11 Interestingly, a connection between PPARα and APAP toxicity was established when it was discovered that pretreatment
with clofibrate, a PPARα activator, INCB024360 protected mice against APAP-induced hepatotoxicity12, 13 and that this protection was PPARα-dependent.14 Furthermore, it was recently reported that toxic doses of APAP inhibit fatty acid β-oxidation and that these effects were significantly reduced in mice lacking the major enzyme responsible for the bioactivation of APAP, CYP2E1, due, in part, to enhanced and persistent activation of PPARα and its target genes.15 Wildtype mice treated with APAP, however, showed suppressed PPARα activity. Thus, PPARα may function to protect mitochondria from ROS that occurs during APAP metabolism and as a natural consequence during fatty acid catabolism. In the present study the protective effects of PPARα activation during APAP-induced hepatotoxicity were further investigated and a role for the PPARα target gene UCP2 in mediating these protective effects explored. ALT, alanine aminotransferase;
APAP, acetaminophen; selleck kinase inhibitor AST aspartate aminotransferase; GSH, glutathione; NAPQI, N-acetyl-p-benzoquinone imine; PPARα, peroxisome proliferator-activated receptor alpha; ROS, reactive oxygen species; UCP2, uncoupling protein 2. Wildtype (C57Bl/6J) and ucp2-null (B6.129-Ucp2tm1Low1/J) mice were obtained from the Jackson Laboratories (Bar Harbor, ME). Ppara-null mice and wildtype counterparts on the 129/Sv background were described previously.16 The PPARα-humanized mouse was described previously.17 All animal experiments were carried out in accordance with the Institute of Laboratory Animal Resources guidelines and approved by the National Cancer Institute Animal Care and Use
Committee. Groups of 6 to 8-week-old male mice were fed Wy-14,643 (0.1%) diet for 24 hours before an intraperitoneal injection of APAP (400 mg/kg) dissolved in saline. All mice were euthanized by CO2 asphyxiation 2 hours, 6 hours, or 24 hours after the APAP dose. Livers were harvested and stored at −80°C before analysis. To MCE公司 assess liver damage, tissue was briefly washed with phosphate-buffered saline (PBS) and fixed in 10% neutral buffered formalin. Necrosis was scored by hematoxylin and eosin (H&E) staining. APAP-induced liver injury was determined by measuring aspartate aminotransferase (AST) and alanine aminotransferase (ALT) catalytic activities in serum using a commercial AST or ALT assay kit (Catachem, Bridgeport, CT). Reduced glutathione (GSH) levels in liver were measured by a glutathione assay kit (Sigma-Aldrich, St. Louis, MO) and liver hydrogen peroxide (H2O2) levels were determined by use of the Peroxidetect kit (Sigma-Aldrich).