Human multidrug resistance protein 4 (MRP4) is a cellular efflux transporter for paracetamol glutathione and cysteine conjugates

Jan B Koenderink,Ab Bilos,Nico P E Vermeulen,Galvin Vredenburg,Frans G M Russel,Jeroen J M W Van Den Heuvel

doi:10.1007/s00204-020-02793-4

Abstract

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.

Highlights

Paracetamol (N-acetyl-para-aminophenol, APAP or acetaminophen) is the most used over-the-counter pain reliever and fever reducer worldwide
The vesicular transport assay was used to determine if APAP, APAP-GSH, and APAP-CYS are transport substrates of MRP1-5, BCRP, P-gp or BSEP
Transport by all transporters followed simple Michaelis–Menten kinetics resulting in Km values for APAP-GSH by MRP1, MRP2 and Multidrug Resistance Protein 4 (MRP4) of 310 ± 70, 880 ± 240 and 190 ± 40 μM, respectively (Fig. 2), and for APAP-CYS by MRP4 of 190 ± 50 μM

Summary

Introduction

Paracetamol (N-acetyl-para-aminophenol, APAP or acetaminophen) is the most used over-the-counter pain reliever and fever reducer worldwide. It is a safe drug with minor side effects, but when overdosed it causes acute liver failure (McGill and Jaeschke 2013). Release of paracetamol adducts with therapeutic doses occurs in the absence of cell lysis (McGill et al 2011, 2013) and serum APAP-CYS concentrations were reported to remain below 1 μM When paracetamol is overdosed and hepatic necrosis occurs, adducts are released as a result of hepatocyte lysis and reach values up to 30 μM (Curry et al 2019). Several studies have shown that serum APAP-CYS could be a useful diagnostic marker for paracetamol overdose in cases of liver injury of unknown or uncertain cause (Curry et al 2019). The long half-life of NAPQI adducts (1–2 days) compared to the short serum half-life of paracetamol is making this a much better option than determining the serum paracetamol concentrations (Curry et al 2019)

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Results

Conclusion