Assessment of APAP-induced liver injury

APAP (Nacalai Tesque) was dissolved in 0.5% (w/v) methylcellulose (Shin-Etsu Chemical, Tokyo, Japan) solution, which was administered to the animals by oral gavage at 800 mg/kg, which is equivalent to twice the daily maximum dose in humans. The animals were divided into ALF (n=3), Fasting (n=5), and RF (n=5) groups according to the feeding conditions before APAP administration. APAP was orally administered to the ALF and Fasting groups at 09:00 and to the RF group at 16:00. After APAP administration, animals in the ALF and Fasting groups were fed freely from 10:00 and then euthanized after overnight fasting, and those in the RF group were given 14 g diet at 17:00 and euthanized after daytime fasting. Twenty-four hours after the administration of APAP, blood samples were drawn into tubes containing a serum-separating agent from an abdominal vein under isoflurane anesthesia (Terumo, Tokyo, Japan). Liver samples were immediately collected, weighed, fixed in 10% neutral-buffered formalin (Wako Pure Chemical Industries), and embedded in paraffin. Paraffin-embedded tissue sections were stained with hematoxylin and eosin (Wako Pure Chemical Industries) for histopathological analysis by light microscopy. Blood samples were centrifuged at 2,000 × g for 15 min at 4°C, and the serum was collected for the measurement of alanine transaminase (ALT) and aspartate transaminase (AST) levels by FUJI DRI-CHEM (Fujifilm, Tokyo, Japan).