In the National Study Council for the care and use of laboratory animals. Experiment style All chemicals have been bought from Sigma-Aldrich (St. Louis, MO) unless otherwise noted. Mice were treated with allopurinol or oxypurinol (one hundred mg/kg in water, p.o.) 18h or 1h prior to APAP (300 mg/kg in warm saline, i.p.) administration. Mice have been fasted overnight and APAP was often administered the following morning. Some mice have been treated with hydralazine (0.1 mg/mL) in 5mM potassium phosphate buffered drinking water, pH 6.0, which can be around 30 mg hydralazine/kg/day. Mice had been euthanized at 0h, 1h, 2h, 4h or 6h right after APAP injection and after that blood and livers had been harvested. Blood was drawn into a heparinized syringe to ascertain alanine aminotransferase (ALT) activity (ALT Reagent Kit, Pointe Scientific, MI). The liver was removed and pieces had been fixed in phosphatebuffered formalin or utilised for mitochondrial isolation. The rest in the liver was snap-frozen in liquid nitrogen and subsequently stored at -80 . Isolation of subcellular fractions The best and caudate lobes from the liver were minced and mechanically disrupted in ice cold isolation buffer (pH 7.four, containing 22 mM mannitol, 70 mM sucrose, 2.5 mM HEPES, 10 mM EDTA, 1 mM EGTA, and 0.1 BSA) with 15 strokes of a tight-fitting motorized Teflon pestle. Cell debris was removed by spinning the homogenate at two,500 x g for 10 min. The resulting supernatant was then centrifuged at 20,000 x g for 10 min to pellet mostly mitochondria. This supernatant was saved because the cytosolic fraction. The mitochondria pellet was washed with isolation buffer, re-pelleted and flash frozen in liquid nitrogen. Both the cytosolic and also the mitochondrial fractions had been stored at -80 . Histology Formalin-fixed tissue samples had been embedded in paraffin and five m sections had been reduce and stained with hematoxylin and eosin (H E) for evaluation of liver necrosis. Aldehyde oxidase (AO) activity assay Liver tissue AO activity was determined spectrophotometrically by utilizing dimethylaminocinnamaldehyde (DMAC) as a substrate at 398nm.NOTA-NHS ester Price The method was performed as previously described (Swenson and Casida, 2013). APAP-cysteine adduct measurement APAP-protein adducts in liver tissue was measured by high-pressure liquid chromatography with electrochemical detection (HPLC-ECD) based on the approach of Muldrew et al. (2002) with modifications (Ni et al., 2012). Western blotting Western blotting was performed applying mouse anti-metallothionein (Dako, Carpinteria, CA), rabbit anti-JNK and rabbit anti-phospho-JNK antibodies (Cell Signaling Technologies,Toxicol Appl Pharmacol.5-Fluorobenzofuran-4-carbaldehyde site Author manuscript; obtainable in PMC 2015 February 01.PMID:23935843 Williams et al.PageDanvers, MA) with horseradish peroxidase-coupled anti-mouse or anti-rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA), as described in detail (Bajt et al., 2000).NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptMeasurement of glutathione Glutathione (GSH) and glutathione disulfide (GSSG) levels in liver tissue were determined making use of a modified Tietze assay (Jaeschke and Mitchell, 1990). In short, tissue was homogenized in three sulfosalicylic acid and centrifuged to get rid of precipitated proteins. Immediately after further dilution with potassium phosphate buffer, the samples have been then assayed having a cycling reaction using glutathione reductase and dithionitrobenzoic acid. To decide GSSG, decreased GSH was initial trapped and removed with N-ethylmaleimide then assayed similarly.
