Impaired protein adduct removal following repeat administration of subtoxic doses of acetaminophen enhances liver injury in fed mice

Acetaminophen (APAP) is a widely used analgesic and is safe at therapeutic doses. However, an overdose of APAP is hepatotoxic and accidental overdoses are increasingly common due to the presence of APAP in several combination medications. Formation of protein adducts (APAP-CYS) is central to APAP-induced liver injury and their removal by autophagy is an essential adaptive response after an acute overdose. Since the typical treatment for conditions such as chronic pain involves multiple doses of APAP over time, this study investigated APAP-induced liver injury after multiple subtoxic doses and examined the role of autophagy in responding to this regimen. Fed male C57BL/6J mice were administered repeated doses (75 mg/kg and 150 mg/kg) of APAP, followed by measurement of adducts within the liver, mitochondria, and in plasma, activation of the MAP kinase JNK, and markers of liver injury. The role of autophagy was investigated by treatment of mice with the autophagy inhibitor, leupeptin. Our data show that multiple treatments at the 150 mg/kg dose of APAP resulted in protein adduct formation in the liver and mitochondria, activation of JNK, and hepatocyte cell death, which was significantly exacerbated by inhibition of autophagy. While repeated dosing with the milder 75 mg/kg dose did not cause mitochondrial protein adduct formation, JNK activation, or liver injury, autophagy inhibition resulted in hepatocyte death even at this lower dose. These data illustrate the importance of adaptive responses such as autophagy in removing protein adducts and preventing liver injury, especially in clinically relevant situations involving repeated dosing with APAP.