Antioxidant activity of the oyster mushroom, Pleurotus ostreatus, on CCl4-induced liver injury in rats

This study was undertaken to investigate the putative antioxidant activity of the oyster mushroom Pleurotus ostreatus on CCl₄-induced liver damage in male Wistar rats. Intraperitoneal administration of CCl₄ (2 ml/kg) to rats for 4 days resulted in significantly elevated (p < 0.05) serum levels of glutamic oxaloacetic transaminase (SGOT), glutamic pyruvate transaminase (SGPT) and alkaline phosphatase (SALP) compared to controls. In the liver, significantly elevated levels (p < 0.05) of malondialdehyde (MDA) and lowered levels (p < 0.05) of reduced glutathione (GSH) were observed following CCl₄ administration. Quantitative and qualitative analysis of catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (Gpx) revealed lower activities of these antioxidant enzymes in the liver of CCl₄-administered rats. An analysis of the isozyme pattern of these enzymes revealed variations in relative concentration presumably due to hepatotoxicity. When rats with CCl₄-induced hepatotoxicity were treated with the extract of P. ostreatus, the serum SGOT, SGPT and SALP levels reverted to near normal, while the hepatic concentration of GSH, CAT, SOD and Gpx were significantly increased (p < 0.05) and that of MDA significantly (p < 0.05) lowered, when compared to CCl₄-exposed untreated rats. Histopathological studies confirmed the hepatoprotective effect conferred by the extract of P. ostreatus. These results suggest that an extract of P. ostreatus is able to significantly alleviate the hepatotoxicity induced by CCl₄ in the rat.     

Effects of clofibrate and indocyanine green on the hepatobiliary disposition of acetaminophen and its metabolites in male CD-1 mice

1. The effects of clofibrate (CFB) and indocyanine green (ICG) on the biliary excretion of acetaminophen (APAP) and its metabolites were investigated. 2. Male CD-1 mice were pretreated with 500?mg CFB/kg, i.p. for 10 days. Controls received corn oil vehicle only. After overnight fasting, common bile duct-cannulated mice were challenged with a non-toxic dose of APAP (1 mmol/kg, i.v.). 3. CFB pretreatment did not affect bile flow rate, nor did it affect the cumulative biliary excretion of APAP and its conjugated metabolites. 4. Additional CFB or corn oil pretreated mice were given 30 μmol indocyanine green (ICG)/kg, i.v., immediately before APAP dosing. ICG is a non-metabolizable organic anion that is completely excreted into the bile through a canalicular transport process for organic anions. 5. ICG significantly decreased the bile flow rate and biliary concentration of APAPglutathione, APAP-glucuronide and APAP-mercapturate within the first hour after dosing without affecting the biliary concentration of APAP. 6. The results indicate thatCFB pretreatment does not affect the total amount of APAP and its metabolites excreted in bile. They also suggest that the biliary excretion of several conjugated metabolites of APAP share the same excretory pathway with the organic anion ICG.     

内毒素血症通过下调HNF4α表达加剧肝损伤

目的 探讨内毒素血症下调肝细胞核因子4α（HNF4α）表达介导肝损伤进展的机制。方法 144只BALB/c小鼠采用随机数字表法分组进行以下实验：（1）四氯化碳（CCl₄）诱导小鼠急性肝损伤。对照组和0.5 mL/kg、1.0 mL/kg、2.0 mL/kg CCl₄组。（2）筛选脂多糖（LPS）干预小鼠的剂量。对照组和0.1 mg/kg、0.5 mg/kg、2.5 mg/kg LPS组。（3）LPS干预CCl₄（1.0 mL/kg）诱导急性肝损伤模型小鼠。对照组、CCl₄组、0.1 mg/kg LPS+CCl₄组、0.5 mg/kg LPS+CCl₄组。每组12只。诱导24 h后处死小鼠,采用酶速率法检测血清丙氨酸转氨酶（ALT）,重氮法检测总胆红素（TBil）水平;Western blot法检测肝组织HNF4α、胱天蛋白酶3剪切体（Cleaved caspase-3）蛋白表达;原位末端标记法检测肝细胞凋亡情况。结果 0.5、1.0、2.0 mL/kg CCl₄组的血清ALT、TBil及肝组织HNF4α、Cleaved caspase-3蛋白表达水平高于对照组,且呈剂量依赖性增高。2.5 mg/kg LPS组血清ALT、TBil及肝组织Cleaved caspase-3蛋白表达水平高于对照组和0.1、0.5 mg/kg LPS组,肝组织HNF4α蛋白表达水平低于对照组和0.1、0.5 mg/kg LPS组（P<0.05）。CCl₄组和0.1、0.5 mg/kg LPS+CCl₄组的血清ALT、TBil,肝组织HNF4α、Cleaved caspase-3蛋白表达水平及肝细胞凋亡指数均高于对照组（P<0.05）;0.1、0.5 mg/kg LPS+CCl₄组的血清ALT、TBil,肝组织Cleaved caspase-3蛋白表达水平及肝细胞凋亡指数高于CCl₄组,HNF4α蛋白表达水平低于CCl₄组（P<0.05）。结论 内毒素血症通过下调HNF4α表达增加肝细胞凋亡,可能是其介导肝损伤进展的机制之一。     

Ameliorative action of cyanobacterial phycoerythrin on CCl(4)-induced toxicity in rats

Carbon tetrachloride (CCl₄) is largely used as solvent in chemical industries. Carbon tetrachloride is also well known for hepatic and renal toxic actions. The in vivo metabolism of carbon tetrachloride to trichloromethyl (CCl₃) and peroxy trichloromethyl (OOCCl₃) radicals has been extensively reported to cause acute liver damage like cirrhosis, steatosis and necrosis. We have evaluated protective action of purified cyanobacterial phycoerythrin (C-PE) on carbon tetrachloride-induced hepatic and renal toxicity in male rats. Rats were orally treated with 25 and 50 mg/kg BW of C-PE along with CCl₄ (50% CCl₄, 0.5 ml/kg BW, intraperitoneally) for 28 consecutive days. Results demonstrated that C-PE dose-responsively ameliorates CCl₄-toxicity by significantly decreasing (P < 0.05) organs weight, aminotransferases, alkaline phosphatase, glucose, lipid profile, creatinine, uric acid and malondialdehyde (MDA) concentrations with rise in body weight, food intake, hemoglobin, protein, bilirubin and FRAP values. Neither C-PE nor CCl₄ influenced on serum minerals. Hepatic and renal tissues showed significant decline (P < 0.05) in malondialdehyde, lipid hydroperoxides and conjugated dienes with rise in SOD, catalase, GPx, GSH, vitamin-E and vitamin-C levels. Presently observed pharmacological effect on CCl₄ toxicity were from tetrapyrrole molecule and to some extent bilirubin biotransformations, as well as metabolic (dietary protein) actions of C-PE.     

Ketoprofen glucuronidation and bile excretion in carbon tetrachloride and alpha-naphthylisothiocyanate induced hepatic injury rats

A pharmacokinetic study was carried out in rats to investigate the effects of experimental hepatic injury on the liver glucuronidation and bile excretion of ketoprofen (KP) and its glucuronides (KPGs). In vivo, KP (20 mg/kg b.w.) was intravenously administered to carbon tetrachloride (CCl₄) or alpha-naphthylisothiocyanate (ANIT) induced hepatic injury male rats. Concentrations of KP and its glucuronides (S-KPG and R-KPG) in plasma and bile were determined by RP-HPLC. It was observed that there was significant difference in the accumulative bile excretion of KPGs between the CCl₄ intoxicated rats and the normal rats (54 ± 18.3% versus 90 ± 6.9%), while it was extremely inhibited in ANIT intoxicated rats (2.0 ± 3.1% versus 90 ± 6.9%). As the result of reduction of KPGs excreted in bile, the area under the curve (AUC_(0–∞)) of KP and KPGs were higher in blood in CCl₄ and ANIT hepatic injury rats than those of the normal rats. Specifically, ANIT caused approximately 10-fold elevation of AUC_(0–∞) of plasma S-KPG. In microsomal incubations experiment, the glucuronyltransferase activity was impaired in CCl₄ and ANIT intoxicated rats. It suggested that the glucuronyltransferase activity was impaired in CCl₄ and ANIT intoxicated rats, while the bile excretion function was suppressed extremely in ANIT intoxicated rats.     

Transport deficient (TR-) hyperbilirubinemic rats are resistant to acetaminophen hepatotoxicity

The biliary excretion of acetaminophen (APAP) is reduced in transport deficient (TR⁻) hyperbilirubinemic rats lacking the multidrug resistance-associated protein 2 (Mrp2). This mutant strain of Wistar rats has impaired biliary excretion of organic anions and increased hepatic glutathione. The rational for this study was to determine if there is an altered risk for liver damage by APAP in the absence of Mrp2. Therefore, the susceptibility of TR⁻ rats to APAP hepatotoxicity was investigated. Male Wistar and TR⁻ rats were fasted overnight before APAP treatment (1 g/kg). Hepatotoxicity was assessed 24 h later by plasma sorbitol dehydrogenase activity and histopathology. In other studies, TR⁻ rats received buthionine sulfoximine before APAP to reduce hepatic glutathione to values similar to those in Wistar rats. mRNA expression of APAP metabolizing enzymes was also measured in naïve animals. Wistar rats treated with APAP showed significant elevations in plasma sorbitol dehydrogenase activity, while no increases in enzyme activity were observed in TR⁻ rats. Histopathology was in agreement. Hepatic non-protein sulfhydryls were significantly lower in Wistar rats receiving APAP than in TR⁻ rats. TR⁻ rats treated with buthionine sulfoximine and APAP showed dramatic increases in hepatotoxicity. TR⁻ rats had increased mRNA expression of several APAP metabolizing enzymes. Mrp2 expression not only is important in biliary excretion, but also influences the toxic potential of reactive intermediates by controlling intrahepatic GSH and possibly drug metabolism.     

Antifungal activity of a new benzothiazole derivative against Candida in vitro and in vivo

The antifungal activity of 6-amino-2-n-pentylthiobenzothiazole (APB) against 26 strains of the genus Candida in vitro was studied. Susceptibility of 17 strains was IC₅₀ ≤ 40 μmol/ml, of 7 strains IC₅₀ = 40−80 μmol/ml and of 2 strains IC₅₀ = 80−200 μmol/ml. Generalized candidosis of mice was treated with APB (doses 50, 100, 250 mg/kg) and ketoconazole (KET, 50 mg/kg of body weight). The optimal dose of APB was shown to be 100 mg/kg; 25% of mice survived after 14 days as compared to control animals. C. albicans was not found in the kidney of the sacrificed mice. 80% of mice survived after KET therapy. However, C. albicans was present in the kidney in an amount of 10⁵−10⁶ CFU/g of tissue. C. albicans did not reappear in the kidney 7 days after the discontinuation of APB treatment, but it was found there after KET therapy.     

Efficacy of 2,3-dimercapto-1-propanesulfonic acid (DMPS) and diphenyl diselenide on cadmium induced testicular damage in mice

The deleterious effect of acute cadmium-intoxication in mice testes was evaluated. Animals received a single dose of CdCl₂ (2.5 or 5 mg/kg, intraperitoneally) and a number of toxicological parameters in mice testes were examined, such as δ-aminolevulinic acid dehydratase (δ-ALA-D) activity, lipid peroxidation, hemoglobin and ascorbic acid contents. Furthermore, the parameters that indicate tissue damage such as plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) were also determined. Thus, a possible protective effect of 2,3-dimercapto-1-propane-sulfonic acid (DMPS) and diphenyl diselenide (PhSe)₂ were studied. The results demonstrated an inhibition of δ-ALA-D activity, a reduction of ascorbic acid and an increase of lipid peroxidation induced by cadmium, indicating testes damage. Furthermore, we observed an increase of plasma LDH, AST and ALT activities. DMPS (400 mol/kg) and (PhSe)₂ (100 μmol/kg) partially protected from the inhibitory effect of 2.5 mg/kg CdCl₂ on δ-ALA-D and from the increase of TBARS (thiobarbituric acid reactive species) levels. (PhSe)₂ therapy was effective in ameliorate ascorbic acid content when the cadmium dose was 2.5 mg/kg. Treatment with DMPS and (PhSe)₂, individually or combined, was inefficient in reducing cadmium-induced plasma LDH and ALT activity increase. The use of combined therapy (DMPS plus (PhSe)₂) proved to be efficient in decreasing cadmium levels in testes and in ameliorating plasma AST activity from animals that received the highest dose of cadmium.     

Effects of carbon tetrachloride on embryonic development studied in the post-implantation rat embryo culture system and in chick embryos in ovo

The effects of carbon tetrachloride on embryonic development were investigated in a mammalian and a non-mammalian system. In the former, whole-rat embryos, taken at 9.5 days of gestation, were exposed in vitro to different concentrations of CCl₄ (10, 100, 300, 600 and 1000 μg/ml) in rat serum with or without a rat liver microsomal activating system (S-9 mix). In the latter system, chick embryos in ovo were exposed to different concentrations of CCl₄ vapour (25, 35 and 75 ppm). When studied in the whole-rat embryo culture system without metabolic activation, concentrations of up to 300 μg CCl₄/ml had no effect on the overall development. Concentrations -600 μg CCl₄/ml affected the somite number, growth and morphology of the embryos, which can be interpreted as general toxicity. In the presence of S-9 mix, toxicity occurred at concentrations -300 μg/ml. In ovo exposure to CCl₄ showed that 25 ppm caused a decrease in the number of somites. At 35 ppm, CCl₄ induced further toxicity, as indicated by reduced somite number and growth and increased malformation rates. The results indicate that effects on morphogenic events appeared in both systems at concentration levels that also affected the overall development and that, independently of the choice of species or route of administration, CCl₄ has no potential to induce specific malformation patterns. The presence of a metabolic system in the rat embryo cultures approximately doubled the toxicity of CCl₄.     

Implication of alterations in intracellular calcium ion homoeostasis in the advent of paracetamol-induced cytotoxicity in primary mouse hepatocyte monolayer cultures

We have examined the fluctuation of free cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) using the fluorescent probe quin-2 during the cytotoxic response induced by low concentrations (100–250 μ ) of the model hepatotoxin paracetamol (APAP) in primary mouse hepatocyte cultures over 5 days. APAP-associated increases in [Ca²⁺]_i were recorded prior to APAP-associated cytotoxicity, and correlated with the subsequent loss of cell viability as measured by intracellular lactate dehydrogenase and K⁺ efflux. Co-incubation with promethazine (1 μ ) or ethyleneglycol-bis-(β-aminoethyl ether)-N,N,N′,N′-tetraacetic 0215 acid (4 m ) attenuated both the APAP-associated [Ca²⁺]_i changes and cytotoxicity. These results support the hypothesis that mobilization of intracellular Ca²⁺ may be an important early event in APAP-induced hepatotoxicity.     

Effects of clofibrate and indocyanine green on the hepatobiliary disposition of acetaminophen and its metabolites in male CD-1 mice

1. The effects of clofibrate (CFB) and indocyanine green (ICG) on the biliary excretion of acetaminophen (APAP) and its metabolites were investigated. 2. Male CD-1 mice were pretreated with 500 mg CFB/kg, i.p. for 10 days. Controls received corn oil vehicle only. After overnight fasting, common bile duct-cannulated mice were challenged with a non-toxic dose of APAP (1 mmol/kg, i.v.). 3. CFB pretreatment did not affect bile flow rate, nor did it affect the cumulative biliary excretion of APAP and its conjugated metabolites. 4. Additional CFB or corn oil pretreated mice were given 30 mumol indocyanine green (ICG)/kg, i.v., immediately before APAP dosing. ICG is a non-metabolizable organic anion that is completely excreted into the bile through a canalicular transport process for organic anions. 5. ICG significantly decreased the bile flow rate and biliary concentration of APAP-glutathione, APAP-glucuronide and APAP-mercapturate within the first hour after dosing without affecting the biliary concentration of APAP. 6. The results indicate that CFB pretreatment does not affect the total amount of APAP and its metabolites excreted in bile. They also suggest that the biliary excretion of several conjugated metabolites of APAP share the same excretory pathway with the organic anion ICG.     

NCX 6560, a nitric oxide-releasing derivative of atorvastatin, inhibits cholesterol biosynthesis and shows anti-inflammatory and anti-thrombotic properties

We compared the lipid-lowering, vasodilating, anti-thrombotic and anti-inflammatory properties of NCX 6560, a novel NO-releasing derivative of atorvastatin, with those of atorvastatin. NCX 6560 and atorvastatin induced similar inhibition of cholesterol biosynthesis in rat smooth muscle cells (IC₅₀ = 1.9 ± 0.4 and 3.9 ± 1.0 μM, respectively). However, in hyperlipidemic mice, a 5-week oral treatment with NCX 6560 (46.8 mg/kg/day, p.o.) was more effective than equivalent atorvastatin (40 mg/kg/day, p.o.) at lowering serum cholesterol (NCX 6560: − 21% vs controls, P < 0.05; atorvastatin: − 14% vs control, P = NS). In norepinephrine-precontracted rabbit aortic rings, NCX 6560-induced vasodilation (EC₅₀ = 53.5 ± 8.3 μM) and in PC12 cells it stimulated cGMP formation (EC₅₀ = 1.8 ± 0.7 μM), while atorvastatin was inactive. In lipopolysaccharide from Escherichia coli (LPS)-treated RAW 264.7 macrophages, NCX 6560 reduced iNOS expression and dimer assembly more efficiently than atorvastatin and inhibited nitrite accumulation (IC₅₀ = 6.7 ± 1.6 μM) and TNF release. U46619- or collagen plus epinephrine-induced platelet pulmonary thromboembolism in mice was reduced by NCX 6560 at 46.8 mg/kg p.o. (mortality: − 44% and − 56% vs vehicle, respectively; P < 0.05), but not by atorvastatin 40 mg/kg, p.o. In the U46619-induced mortality model, isosorbide mononitrate (ISMN) (20 mg/kg, p.o.), a pure NO-donor, was also active (mortality: − 40%, P < 0.05). NCX 6560 significantly reduced ex vivo platelet adhesion to collagen at high shear (− 31 ± 1.3% vs vehicle), and so did ISMN (− 33.3 ± 1.7% vs vehicle). Atorvastatin was ineffective. NCX 6560, but not atorvastatin, reduced blood pressure in eNOS knockout mice (− 16%, P < 0.001 vs vehicle), an effect not observed in wild type mice. On the contrary, ISMN provoked a significant drop of blood pressure both in wild type (− 20%, P < 0.05 vs vehicle) and in eNOS−/− mice (− 21%, P < 0.05 vs vehicle). In conclusion, NCX 6560 exerts greater lipid-lowering, anti-thrombotic and anti-inflammatory effects than atorvastatin, due to a large extent to NO release.     

Partial structural determination of hepatotoxic peptides from Microcystis aeruginosa (cyanobacterium) collected in ponds of central China

, , , and . Partial structural determination of hepatotoxic peptides from Microcystis aeruginosa (cyanobacterium) collected in ponds of central China. Toxicon 26, 1213–1217, 1988.—Waterbloom samples of the colonial cyanobacterium Microcystis aeruginosa, collected in fish ponds at the Hydrobiological Institute, Wuhan, People's Republic of China, were hepatotoxic to mice. Lyophilized cells had an ₅₀ (i.p. mouse; 40 mg/kg) and signs of poisoning similar to that reported for other cyanobacterial hepatotoxic peptides. Two toxins, with an ₅₀ (i.p. mouse) of 40 and 150 μg/kg, were isolated using gel filtration and high performance liquid chromatography. The amino acid composition and mol.wt (994) of the 40 μg/kg toxin was the same as that for microcystin-LR, while the 150 μg/kg toxin had an amino acid composition and mol.wt (1048) different from any of the reported cyanobacteria heptapeptide toxins reported to date.     

Polysaccharides isolated from Ganoderma lucidum occurring in Southern parts of India, protects radiation induced damages both in vitro and in vivo

The in vivo and in vitro radioprotective property of the polysaccharides isolated from Ganoderma lucidum were determined by survival studies, induction of micronucleus in reticulocytes of mice, strand breaks in plasmid pBR322 DNA and inhibition of lipid peroxidation (TBARS assay). Polysaccharides were administered as a single dose after whole body exposure to 10 Gy ⁶⁰Co γ-radiation to Swiss albino mice. At a dose of 500 μg/kg body wt, the polysaccharides were most effective in protecting animals from radiation induced loss of lethality. Administration of 500 μg/kg body wt to animal exposed to 10 Gy gamma radiation resulted in more than 60% survival on the 30th day compared to the dose of 300 mg/kg/body wt administration of amifostine, a clinically used radioprotective drug. The induction of micronuclei was reduced by the administration of polysaccharides. The decrease in micronuclei induction was dose dependent. Thus following 4 Gy exposure the micronuclei in polychromatic erythrocytes (MNCE) was reduced from 28.16 ± 3.049 to 16.0243 ± 2.074 and 6.30 ± 2.422 by polysaccharides at doses of 250 μg/kg body wt and 500 μg/kg body wt, respectively, and to 10.4 ± 2.581 by amifostine at a dose of 300 mg/kg body wt. The results indicate the significant protective effect of Ganoderma polysaccharides against radiation induced damages. The findings thus suggest the potential use of Ganoderma polysaccharides as novel radioprotective agent.     

Green tea extract can potentiate acetaminophen-induced hepatotoxicity in mice

Green tea extract (GTE) has been advocated as a hepatoprotective compound and a possible therapeutic agent for acetaminophen (APAP) overdose. This study was conducted to determine if GTE can provide protection against APAP-induced hepatotoxicity. Three different exposure scenarios were tested. The first involved administering APAP (150 mg/kg, orally) to mice followed 6 h later by GTE (500 or 1000 mg/kg). The other two involved administering GTE prior to the APAP dose. GTE (500 or 1000 mg/kg, orally) was administered 3 h prior to APAP (200 mg/kg, orally) or for three consecutive days (once-daily) followed by APAP (300 mg/kg) on the fourth day. Indices of hepatotoxicity were assessed 24 h after the APAP dose. GTE potentiated APAP-induced hepatotoxicity when administered after the APAP dose. GTE caused significant glutathione depletion and this effect likely contributed to the observed potentiation. In contrast, GTE provided protection against APAP-induced hepatotoxicity when administered prior to the APAP dose. GTE dramatically decreased APAP covalent binding to protein indicating that less reactive metabolite was available to cause hepatocellular injury. These results highlight the potential for drug-dietary supplement interactions and the importance of testing multiple exposure scenarios to adequately model different types of potential interactions.     

Interspecies differences in acetaminophen sensitivity of human, rat, and mouse primary hepatocytes

Most of the experiments studying acetaminophen (APAP) induced hepatotoxicity were performed using moue as model specie, right because its high sensitivity. While the toxic responses can be called forth easily in mice, the human relevancy of these results is questionable. In this study human, rat, and mouse primary hepatocytes were treated with increasing concentrations of APAP, and cell viability was measured by MTT cytotoxicity assay. Pronounced interspecies differences were obtained in cell viability following 24 h of APAP treatment starting at 24 h after seeding (EC₅₀: 3.8 mM, 7.6 mM, and 28.2 mM, in mouse, rat, and human hepatocyte culture, respectively). The longer time of culturing highly increased the resistance of hepatocytes of all species investigated. In rat hepatocyte culture EC₅₀ values were 6.0 mM, 12.5 mM, and 18.8 mM, when starting APAP treatment after 24, 48, and 72 h of seeding. Although N-acetylbenzoquinoneimine, a minor metabolite of APAP, which is mainly formed by CYP2E1 at high APAP concentration in every species studied, is thought to initiate the toxic processes, no correlation was found between CYP2E1 activities and hepatocyte sensitivity of different species. We conclude that the toxicity induced by APAP overdose highly depends on the animal model applied.     

Changes in susceptibility to acetaminophen-induced liver injury by the organic anion indocyanine green.

The non-metabolizable organic anion indocyanine green (ICG) has been shown previously to reduce markedly the biliary secretion of acetaminophen, particularly the glutathione conjugate of APAP (APAP-GSH), suggesting that this APAP metabolite may compete with other xenobiotics for excretion into the bile via a canalicular organic anion transport process. This study was conducted to determine whether changes in the biliary disposition of APAP induced by ICG could lead to alterations in susceptibility to APAP hepatotoxicity. To investigate this, groups of overnight-fasted male CD-1 mice received 30 micromol ICG/kg, intravenously, immediately prior to APAP dosing (500 mg/kg, ip). Controls were given propylene glycol vehicle. Mice were killed at 4 h after APAP challenge for immunochemical analysis of cytosolic protein arylation and determination of non-protein sulfhydryl (NPSH) depletion, or at 12 and 24 h for biochemical and histological assessment of liver injury. Elevated plasma sorbitol dehydrogenase activity and centrilobular hepatocellular necrosis was present in control mice receiving APAP at 12 and 24 h. Treatment with ICG did not alter susceptibility to APAP toxicity when measured at 12 h after challenge. However, the severity of histologic lesions in the ICG-APAP group was significantly lower at 24 h after challenge. Furthermore, treatment with ICG did not alter APAP-induced glutathione depletion or cytosolic protein arylation. These data suggest that the organic anion ICG has a protective effect on APAP toxicity that promotes a faster recovery from liver injury.     

Hepatic subcellular distribution of manganese in manganese and manganese-bilirubin induced cholestasis

Administration of non-cholestatic doses of manganese (Mn2+) followed by injection of bilirubin (BR) results in a severe reduction in rat bile flow. Male Sprague-Dawley rats were given various doses of Mn2+ (2, 4.5, 8, and 18 mg/kg, i.v.) and killed 0.25, 1, 3, or 5 hr later. 54Mn2+ was used to evaluate Mn2+ content (micrograms/g protein) in different liver fractions: homogenate, mitochondria, microsomes, cytoplasm, nuclei-membrane fraction and liver cell plasma membrane fractions, one containing bile canalicular complexes (LCPM-BCM), the other containing sinusoidal membranes (LCPM-PM). In LCPM-BCM and LCPM-PM, two time-related patterns of Mn2+ content were observed. With non-cholestatic doses (2, 4.5, and 8 mg/kg), Mn2+ content decreased with time and rarely exceeded 50 micrograms/g protein. With 18 mg/kg (a cholestatic dose), Mn2+ content increased with time and reached values over 100 micrograms/g protein (3-5 hr), reflecting possible modification in membrane structure. BR caused a marked increase in Mn2+ content (at a dose of 4.5 mg Mn2+/kg) in LCPM-BCM (240%), approaching values seen with 18 mg Mn2+/kg, whereas in LCPM-PM it was less striking (50%). These and other results obtained with various treatments (cholestatic and non-cholestatic) suggest than Mn2+ concentration in bile canalicular membranes is a critical factor in both forms of cholestasis, and that BR can facilitate Mn2+ incorporation in the bile canalicular membrane.     

Role of CYP3A and CYP2E1 in alcohol-mediated increases in acetaminophen hepatotoxicity: comparison of wild-type and Cyp2e1(-/-) mice.

CYP2E1 is widely accepted as the sole form of cytochrome P450 responsible for alcohol-mediated increases in acetaminophen (APAP) hepatotoxicity. However, we previously found that alcohol [ethanol and isopentanol (EIP)] causes increases in APAP hepatotoxicity in Cyp2e1(-/-) mice, indicating that CYP2E1 is not essential. Here, using wild-type and Cyp2e1(-/-) mice, we investigated the relative roles of CYP2E1 and CYP3A in EIP-mediated increases in APAP hepatotoxicity. We found that EIP-mediated increases in APAP hepatotoxicity occurred at lower APAP doses in wild-type mice (300 mg/kg) than in Cyp2e1(-/-) mice (600 mg/kg). Although this result suggests that CYP2E1 has a role in the different susceptibilities of these mouse lines, our findings that EIP-mediated increases in CYP3A activities were greater in wild-type mice compared with Cyp2e1(-/-) mice raises the possibility that differential increases in CYP3A may also contribute to the greater APAP sensitivity in EIP-pretreated wild-type mice. At the time of APAP administration, which followed an 11 h withdrawal from the alcohols, alcohol-induced levels of CYP3A were sustained in both mouse lines, whereas CYP2E1 was decreased to constitutive levels in wild-type mice. The CYP3A inhibitor triacetyloleandomycin (TAO) decreased APAP hepatotoxicity in EIP-pretreated wild-type and Cyp2e1(-/-) mice. TAO treatment in vivo resulted in inhibition of microsomal CYP3A-catalyzed activity, measured in vitro, with no inhibition of CYP1A2 and CYP2E1 activities. In conclusion, these findings suggest that both CYP3A and CYP2E1 contribute to APAP hepatotoxicity in alcohol-treated mice.     

Histamine H3 receptor modulates nociception in a rat model of cholestasis

Cholestasis is associated with changes including analgesia. The histaminergic system regulates pain perception. The involvement of histamine H₃ receptors in modulation of nociception in a model of elevated endogenous opioid tone, cholestasis, was investigated in this study using immepip and thioperamide as selective H₃ receptor agonist and antagonist respectively. Cholestasis was induced by ligation of main bile duct using two ligatures and transsection the duct between them. Cholestatic rats had increased tail-flick latencies (TFLs) compared to non-cholestatics. Administration of immepip (5 and 30 mg/kg) and thioperamide (10 and 20 mg/kg) to the cholestatic groups significantly increased and decreased TFLs compared to the saline treated cholestatic group. Immepip antinociception in cholestatic animals was attenuated by co-administration of naloxone. Immepip and thioperamide injections into non-cholestatic animals did not alter TFLs. At the doses used here, none of the drugs impaired motor coordination, as revealed by the rotarod test. The present data show that the histamine H₃ receptor system may be involved in the regulation of nociception during cholestasis in rats.