Role of extracellular vesicles in release of protein adducts after acetaminophen-induced liver injury in mice and humans.

Formation of acetaminophen (APAP) protein adducts are a critical feature of APAP hepatotoxicity, and circulating protein adducts have recently been utilized in bioassays for identification of APAP overdose in humans. Despite their clinical significance, mechanisms of adduct release into the circulation are not well understood. Extracellular vesicles (EVs) are discrete membrane bound vesicles, which package cellular cargo and function in extracellular transport. Clarification of their role in transport of APAP adducts is relevant since adduct packaging within these vesicles could shield them from detection by antibody based methods, resulting in under-estimating adduct levels. Hence, this study evaluated EV release after APAP overdose in primary mouse hepatocytes and human HepaRG cells in vitro, in mice and APAP overdose patients in vivo and examined their role in transport of APAP-protein adducts. EVs were characterized by size and protein composition and the levels of APAP-protein adducts were measured. Significant elevations in circulating EV numbers were observed 6 h after APAP overdose in vivo and by 4 h in primary mouse hepatocytes in culture. EVs were also elevated in media from HepaRG cells by 24 h after APAP exposure, an effect recapitulated in APAP overdose patients, where EV numbers were elevated compared to healthy controls. Although APAP-protein adducts were elevated in circulation and media parallel to the increased exosome release, no detectable adducts were observed within EVs. This suggests that although APAP overdose enhances EV release from hepatocytes in mice and humans, it is not a significant mechanism of release of APAP protein adducts into circulation.