Molecular mechanisms of hepatotoxic cholestasis by clavulanic acid: Role of NRF2 and FXR pathways

Treatment of β-lactamase positive bacterial infections with a combination of amoxicillin (AMOX) and clavulanic acid (CLAV) causes idiosyncratic drug-induced liver injury (iDILI) in a relevant number of patients, often with features of intrahepatic cholestasis. This study aims to determine serum bile acid (BA) levels in amoxicillin/clavulanate (A+C)-iDILI patients and to investigate the mechanism of cholestasis by A+C in human in vitro hepatic models. In six A+C-iDILI patients, significant elevations of serum primary conjugated BA definitely demonstrated A+C-induced cholestasis. In cultured human Upcyte hepatocytes and HepG2 cells, CLAV was more cytotoxic than AMOX, and, at subcytotoxic concentrations, it altered the expression of more than 1,300 genes. CLAV, but not AMOX, downregulated the expression of key genes for BA transport (BSEP, NTCP, OSTα and MDR2) and synthesis (CYP7A1 and CYP8B1). CLAV also caused early oxidative stress, with reduced GSH/GSSG ratio, along with induction of antioxidant nuclear factor erythroid 2-related factor 2 (NRF2) target genes. Activation of NRF2 by sulforaphane also resulted in downregulation of NTCP, OSTα, ABCG5, CYP7A1 and CYP8B1. CLAV also inhibited the BA-sensor farnesoid X receptor (FXR), in agreement with the downregulation of FXR targets BSEP, OSTα and ABCG5. We conclude that CLAV, the culprit molecule in A+C, downregulates several key biliary transporters by modulating NRF2 and FXR signaling, thus likely promoting intrahepatic cholestasis. On top of that, increased ROS production and GSH depletion may aggravate the cholestatic injury by A+C. Mechanisms involved in CLAV-induced cholestasis. Schematic representation of a human hepatocyte with basolateral and canalicular biliary transporters. Blue lines with arrows represent the genes of biliary transporters. Green-pointed and red-blocked arrows represent activating or repressing events. An “X” through a point arrow represents a loss of activation. Green and red ovals represent proteins that result activated/upregulated or inhibited/downregulated by CLAV. ROS. Reactive oxygen species. BA: bile acids, PL: phospholipids, Chol: cholesterol, GS-conj: glutathione conjugates, Bil: bilirubin, GSSG, oxidized glutathione. [Display omitted] •Amoxicillin-clavulanate raises serum primary conjugated bile acids in DILI patients.•Clavulanate is the culprit of cholestasis by amoxicillin-clavulanate in hepatocytes.•Clavulanate downregulates expression of bile acid transporter and synth