The modulation of transcriptional expression and inhibition of multidrug resistance associated protein 4 (MRP4) by analgesics and their primary metabolites

Renato J Scialis; Carolina I Ghanem; José E Manautou

doi:10.1016/j.crtox.2020.04.002

The modulation of transcriptional expression and inhibition of multidrug resistance associated protein 4 (MRP4) by analgesics and their primary metabolites

Curr Res Toxicol. 2020 Apr 30:1:34-41. doi: 10.1016/j.crtox.2020.04.002. eCollection 2020 Jun 10.

Authors

Renato J Scialis^{1

2

3}, Carolina I Ghanem^{1

2

3}, José E Manautou^{1

2

3}

Affiliations

¹ University of Connecticut, School of Pharmacy, Department of Pharmaceutical Sciences, Storrs, CT 06269, USA.
² Instituto de Investigaciones Farmacológicas (ININFA), Facultad de Farmacia y Bioquímica, CONICET, Universidad de Buenos Aires, Buenos Aires, Argentina.
³ Cátedra de Fisiopatología, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Buenos Aires, Argentina.

Abstract

During the course of a toxic challenge, changes in gene expression can manifest such as induction of metabolizing enzymes as a compensatory detoxification response. We currently report that a single 400 mg/kg acetaminophen (APAP) dose to C57BL/6J mice led to an increase in multidrug resistance-associated (Mrp) 4 (Abcc4) mRNA 12 h after administration. Alanine aminotransferase, as a marker of liver injury, was also elevated indicating hepatotoxicity had occurred. Therefore, induction of Mrp4 mRNA was likely attributable to APAP-induced liver injury. Mrp4 has been shown to be upregulated during oxidative stress, and it is well-established that APAP overdose causes oxidative stress due to depletion of glutathione. Given the importance of Mrp4 upregulation as an adaptive response during cholestatic and oxidative liver injury, we next investigated the extent by which human MRP4 can be inhibited by the analgesics, APAP, diclofenac (DCF), and their metabolites. Using an in vitro assay with inside out human MRP4 vesicles, we determined that APAP-cysteine inhibited MRP4-mediated transport of leukotriene C₄ with an apparent IC₅₀ of 125 μM. APAP-glutathione also attenuated MRP4 activity though it achieved only 28% inhibition at 300 μM. Diclofenac acyl glucuronide (DCF-AG) inhibited MRP4 transport by 34% at 300 μM. The MRP4 in vitro inhibition occurs at APAP-cysteine and DCF-AG concentrations seen in vivo after toxic doses of APAP or DCF in mice, hence the findings are important given the role that Mrp4 serves as a compensatory response during oxidative stress following toxic challenge.

Keywords: ALT, alanine aminotransferase; AMP, adenosine monophosphate; APAP, acetaminophen; APAP-CYS, acetaminophen cysteine; APAP-GLU, acetaminophen glucuronide; APAP-NAC, acetaminophen N-acetylcysteine; APAP-SUL, acetaminophen sulfate; ATP, adenosine triphosphate; Acetaminophen; DCF, diclofenac; DCF-AG, diclofenac acyl glucuronide; Diclofenac; Fmo, flavin containing monooxygenase; IS, internal standard; Inhibition; LTC4, leukotriene C4; MRP, multidrug resistance-associated protein; MRP4; Metabolite; OH-DCF, 4′-hydroxy diclofenac; PGE2, prostaglandin E2.