In each case, a negative control (non-immune serum) was used

In each case, a negative control (non-immune serum) was used. formation, 3-nitrotyrosine protein adducts, and inducible nitric oxide synthase were increased in the Jo2 plus ethanol group. The enhanced hepatotoxicity of Jo2 plus ethanol and the elevated oxidative stress and TNF levels were lower in CYP2E1 knockout mice compared to wild-type mice expressing CYP2E1 but higher than saline controls. Toxicity also declined in mice treated with gadolinium chloride, an inhibitor of the inducible nitric oxide synthase or the antioxidant, N-acetyl-L-cysteine. These data show acute ethanol pretreatment is usually capable of elevating hepatic apoptosis and liver injury induced by Jo2 Fas Loxapine Succinate agonistic antibody. The enhanced hepatotoxicity entails increased oxidative and nitrosative stress, and appears to be mediated by CYP2E1-dependent but also CYP2E1-impartial mechanisms. apoptosis detection kit (Serological Corp., Atlanta, GA). Under light microscopy the numbers of TUNEL positive cells were counted per high-powered field (400 magnification) with 20 random fields counted per liver. Caspase-8 and caspase-3 activities were decided in liver tissue homogenates by measuring proteolytic cleavage of the proluminescent substrates Z-IETD-AFC or AC-DEVD-AMC (Calbiochem, La Jolla, CA). The fluorescence was decided based on the amount of released AFC (caspase-8, ex=400, em=505) or AMC (caspase-3, ex=380, em=460). The results were expressed as arbitrary models of fluorescence (AUF) per milligram of protein. Treatment with Potential Protectants against Jo2 plus Ethanol Toxicity Male C57BL/6 mice, weighing 20C23 g, were divided into groups which received either saline (Sal group, n=4) or Jo2 following ethanol pretreatment (Eth/Jo2 group, n=8) or Jo2 following ethanol pretreatment plus potential protectants: gadolinium chloride (50 mg/kg body weight) (Eth/Jo2/GdCl3 group, n=10); pentoxifylline Loxapine Succinate (100mg/kg) (Eth/Jo2/PTX group, n=10); N-[(3-aminomethyl) benzyl] acetamidine (10 mg/kg body weight) (Eth/Jo2/1400W group, n=10) or N-acetyl-L-cysteine (150 mg/kg body weight) (Eth/Jo2/NAC group, n=10), respectively. Mice were injected intraperitoneally with the above potential protectants 30 min prior to Jo2 treatment. At 10 hours after administration of Jo2 or saline, mice were sacrificed for collecting serum and liver tissue for ALT, TNF- assay and pathological observation. GdCl3 was injected IP since this mode of administration was effective in preventing concanavalin- or endotoxin-induced liver injury [34,35]. Jo2 Toxicity in CYP2E1-Null Mice CYP2E1 knockout (?/?) mice, values of less than 0.05 were considered statistically significant. The number of mice is usually indicated in the Story to Figures. Results Serum Transaminases and Liver Pathological Changes In the Eth/Jo2 group, serum ALT and AST activities were significantly higher than that in the Jo2 or Eth alone group. In the Jo2 or Eth alone group, serum ALT and AST activities were slightly but not Loxapine Succinate significantly higher than that in the Sal (Fig.1A, 1B). Severe pathological changes were observed in the Eth/Jo2 group in which many hepatocytes displayed massive acidophilic necrosis or apoptosis, cytolysis and focal infiltration of inflammatory cells in the central zone of the hepatic lobule (Fig.1C4). There was only moderate or moderate pathological changes Loxapine Succinate in hepatocytes including limited necrosis in the Jo2 group (Fig.1C2) and vacuolar degeneration in the Eth group (Fig.1C3). In the Sal group, there were no obvious pathological changes (Fig.1C1). Open in a separate windows Fig.1 Levels of serum ALT, AST and histopathological changes of liver after treatment with Jo2 plus ethanol. (A) serum ALT and (B) serum AST. (C1) Liver showed normal morphology (HE200). (C2) Liver showed slightly sinusoid dilation and congestion, limited acidophilic degeneration and Loxapine Succinate necrosis of hepatocytes in the centrilobular zone (arrows, HE200). (C3) Liver showed slight congestion and large amounts of microvesicular fatty degeneration (arrows, HE200). (C4) Liver showed considerable acidophilic necrosis, focal hemorrhages, cytolysis and focal infiltration of inflammatory cells in the central zone of the hepatic lobule (arrows, HE200). Data are the meanSEM for 6C8 mice. ** and ## compared to Jo2 or to Eth groups, apoptosis detection kit. * significantly different from the Jo2 group, of 4-HNE was mainly found in cytoplasm of hepatocytes in the centrilobular zone of the liver. The 4-HNE levels were highest in the Eth/Jo2 group (+++) (Fig.3D3); smaller increases were also found in the Jo2 (++) (Fig.3D1) and Eth group (+) (Fig.3D2). Total GSH levels decreased in the Jo2 (?29%), Eth (?39%) and Eth/Jo2 (?48%) groups compared to the Sal group. GSH levels were least expensive in the Eth/Jo2 group, but there were no significant differences in the Eth/Jo2 group compared to the Eth group (Fig.3E). Catalase activity did not switch Rabbit Polyclonal to Collagen XII alpha1 in the Jo2, Eth or Eth/Jo2 group compared to the Sal group (data not shown). Open in a separate windows Fig.3 Protein carbonyl, 3-NT protein adducts, lipid peroxidation and GSH levels. (A) The protein carbonyl level was assayed in liver homogenates using 20 g of protein.