Ainst intestinal and later also hepatic ischemia-reperfusion injury (Parks et al., 1982; Nordstrom et al., 1985). It was hypothesized that inhibition of xanthine oxidase by allopurinol, that is a hypoxanthine analogue, inhibits the post-ischemic oxidant anxiety and attenuates reperfusion injury. Even though this mechanism of protection has been questioned (Jaeschke, 2002), the reality remained that allopurinol effectively protected against reperfusion injury (Peglow et al., 2011). As well as ischemia-reperfusion models, allopurinol also protected against acetaminophen hepatotoxicity (Jaeschke, 1990; Tirmenstein and Nelson, 1990). These early studies demonstrated that the dose of allopurinol necessary to inhibit drug toxicity was 5 to 10-fold larger than the dose necessary to inhibit xanthine oxidase (Jaeschke, 1990). Also, depending on GSH depletion kinetics just after APAP overdose, allopurinol didn’t look to inhibit reactive metabolite formation (Jaeschke, 1990). These conclusions were supported by direct proof that allopurinol can be a poor P450 substrate and is mostly metabolized to oxypurinol inside the liver by aldehyde oxidase (Breithaupt and Tittel, 1982). Since allopurinol inhibited the mitochondrial oxidant stress and peroxynitrite formation just after APAP overdose (Jaeschke, 1990; Knight et al., 2001; Knight and Jaeschke, 2002), it was hypothesized that allopurinol acted as a radical scavenger. Having said that, this hypothesis was not confirmed (Zimmermann et al., 1988). Definitely from these findings the mechanism of protection isn’t just the potential of allopurinol to inhibit xanthine oxidase or scavenge reactive oxygen. Consequently, we hypothesized that allopurinol must alter intracellular signaling pathways or up-regulate the expression of cytoprotective genes. To test this hypothesis we utilised the in vivo mouse model of APAP overdose with and without the need of allopurinol pretreatment to investigate the early events in liver injury.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptToxicol Appl Pharmacol. Author manuscript; available in PMC 2015 February 01.Williams et al.PageMATERIALS AND METHODSAnimalsNIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptMale C3HeB/FeJ mice (82 weeks old) purchased from Jackson Laboratories (Bar Harbor, ME) were employed in our experiments.Brassicasterol MedChemExpress The mice have been kept in an environmentally controlled room using a 12 h light/dark cycle and cost-free access to meals and water. All experimental protocols have been approved by the Institutional Animal Care and Use Committee on the University of Kansas Medical Center and followed the criteria in the National Research Council for the care and use of laboratory animals.Shogaol supplier Experiment design All chemicals had been purchased from Sigma-Aldrich (St.PMID:24423657 Louis, MO) unless otherwise noted. Mice have been 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 had been fasted overnight and APAP was generally administered the following morning. Some mice had been treated with hydralazine (0.1 mg/mL) in 5mM potassium phosphate buffered drinking water, pH 6.0, that is around 30 mg hydralazine/kg/day. Mice were euthanized at 0h, 1h, 2h, 4h or 6h immediately after APAP injection then blood and livers had been harvested. Blood was drawn into a heparinized syringe to figure out alanine aminotransferase (ALT) activity (ALT Reagent Kit, Pointe Scientific, MI). The liver was removed and pieces were fixed in phosphat.
