Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS, USA.
Department of Medical Toxicology, Banner - University Medical Center Phoenix, Phoenix, AZ, USA; Department of Medicine, and Division of Clinical Data Analytics and Decision Support, University of Arizona College of Medicine - Phoenix, Phoenix, AZ, USA.
Toxicol Lett. 2019 Feb;301:125-132. doi: 10.1016/j.toxlet.2018.11.005. Epub 2018 Nov 15.
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.
乙酰氨基酚（APAP）蛋白加合物的形成是 APAP 肝毒性的一个关键特征，最近循环蛋白加合物已被用于生物测定法以鉴定人类的 APAP 过量。尽管它们具有临床意义，但加合物释放到循环中的机制尚未得到很好的理解。细胞外囊泡（EVs）是离散的膜结合囊泡，其包装细胞货物并在细胞外运输中发挥作用。阐明它们在 APAP 加合物转运中的作用很重要，因为这些囊泡中加合物的包装可能会使它们免受基于抗体的方法的检测，从而导致低估加合物水平。因此，本研究评估了 APAP 过量后在原代小鼠肝细胞和人 HepaRG 细胞中的 EV 释放，在小鼠和 APAP 过量患者体内，并研究了它们在 APAP-蛋白加合物转运中的作用。通过大小和蛋白质组成对 EV 进行了表征，并测量了 APAP-蛋白加合物的水平。在体内 APAP 过量后 6 小时和在原代小鼠肝细胞培养物中 4 小时观察到循环 EV 数量的显着增加。在 HepaRG 细胞暴露于 APAP 后 24 小时的培养基中，EV 也升高，这在 APAP 过量患者中得到了重现，与健康对照组相比，EV 数量升高。尽管循环中和培养基中的 APAP-蛋白加合物与外泌体释放的增加平行升高，但在 EV 内未观察到可检测的加合物。这表明，尽管 APAP 过量会增强小鼠和人类肝细胞中 EV 的释放，但它不是将 APAP 蛋白加合物释放到循环中的重要机制。
