Paracetamol (acetaminophen, APAP) overdose is a leading cause of acute drug-induced liver failure. APAP hepatotoxicity is mediated by the reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI). NAPQI is inactivated by conjugation with glutathione (GSH) to APAP-GSH, which is further converted into its cysteine derivative APAP-CYS. Before necrosis of hepatocytes occurs, APAP-CYS is measurable in plasma of the affected patient and it has been proposed as an early biomarker of acetaminophen toxicity. APAP-GSH and APAP-CYS can be extruded by hepatocytes, but the transporters involved are unknown. In this study we examined whether ATP-binding cassette (ABC) transporters play a role in the cellular efflux of APAP, APAP-GSH, and APAP-CYS. The ABC transport proteins P-gp/ABCB1, BSEP/ABCB11, BCRP/ABCG2, and MRP/ABCC1-5 were overexpressed in HEK293 cells and membrane vesicles were produced. Whereas P-gp, BSEP, MRP3, MRP5, and BCRP did not transport any of the compounds, uptake of APAP-GSH was found for MRP1, MRP2 and MRP4. APAP-CYS appeared to be a substrate of MRP4 and none of the ABC proteins transported APAP. The results suggest that the NAPQI metabolite APAP-CYS can be excreted into plasma by MRP4, where it could be a useful biomarker for APAP exposure and toxicity. Characterization of the cellular efflux of APAP-CYS is important for its development as a biomarker, because plasma concentrations might be influenced by drug-transporter interactions and upregulation of MRP4.

扑热息痛（对乙酰氨基酚，APAP）过量是急性药物性肝衰竭的主要原因。APAP的肝毒性由反应性代谢物N-乙酰基-p介导-苯醌亚胺（NAPQI）。NAPQI通过与谷胱甘肽（GSH）与APAP-GSH结合而失活，然后进一步转化为其半胱氨酸衍生物APAP-CYS。在肝细胞坏死发生之前，APAP-CYS在受影响患者的血浆中可测量，并且已被提议作为对乙酰氨基酚毒性的早期生物标志物。APAP-GSH和APAP-CYS可以被肝细胞挤出，但是所涉及的转运蛋白尚不清楚。在这项研究中，我们检查了ATP结合盒（ABC）转运蛋白是否在APAP，APAP-GSH和APAP-CYS的细胞外排中发挥作用。ABC转运蛋白P-gp / ABCB1，BSEP / ABCB11，BCRP / ABCG2和MRP / ABCC1-5在HEK293细胞中过表达，并产生膜囊泡。尽管P-gp，BSEP，MRP3，MRP5和BCRP没有转运任何化合物，但发现MRP1吸收了APAP-GSH，MRP2和MRP4。APAP-CYS似乎是MRP4的底物，没有ABC蛋白转运APAP。结果表明，NAPQI代谢产物APAP-CYS可通过MRP4排入血浆，这可能是APAP暴露和毒性的有用生物标记。APAP-CYS细胞外排的特性对其作为生物标记物的发展非常重要，因为血浆浓度可能受药物-转运蛋白相互作用和MRP4上调的影响。