Acetaminophen Hepatotoxicity in Humans and Mice

Abstract

Acetaminophen (APAP) is a popular analgesic and antipyretic. Most of a therapeutic dose is glucuronidated or sulfated and excreted. A small amount is converted by cytochromes P450 to the reactive electrophile N-acetyl-p-benzoquinone imine (NAPQI). Fortunately, NAPQI can be detoxified by conjugation with glutathione (GSH). However, after an overdose the glucuronidation and sulfation pathways are overwhelmed, resulting in formation of excess NAPQI which depletes GSH and binds proteins. This causes mitochondrial dysfunction and oxidative stress. Oxidative stress activates the c-Jun N-terminal kinase, which translocates to mitochondria and exacerbates the injury. The result is hepatocyte death. Though well-established in mice, less work has been done with human models. Our goal was to further investigate the role of mitochondria in mice and to begin studying the mechanisms of hepatotoxicity in humans. A comparison of rats and mice supported the role of mitochondria in mice. Using the human liver cell line HepaRG, we found that protein binding, loss of mitochondrial potential, and oxidative stress preceded injury. Finally, using novel mechanistic plasma biomarkers, we have provided evidence that mitochondrial damage may also occur in APAP overdose patients, leading to oncotic necrosis. Recently, it was proposed that serum APAP-protein adducts can be used to diagnose APAP overdose. However, little work has been done to characterize the dose-response and timecourse of this parameter. We found that liver GSH depletion isn't required for protein binding in mice and that binding occurred without toxicity. Importantly, APAP-protein adducts could be measured in plasma without liver injury. The mechanism by which this occurs likely involves secretion of proteins adducted within hepatocytes, though other mechanisms couldn't be ruled out. Finally, liver injury caused by ischemia-reperfusion increased APAP-protein adducts in mouse plasma after a subtoxic dose. Our data support the use of APAP-protein adducts in plasma, but urge consideration of potential confounding factors.