine the protective role of crude polysaccharide from brown seaweed Sargassum polycystum against acetaminophen-induced abnormality in blood glucose, serum albumin/globulin ratio, and liver glycogen, lactate, and pyruvate. Liver and renal tissue histology was performed to confirm the efficacy of Sargassum polysaccharide. A toxic dose of acetaminophen (800 mg/kg body weight intraperitoneally) induced severe abnormality in all basic parameters with apparent toxicity in liver (enlargement of hepatocytes, loss of cytoplasmic content with disruption in the hepatic plates and sinusoidal dilation) and renal tissue (glomerular damage with congestion of tubules). The isolated liver cells were stained with acridine orange and examined under fluorescence microscope, which revealed that the acetaminophen induced significant damage. In contrast, the rats pretreated with Sargassum polysaccharide (200 mg/kg body weight) daily for 3 weeks did not show liver and renal tissue with these severe aberrations induced by acetaminophen. Histology results were also consistent with analyzed basic biochemical parameters, which confirmed the effectiveness of the crude polysaccharide against acetaminophen-induced abnormality in rats.

The protective effect of salidroside (SDS) isolated from Rhodiola sachalinensis A. BOR. (Crassulaceae), was investigated in acetaminophen (APAP)-induced hepatic toxicity mouse model in comparison to N-acetylcysteine (NAC). Drug-induced hepatotoxicity was induced by an intraperitoneal (i.p.) injection of 300 mg/kg (sub-lethal dose) of APAP. SDS was given orally to mice at a dose of 50 or 100 mg/kg 2 h before the APAP administration in parallel with NAC. Mice were sacrificed 12 h after the APAP injection to determine aspartate aminotransferase (AST), alanine aminotransferase (ALT), and tumor necrosis factor-alpha (TNF-alpha) levels in serum and glutathione (GSH) depletion, malondialdehyde (MDA) accumulation, and caspase-3 expression in liver tissues. SDS significantly protected APAP-induced hepatotoxicity for SDS improved mouse survival rates better than NAC against a lethal dose of APAP and significantly blocked not only APAP-induced increases of AST, ALT, and TNF-alpha but also APAP-induced GSH depletion and MDA accumulation. Histopathological and immunohistochemical analyses also demonstrated that SDS could reduce the appearance of necrosis regions as well as caspase-3 and hypoxia inducible factor-1alpha (HIF-1alpha) expression in liver tissue. Our results indicated that SDS protected liver tissue from the APAP-induced oxidative damage via preventing or alleviating intracellular GSH depletion and oxidation damage, which suggested that SDS would be a potential antidote against APAP-induced hepatotoxicity.

Cuscuta chinensis is a commonly used traditional Chinese medicine to nourish the liver and kidney. Due to the poor water solubility of its major constituents such as flavonoids and lignans, its absorption upon oral administration could be limited. The purpose of the present study was to use the nanosuspension method to prepare C. chinensis nanoparticles (CN), and to compare the hepatoprotective and antioxidant effects of C. chinensis ethanolic extract (CE) and CN on acetaminophen-induced hepatotoxicity in rats. An oral dose of CE at 125 and 250 mg/kg and CN at 25 and 50mg/kg showed a significant hepatoprotective effect relatively to the same extent (P<0.05) by reducing levels of aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase. These biochemical assessments were supported by rat hepatic biopsy examinations. In addition, the antioxidant activities of CE and CN both significantly increased superoxide dismutase, catalase, glutathione peroxidase, and reduced malondialdehyde (P<0.05). Moreover, the results also indicated that the hepatoprotective and antioxidant effects of 50 mg/kg CN was effectively better than 125 mg/kg CE (P<0.05), and an oral dose of CN that is five times as less as CE could exhibit similar levels of outcomes. In conclusion, we suggest that the nanoparticles system can be applied to overcome other water poorly soluble herbal medicines and furthermore to decrease the treatment dosage.

The substantial therapeutic potential of tempol (4-hydroxy-2,2,6,6-tetramethyl-1-piperidinyloxy) and related cyclic nitroxides as antioxidants has stimulated innumerous studies of their reactions with reactive oxygen species. In comparison, reactions of nitroxides with nitric oxide-derived oxidants have been less frequently investigated. Nevertheless, this is relevant because tempol has also been shown to protect animals from injuries associated with inflammatory conditions, which are characterized by the increased production of nitric oxide and its derived oxidants. Here, we review recent studies addressing the mechanisms by which cyclic nitroxides attenuate the toxicity of nitric oxide derived oxidants. As an example, we present data showing that tempol protects mice from acetaminophen-induced hepatotoxicity and discuss the possible protection mechanism. In view of the summarized studies, it is proposed that nitroxides attenuate tissue injury under inflammatory conditions mainly because of their ability to react rapidly with nitrogen dioxide and carbonate radical. In the process the nitroxides are oxidized to the corresponding oxammonium cation, which, in turn, can be recycled back to the nitroxides by reacting with upstream species, such as peroxynitrite and hydrogen peroxide, or with cellular reductants. An auxiliary protection mechanism may be down-regulation of inducible nitric oxide synthase expression. The possible therapeutic implications of these mechanisms are addressed.

Multiple analgesic-drug combinations are commonly used in the management of acute and chronic pain in humans during multimodal or balanced analgesia. At present, these combinations are used empirically in clinical practice and are considered to be beneficial for the patient. Interactions between two antinociceptive drugs have been thoroughly examined, but the nature of interactions between three analgesics has not been studied. The antinociceptive interaction of ketoprofen (K) with a mixture of morphine (M) and paracetamol (P) was evaluated using a model of visceral acute tonic pain, the acetic-acid writhing test in mice. The i.p. administration of the combination M/P+K resulted in a significant potentiation of the antinociception induced either by K or by the synergic two-drug mixtures M/K, P/K and M/P. The effect of opioid, cholinergic, adrenergic and serotonergic antagonists on the analgesic interaction was assessed. The pretreatment of mice with atropine (1 mg/kg) did not produce any change in the synergistic interaction of the triple combination. The pretreatment with naltrexone (1 mg/kg) or tropisetron (1 mg/kg) reduced the intensity of M/P+K synergic interaction, while prazosin (0.1 mg/kg) significantly potentiated the synergy. The findings of this work suggest that the two major pathways of descending inhibitory systems, noradrenergic and serotonergic are significantly involved in the mechanism of the antinociceptive synergy induced by the triple combination. On the other hand, opioid pathways also seem to participate, since pretreatment with naltrexone reduced the synergy. In conclusion, the triple combination M/P+K induced a strong synergistic antinociceptive effect, which could be of interest for optimal multimodal clinical analgesia.

A mixture of leucine and zinc sulfate (4 : 1; 100 mg/kg body weight) produces a hepatoprotective effect, preventing the ultrastructural injury of hepatic tissue and the disturbance of free amino acid metabolism caused by a toxic dose of paracetamol.

Background and Purpose: To follow up in vitro evidence that Delta(9)-tetrahydrocannabivarin extracted from cannabis (eDelta(9)-THCV) is a CB(1) receptor antagonist by establishing whether synthetic Delta(9)-tetrahydrocannabivarin (O-4394) and Delta(8)-tetrahydrocannabivarin (O-4395) behave as CB(1) antagonists in vivo., Experimental Approach: O-4394 and O-4395 were compared with eDelta(9)-THCV as displacers of [(3)H]-CP55940 from specific CB(1) binding sites on mouse brain membranes and as antagonists of CP55940 in [(35)S]GTPgammaS binding assays performed with mouse brain membranes and of R-(+)-WIN55212 in mouse isolated vasa deferentia. Their ability to antagonize in vivo effects of 3 or 10 mg kg(-1) (i.v.) Delta(9)-tetrahydrocannabinol in mice was then investigated., Key Results: O-4394 and O-4395 exhibited similar potencies to eDelta(9)-THCV as displacers of [(3)H]-CP55940 (K (i)=46.6 and 64.4 nM, respectively) and as antagonists of CP55940 in the [(35)S]GTPgammaS binding assay (apparent K (B)=82.1 and 125.9 nM, respectively) and R-(+)-WIN55212 in the vas deferens (apparent K (B)=4.8 and 3.9 nM respectively). At i.v. doses of 0.1, 0.3, 1.0 and/or 3 mg kg(-1) O-4394 and O-4395 attenuated Delta(9)-tetrahydrocannabinol-induced anti-nociception (tail-flick test) and hypothermia (rectal temperature). O-4395 but not O-4394 also antagonized Delta(9)-tetrahydrocannabinol-induced ring immobility. By themselves, O-4395 and O-4394 induced ring immobility at 3 or 10 mg kg(-1) (i.v.) and antinociception at doses above 10 mg kg(-1) (i.v.). O-4395 also induced hypothermia at 3 mg kg(-1) (i.v.) and above., Conclusions and Implications: O-4394 and O-4395 exhibit similar in vitro potencies to eDelta(9)-THCV as CB(1) receptor ligands and as antagonists of cannabinoid receptor agonists and can antagonize Delta(9)-tetrahydrocannabinol in vivo.

The insulin-like growth factor (IGF) system and type-I IGF receptor (IGF-IR) signaling are involved in protecting against chemotherapeutic drug-induced cell death in human hepatoma cells. Acetaminophen (AAP) hepatotoxicity is the leading cause of liver failure, and the prevention of AAP-induced cell death has been the focus of many studies. We determined whether IGF-I could protect against AAP-induced cell death in Chang liver cells and investigated the protective mechanism. Based on the results of MTS assays, LDH release assays, Hoechst 33342 cell staining, and DNA fragmentation experiments, AAP induced cell death in a dose-dependent manner. According to Western blot analysis, treatment with AAP increased the level of poly(ADP-ribose) polymerase (PARP) fragments in cells compared with that in control cells; however, caspase-3, a critical signaling molecule in apoptosis, was not activated after AAP overdose. Moreover, combined treatment with AAP and IGF-I inhibited PARP cleavage, which was consistent with the ability of IGF-I to restore the level of glutathione (GSH) and cell viability in GSH and MTS assays, respectively. We investigated whether the protective effect of IGF-I against AAP cytotoxicity is related to the extracellular signal-related kinase ERK1/2, which is generally activated by mitogenic and proliferative stimuli such as growth factors. Compared with AAP treatment alone, IGF-I and AAP co-treatment increased ERK1/2 phosphorylation but inhibited PARP cleavage. Thus ERK1/2 activation is instrumental in the protective effect of IGF-I against AAP-induced cell death in Chang liver cells.

Adsorption characteristics of medicinal carbon powder (JP 14) for acetaminophen were examined at 37 degrees C using conventional incubation in an attempt to obtain an effective oral dosage form. Hydroxypropyl cellulose (HPC) and maltitol (MT), being able to act as a binding agent, were tested as additives. Tablets of medicinal carbon were produced by the wet granulation method. The rate and extent of adsorption of the medicinal carbon powder were roughly similar in water, JP 14 1st fluid (pH 1.2) and JP 14 2nd fluid (pH 6.8). The relationship between concentrations of free and adsorbed acetaminophen indicated that the adsorption followed the Langmuir mode. The maximal adsorption of acetaminophen in water was 0.219 g per gram medicinal carbon powder, little influenced by the addition of MT, but slightly reduced by the addition of HPC. The tablet prepared using MT as a binding agent displayed a favorable hardness and adequate disintegration time. The tablet showed good adsorption potential for acetaminophen, though the adsorption rate and extent of the tablet were reduced to some extent as compared with powder.

The pineal gland and its main hormone, melatonin (MLT), are involved in a variety of physiological processes. MLT is a member of the indolamine family and has significant antioxidative activity. Acetaminophen (AA) is the most widely used medication in the world, both by prescription and over the counter. In large doses, AA is hepatotoxic causing oxidative stress and lipid peroxidation. Therefore, antioxidants have been used to protect against the toxicity of AA. Here, we examined in vitro and in vivo the protective effects of MLT against AA-induced toxicity in mice. MLT (100 microM) had a significant protective effect on the AA (7 mM)-induced loss of cell viability in mouse primary cultured hepatocytes as determined using the 3H-thymidine incorporation assay and MTT assay. The AA-induced generation of reactive oxygen species (ROS) peaked at 6 h and was followed by an increase in lipid peroxidation at 12 h in hepatocytes. MLT (0.1, 1, 10 or 100 microM) dose-dependently attenuated the increase in both production of ROS and lipid peroxidation by AA. Similarly, in vivo, AA (400, 600 or 800 mg/kg, intraperitoneally)-induced mortality and hepatotoxicity were significantly decreased by MLT (10 mg/kg, subcutaneously). Pretreatment with MLT had a greater protective effect on the hepatotoxicity of AA than post-treatment. However, MLT had no protective effect on the antipyretic effect or antinociception caused by AA. These results suggest that MLT is potentially useful for preventing AA-induced toxicity, but not the antipyretic effect or antinociception caused by AA.

Geranylgeranylacetone (GGA), an anti-ulcer drug, has been reported to induce heat shock protein (HSP) 70 in several animal organs. The present study was performed to determine whether GGA protects mouse liver against acetaminophen (APAP)-induced injury and whether it has potential as a therapeutic agent for APAP overdose. Hepatic damage was induced by single oral administration of APAP (500 mg/kg). GGA at 400 mg/kg was given orally 4 or 8h before, or 0.5h after APAP administration. Treatment of mice with GGA 4h before or 0.5h after APAP administration suppressed increases in transaminase activities and ammonia content in blood as well as hepatic necrosis. Such GGA treatment significantly increased hepatic HSP70 accumulation after APAP administration. Furthermore, GGA inhibited increases in hepatic lipid peroxide content and hepatic myeloperoxidase activity after APAP administration. In contrast, GGA neither inhibited hepatic cytochrome P450 2E1 activity nor suppressed hepatic glutathione depletion after APAP administration. The protective effect of GGA treatment 4h before APAP on hepatotoxicity induced by APAP was completely inhibited with quercetin, known as an HSP inhibitor. In conclusion, GGA has been identified as a new antidote to APAP injury, acting by induction of HSP70. The potential of GGA as a therapeutic tool is strongly supported by its ability to inhibit hepatic injury even when administered after ingestion of APAP.

Freshly isolated mouse hepatocytes were used to determine the role of mitochondrial permeability transition (MPT) in acetaminophen (APAP) toxicity. Incubation of APAP (1 mM) with hepatocytes resulted in cell death as indicated by increased alanine aminotransferase in the media and propidium iodide fluorescence. To separate metabolic events from later events in toxicity, hepatocytes were preincubated with APAP for 2 h followed by centrifugation of the cells and resuspension of the pellet to remove the drug and reincubating the cells in media alone. At 2 h, toxicity was not significantly different between control and APAP-incubated cells; however, preincubation with APAP followed by reincubation with media alone resulted in a marked increase in toxicity at 3 to 5 h that was not different from incubation with APAP for the entire time. Inclusion of cyclosporine A, trifluoperazine, dithiothreitol (DTT), or N-acetylcysteine (NAC) in the reincubation phase prevented hepatocyte toxicity. Dichlorofluorescein fluorescence increased during the reincubation phase, indicating increased oxidative stress. Tetramethylrhodamine methyl ester perchlorate fluorescence decreased during the reincubation phase indicating a loss of mitochondrial membrane potential. Inclusion of cyclosporine A, DTT, or NAC decreased oxidative stress and loss of mitochondrial membrane potential. Confocal microscopy studies with the dye calcein acetoxymethyl ester indicated that MPT had also occurred. These data are consistent with a hypothesis where APAP-induced cell death occurs by two phases, a metabolic phase and an oxidative phase. The metabolic phase occurs with GSH depletion and APAP-protein binding. The oxidative phase occurs with increased oxidative stress, loss of mitochondrial membrane potential, MPT, and toxicity.

This study was performed to determine the optimal reversal dosage of atipamezole on medetomidine-ketamine combination anesthesia. The subject rabbits were divided into five groups (n=5/group), and all were anesthetized with intravenous medetomidine (0.35 mg/kg) and ketamine (5 mg/kg). Atipamezole was administered intravenously 35 min after administration of the medetomidine-ketamine mixture, at doses of a quarter, a half, equal, or two times higher than the preceding medetomidine -ketamine dose according to experimental group. Heart rate (HR), mean arterial pressure (MAP), respiratory rate (RR) and rectal temperature (RT) were measured every five minutes and the mean arousal time (MAT) was also recorded. This study revealed that the optimal atipamezole dosage to achieve reversal effects is equal to or double the dose of medetomidine. At these dosages, HR and MAP significantly recovered and MAT was significantly shortened with no side effects being observed (p<0.05).

The acute treatment of rats and mice with a hydroalcoholic extract from the seeds of Dioclea grandiflora (EHDg) at doses of 250 and 500 mg/kg, by intraperitoneal or oral administration, produced a significant antinociceptive effect in the tail flick and hot plate tests, an effect which was inhibited by naloxone. EHDg given to mice daily for 30 days at a dose of 500 mg/kg, did not cause any observable toxic effect nor any alteration in the pattern of antinociceptive response by the tail immersion test during the course of this treatment. These results suggest that EHDg has a central antinociceptive action devoid of tolerance effect typical of opioid drugs.

The nitric oxide (NO) donor, O(2)-vinyl 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate (V-PYRRO/NO), is metabolized by P450 enzymes to release NO in the liver and possibly other tissues. V-PYRRO/NO has been shown to be hepatoprotective, but little is known about its effect in the kidney, another organ rich in P450s. Thus, mice were given V-PYRRO/NO (0.4-5.4 mg/ml, 8 microl/h) before and/or after a nephrotoxic dose of acetaminophen (APAP; 600 mg/kg, i.p.) to examine its nephroprotective effects. V-PYRRO/NO administration significantly reduced APAP-induced nephrotoxicity in a dose- and time-dependent manner, as evidenced by mitigation of increased blood urea nitrogen levels and by amelioration of renal pathology, specifically interstitial congestion, proximal tubular cell degeneration and necrosis. The best protection was observed at the highest dose (5.4 mg/ml) and with V-PYRRO/NO pretreatment (4-16 h). Implanting V-PYRRO/NO pumps simultaneously with APAP also attenuated APAP nephrotoxicity. The protection is probably not due to a decreased APAP toxication metabolism, as similar depletion of renal glutathione levels was observed regardless of V-PYRRO/NO treatment. APAP-induced renal lipid peroxidation was reduced by V-PYRRO/NO, as determined by the concentrations of hydroxynonenals and malondialdehyde. In summary, this study demonstrates that the NO donor V-PYRRO/NO is effective in blocking APAP-induced nephrotoxicity in mice. The protection is probably due to multiple mechanisms involving attenuation of APAP-induced congestion and lipid peroxidation in the kidney.

Acetaminophen (AA) is a commonly used analgesic and antipyretic drug; however, when used in high doses, it causes fulminant hepatic necrosis and nephrotoxic effects in both humans and experimental animals. It has been reported that the toxic effects of AA are the result of oxidative reactions that take place during its metabolism. In this study we investigated if melatonin, vitamin E or N-acetylcysteine (NAC) are protective against AA toxicity in mice. The doses of the antioxidants used were as follows: melatonin (10 mg/kg), vitamin E (30 mg/kg) and NAC (150 mg/kg). Blood urea nitrogen (BUN), serum creatinine, alanine aminotransferase (ALT), aspartate aminotransferase (AST) levels in blood, and glutathione (GSH), malondialdehyde (MDA), oxidized protein levels and myeloperoxidase (MPO) activity in liver and kidney tissues were measured. BUN and serum creatinine, ALT and AST levels which were increased significantly following AA treatment decreased significantly after pretreatment with either vitamin E, melatonin or NAC; however, they were not reduced to control levels. ALT and AST levels were significantly higher at 4 hr compared with the 24 hr levels after AA administration. However, BUN and creatinine levels were significantly elevated only at 24 hr. GSH levels were reduced while MDA, MPO and oxidized protein levels were increased significantly following AA administration. These changes were reversed by pretreatment with either melatonin, vitamin E or NAC. Liver toxicity was higher at 4 hr, whereas nephrotoxicity appeared to be more severe 24 hr after treatment with AA. Vitamin E was the least efficient agent in reversing AA toxicity while melatonin, considering it was given as at lower dose than either vitamin E or NAC, was the most effective. This may be the result of the higher efficacy of melatonin in scavenging various free radicals and also because of its ability in stimulating the antioxidant enzymes.

Streptozotocin (STZ)-induced diabetic (DB) mice challenged with single ordinarily lethal doses of acetaminophen (APAP), carbon tetrachloride (CCl4), or bromobenzene (BB) were resistant to all three hepatotoxicants. Mechanisms of protection against APAP hepatotoxicity were investigated. Plasma alanine aminotransferase, aspartate aminotransferase, and liver histopathology revealed significantly lower hepatic injury in DB mice after APAP administration. HPLC analysis of plasma and urine revealed lower plasma t1/2, increased volume of distribution (Vd), and increased plasma clearance (CLp) of APAP in the DB mice and no difference in APAP-glucuronide, a major metabolite in mice. Interestingly, covalent binding of 14C-labeled APAP to liver target proteins; arylation of APAP to 58, 56, and 44 kDa acetaminophen binding proteins (ABPs); and glutathione (GSH) depletion in the liver did not differ between nondiabetic (non-DB) and DB mice in spite of downregulated hepatic microsomal CYP2E1 and 1A2 proteins in the DB mice, known to be involved in bioactivation of APAP. Compensatory cell division measured via 3H-thymidine pulse labeling and immunohistochemical staining for proliferating cell nuclear antigen (PCNA) indicated earlier onset of S-phase in the DB mice after exposure to APAP. Antimitotic intervention of liver cell division by colchicine (CLC) after administration of APAP led to significantly higher mortality in the DB mice suggesting a pivotal role of liver cell division and tissue repair in the protection afforded by diabetes. In conclusion, the resistance of DB mice against hepatotoxic and lethal effects of APAP appears to be mediated by a combination of enhanced APAP clearance and robust compensatory tissue repair.

We previously observed that (trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide (U50,488H) promoted internalization and phosphorylation of the FLAG-tagged human kappa opioid receptor (FLAG-hkor) stably expressed in Chinese hamster ovary (CHO) cells. In this study, we compared regulation of the FLAG-hkor expressed in CHO cells by U50,488H, dynorphin A, etorphine, and levorphanol, which were potent full agonists as determined by stimulation of guanosine 5'-O-(3-[(35)S]thio)triphosphate binding. Using fluorescence flow cytometry, we found that dynorphin A(1-17), like U50,488H, promoted internalization of the FLAG-hkor in a time- and dose-dependent manner. The antagonists naloxone and norbinaltorphimine, having no effect on FLAG-hkor internalization, effectively blocked dynorphin A(1-17)- and U50,488H-induced internalization. Interestingly, the full agonists etorphine and levorphanol did not cause internalization of the FLAG-hkor but significantly reduced dynorphin A(1-17)- and U50,488H-induced internalization in a dose-dependent manner. Immunofluorescence staining of FLAG-hkor yielded similar results. Dynorphin A(1-17) and U50,488H enhanced phosphorylation of FLAG-hkor to a greater extent than etorphine, but levorphanol did not increase FLAG-hkor phosphorylation. Etorphine or levorphanol decreased dynorphin- or U50,488H-induced phosphorylation. It is likely that conformations of the hkor required for phosphorylation and initiation of internalization are different from those for activation of G proteins. We also examined whether the four agonists had differential effects on superactivation of adenylate cyclase. Pretreatment with U50,488H, dynorphin A(1-17), or etorphine enhanced forskolin-stimulated adenylate cyclase activity to approximately 200 to 250% of the control, whereas levorphanol pretreatment did not result in significant adenylate cyclase superactivation. Thus, the degree of superactivation caused by an agonist is unrelated to its ability to promote internalization of the hkor.

L-Cysteine-glutathione disulfide, a ubiquitous substance present in mammalian cells, was shown to be highly effective in protecting mice against acetaminophen-induced hepatotoxicity. Since the corresponding D-cysteine-glutathione disulfide was totally ineffective in this regard, an enzymatic mechanism that provides glutathione directly to cells is postulated., (Copyright 2003 Wiley Periodicals, Inc. J Biochem Mol Toxicol 17:95-97, 2003; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.10069)

We have previously shown that fructose-1,6-diphosphate (FDP) stimulates the synthesis of nitric oxide probably by stimulating the hepatic inducible nitric oxide synthase (iNOS). The aim of the present study was to evaluate the hepatoprotective role of FDP in acetaminophen-induced liver injury and whether this hepatoprotective effect is mediated by nitric oxide. Liver injury was induced in adult Sprague-Dawley rats by the administration of acetaminophen (1.6 g/kg by gavage) 10 min prior to the intraperitoneal injection of either FDP or normal saline. Liver injury was assessed by alanine aminotransferase (ALT) activity in the serum. iNOS and malondialdehyde (MDA) levels were determined in liver homogenates. Acetaminophen produced striking elevations of serum ALT, high MDA levels and a profound decrease in the liver iNOS. Administration of FDP attenuated the ALT and MDA elevations and prevented the liver iNOS depletion caused by acetaminophen. Pretreatment of the animals with the iNOS inhibitor L-NAME abolished this hepatoprotection. These findings suggest that FDP protects against acetaminophen-induced liver injury, at least partly, by stimulating production of nitric oxide.

Acetaminophen (AAP) overdose causes formation of nitrotyrosine, a footprint of peroxynitrite, in centrilobular hepatocytes. The importance of peroxynitrite for the pathophysiology, however, is unclear. C3Heb/FeJ mice were treated with 300 mg/kg AAP. To accelerate the restoration of hepatic glutathione (GSH) levels as potential endogenous scavengers of peroxynitrite, some groups of animals received 200 mg of GSH/kg i.v. at different time points after AAP. AAP induced severe liver cell damage at 6 h. Total liver and mitochondrial glutathione levels decreased by >90% at 1 h but recovered to 75 and 45%, respectively, of untreated values at 6 h after AAP. In addition, the hepatic and mitochondrial glutathione disulfide (GSSG) content was significantly increased over baseline, suggesting a mitochondrial oxidant stress. Moreover, centrilobular hepatocytes stained for nitrotyrosine. Treatment with GSH at t = 0 restored hepatic GSH levels and completely prevented the mitochondrial oxidant stress, peroxynitrite formation, and liver cell injury. In contrast, treatment at 1.5 and 2.25 h restored hepatic and mitochondrial GSH levels but did not prevent the increase in GSSG formation. Nitrotyrosine adduct formation and liver injury, however, was substantially reduced. GSH treatment at 3 h after AAP was ineffective. Similar results were obtained when these experiments were repeated with glutathione peroxidase-deficient animals. Our data suggest that early GSH treatment (t = 0) prevented cell injury by improving the detoxification of the reactive metabolite of AAP. Delayed GSH treatment enhanced hepatic GSH levels, which scavenged peroxynitrite in a spontaneous reaction. Thus, peroxynitrite is an important mediator of AAP-induced liver cell necrosis.

Previous studies have shown that the kappa-opioid effects are sensitive to pertussis toxin (PTX) and affected by Ca(2+) fluxes. However, the possible involvement of Ca(2+) channels in PTX-induced inhibition of kappa-opioid effects has not been reported. The effect of intracerebroventricular (i.c.v.) treatment of pertussis toxin (1 microg/rat, PTX) or saline on the kappa-opioid agonist, U-50,488H (U5H) induced tail-flick analgesia and hypothermia in rats was determined. The effect of nimodipine (NIM), a dihydropyridine (DHP)-sensitive Ca(2+) channel blocker (CCB), on PTX-induced modulation of U5H effects was examined. The DHP ligand, [3H]PN200-110 binding was also determined in both PTX and saline treated rats to study the possible involvement of L-type Ca(2+) channels in PTX modulation of kappa-opioid agonist effects. The analgesia and change in colonic temperature were determined using tail-flick analgesiometer and telethermometer, respectively. U5H (40 mg/kg, i.p.) produced significant analgesic and hypothermic responses. PTX treatment significantly (P<0.01) antagonized the analgesic and hypothermic effects of U5H. Acute pretreatment of NIM (1 mg/kg, i.p.) 15 min prior significantly (P<0.01) reversed the PTX-induced antagonism of U5H effects. In the binding study, PTX treatment (72 h before) resulted in a significant (P<0.005) upregulation (+45% vs. saline control) of DHP binding (B(max)) with no change in affinity (K(d)). The results showed significant upregulation of DHP binding in accordance with PTX-induced antagonism of U5H effects and this blockade was reversed by NIM. Thus, present results suggest that U5H-induced analgesia and hypothermia may be mediated through PTX-sensitive transducer G-proteins (G(i/o)) coupled to L-type Ca(2+) channels.

Administration of acetaminophen (ACP, 400 mg/kg, i.p.) to fasted, male Swiss-Webster mice caused a rapid 90% decrease in total hepatic glutathione (GSH) and a 58% decrease in mitochondrial GSH by 2 h post ACP. This was followed by a time-dependent decrease (72%) in hepatic AdoMet synthetase activity and rise in plasma ALT levels (>10000 U/l) at 24 h post ACP treatment. AdoMet synthetase activity was maintained at 82, 78 and 60% of controls, respectively, by the cysteine prodrugs PTCA, CySSME and NAC. Total hepatic and mitochondrial GSH levels were also protected from severe ACP-induced depletion by CySSME and MTCA. These results suggest that the maintenance of GSH homeostasis by cysteine prodrugs can protect mouse hepatic AdoMet synthetase, a sulfhydryl enzyme whose integrity is dependent on GSH, as well as the liver itself from the consequences of oxidative stress elicited by toxic metabolites of xenobiotics.

In the present study, an attempt has been made to investigate whether the potentiating effect of U-50,488H (U50)-induced analgesia by ginseng total saponin (GTS) is playing a role in inhibiting the tolerance to U50-induced analgesia as measured using the tail-flick test in mice. GTS (100 and 200 mg/kg i.p.), on acute administration, potentiated the U50 (40 mg/kg i.p.)-induced analgesia in U50-naive mice. Twice daily administration of U50 (40 mg/kg i.p.) for 6 days resulted in tolerance to U50-induced analgesia in mice. Chronic administration (Days 4-6) of GTS (50, 100, and 200 mg/kg i.p.) to U50-tolerant mice dose-dependently inhibited the tolerance to U50-induced analgesia. On the other hand, chronic administration of GTS (50, 100, and 200 mg/kg i.p.) dose-dependently potentiated the U50-induced analgesia in U50-naive mice. The dose-response curve to U50-induced analgesia in U50-tolerant mice was shifted rightward (2.6-fold) as compared to U50-naive mice, indicating the development of tolerance to U50-induced analgesia. GTS (100 mg/kg i.p. o.d.), on chronic administration, prevented the rightward shift of dose-response curve to U50-induced analgesia in U50-tolerant mice, whereas in U50-naive mice it resulted in leftward shift (0.6-fold). It can be concluded that acute and chronic administration of GTS potentiates the U50-induced analgesia in U50-naive mice. The potentiating effect of GTS on U50-induced analgesia may be partially responsible in the inhibition of tolerance to U50-induced analgesia in mice.

Sixteen captive and wild-caught American alligators (Alligator mississippiensis), seven juveniles (< or = 1 m total length [TL]; 6.75 +/- 1.02 kg), and nine adults (> or = 2 m TL; 36.65 +/- 38.85 kg), were successfully anesthetized multiple times (n = 33) with an intramuscular (i.m.) medetomidine-ketamine (MK) combination administered in either the triceps or masseter muscle. The juvenile animals required significantly larger doses of medetomidine (x = 220.1 +/- 76.9 microg/kg i.m.) and atipamezole (x = 1,188.5 -/+ 328.1 microg/kg i.m.) compared with the adults (medetomidine, x = 131.1 +/- 19.5 microg/kg i.m.; atipamezole, x = 694.0 +/- 101.0 microg/kg i.m.). Juvenile alligators also required higher (statistically insignificant) doses of ketamine (x = 10.0 +/- 4.9 mg/kg i.m.) compared with the adult animals (x = 7.5 +/- 4.2 mg/kg i.m.). The differences in anesthesia induction times (juveniles, x = 19.6 +/- 8.5 min; adults, x = 26.6 +/- 17.4 min) and recovery times (juveniles, x = 35.4 +/- 22.1 min; adults, x = 37.9 +/- 20.2 min) were also not statistically significant. Anesthesia depth was judged by the loss of the righting, biting, corneal and blink, and front or rear toe-pinch withdrawal reflexes. Recovery in the animals was measured by the return of reflexes, open-mouthed hissing, and attempts to high-walk to the opposite end of the pen. Baseline heart rates (HRs) were significantly higher in the juvenile animals (x = 37 +/- 4 beats/min) compared with the adults (x = 24 +/- 5 bpm). However, RRs (juveniles, x = 8 +/- 2 breaths/min; adults, x = 8 +/- 2 breaths/min) and body temperatures (juveniles, x = 24.1 +/- 1.1 degrees C; adults, x = 25.2 +/- 1.2 degrees C) did not differ between the age groups. In both groups, significant HR decreases were recorded within 30-60 min after MK administration. Cardiac arrhythmias (second degree atrio-ventricular block and premature ventricular contractions) were seen in two animals but were not considered life-threatening. Total anesthesia times ranged from 61-250 min after i.m. injection. Although dosages were significantly different between the age groups, MK and atipamezole provided safe, effective, completely reversible anesthesia in alligators. Drug-dosage differences appear to be related to metabolic differences between the two size-classes, requiring more research into metabolic scaling as a method of calculating anesthetic dosages.

Acetaminophen (AAP) hepatotoxicity, resulting in centrilobular necrosis, is frequently encountered following suicidal attempts, especially by adolescents, but also after its excessive use in infants. The subcellular and molecular sequences leading to hepatocellular cell death are not yet clear. We therefore investigated AAP hepatotoxicity by using cultured hepatoma-derived cells (Hep3B) exposed to AAP and N-acetylcysteine (NAC), used as a protective agent. Specifically, we studied the role of apoptosis and oxidative damage as putative mechanisms of AAP-associated cytotoxicity. Hep3B cells were exposed to AAP (5-25 mM) and NAC (5 mM) for different time periods. Cell viability was assessed by the Alamar Blue Reduction Test and LDH. Oxidative damage was evaluated by measuring reactive oxygen species (ROS) and glutathione. AAP-induced apoptosis was investigated by flow cytometry and transmission electron microscopy. We found that: 1. In Hep3B cells, AAP causes a time- and concentration-dependent cytotoxic effect, leading to oxidative stress, mitochondrial dysfunction, alterations of membrane permeability and apoptosis; 2. In the course of AAP cytotoxicity, the generation of ROS appears as an early event which precedes decrease of viability, LDH leakage, glutathione depletion and apoptosis; 3. NAC protects Hep3B cells from AAP-induced oxidative injury, but does not prevent apoptosis.

Treatment with the triester of S-(1,2-dicarboxyethyl)glutathione (DCE-GS) prevented the hepatotoxicity induced by acetaminophen via elevation of the glutathione (GSH) level in rat hepatocytes. This elevation of the GSH level in rat hepatocytes by DCE-GS triester was dose- and time-dependent (2.1-fold in 24 h with 0.5 mm). DCE-GS triester increased the GSH level much more effectively than GSH, DCE-GS, and DCE-GS monoester and diester. Furthermore, the activity of y-glutamylcysteine synthetase (gamma-GCS), the rate-limiting enzyme in GSH biosynthesis, was also increased by DCE-GS triester treatment (1.4-fold in 24 h with 1.0 mm). In contrast, with a rat liver homogenate, DCE-GS increased the y-GCS activity, whereas DCE-GS triester had no effect on this activity. These results suggested that DCE-GS triester, which is transported into hepatocytes much more effectively than DCE-GS and other DCE-GS esters due to its greater lipophilicity, was hydrolyzed to DCE-GS, and then the DCE-GS produced increased the GSH level via activation of gamma-GCS in rat hepatocytes.

The intravenous administration of synthetic cannabinoid agonists was recently shown to dose dependently increase acetylcholine release from the rat prefrontal cortex and hippocampus (Eur. J. Pharmacol. 401 (2000) 179]. We report here that the active ingredient of cannabis preparations, delta9-tetrahydrocannabinol, administered at 10, 37.5, 75 and 150 microg/kg, dose dependently stimulated acetylcholine release from rat prefrontal cortex and hippocampus estimated by means of in vivo brain microdialysis with vertical concentric probes. At these doses, delta9-tetrahydrocannabinol induced behavioural stimulation. The administration of the CB1 receptor antagonist, ([N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3carboxamide]HCl) SR 141716A (200 microg/kg i.p.) significantly reduced the effect of delta9-tetrahydrocannabinol (75 microg/kg i.v.) on acetylcholine release from rat prefrontal cortex and hippocampus.