Endoplasmic reticulum stress precedes oxidative stress in antibiotic-induced cholestasis and cytotoxicity in human hepatocytes

Free Radic Biol Med. 2018 Feb 1;115:166-178. doi: 10.1016/j.freeradbiomed.2017.11.017. Epub 2017 Dec 2.

Abstract

Endoplasmic reticulum (ER) stress has been associated with various drug-induced liver lesions but its participation in drug-induced cholestasis remains unclear. We first aimed at analyzing liver damage caused by various hepatotoxic antibiotics, including three penicillinase-resistant antibiotics (PRAs), i.e. flucloxacillin, cloxacillin and nafcillin, as well as trovafloxacin, levofloxacin and erythromycin, using human differentiated HepaRG cells and primary hepatocytes. All these antibiotics caused early cholestatic effects typified by bile canaliculi dilatation and reduced bile acid efflux within 2h and dose-dependent enhanced caspase-3 activity within 24h. PRAs induced the highest cholestatic effects at non cytotoxic concentrations. Then, molecular events involved in these lesions were analyzed. Early accumulation of misfolded proteins revealed by thioflavin-T fluorescence and associated with phosphorylation of the unfolded protein response sensors, eIF2α and/or IRE1α, was evidenced with all tested hepatotoxic antibiotics. Inhibition of ER stress markedly restored bile acid efflux and prevented bile canaliculi dilatation. Downstream of ER stress, ROS were also generated with high antibiotic concentrations. The protective HSP27-PI3K-AKT signaling pathway was activated only in PRA-treated cells and its inhibition increased ROS production and aggravated caspase-3 activity. Overall, our results demonstrate that (i) various antibiotics reported to cause cholestasis and hepatocellular injury in the clinic can also induce such effects in in vitro human hepatocytes; (ii) PRAs cause the strongest cholestatic effects in the absence of cytotoxicity; (iii) cholestatic features occur early through ER stress; (iv) cytotoxic lesions are observed later through ER stress-mediated ROS generation; and (v) activation of the HSP27-PI3K-AKT pathway protects from cytotoxic damage induced by PRAs only.

Keywords: Bile acid efflux; Bile canaliculi deformation; Drug-induced liver injury; Heat shock protein; HepaRG cells; Oxidative stress; PI3K-AKT; Unfolded protein response.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Anti-Bacterial Agents / administration & dosage
  • Anti-Bacterial Agents / adverse effects*
  • Apoptosis*
  • Caspase 3 / metabolism
  • Cell Line
  • Chemical and Drug Induced Liver Injury / metabolism*
  • Cholestasis / chemically induced
  • Cholestasis / metabolism*
  • Endoplasmic Reticulum Stress*
  • Endoribonucleases / metabolism
  • HSP27 Heat-Shock Proteins / metabolism
  • Hepatocytes / physiology*
  • Humans
  • Oxidative Stress*
  • Phosphorylation
  • Protein-Serine-Threonine Kinases / metabolism
  • Proto-Oncogene Proteins c-akt / metabolism
  • Reactive Oxygen Species / metabolism
  • Signal Transduction
  • Unfolded Protein Response
  • eIF-2 Kinase / metabolism

Substances

  • Anti-Bacterial Agents
  • HSP27 Heat-Shock Proteins
  • Reactive Oxygen Species
  • EIF2AK1 protein, human
  • ERN1 protein, human
  • Protein-Serine-Threonine Kinases
  • Proto-Oncogene Proteins c-akt
  • eIF-2 Kinase
  • Endoribonucleases
  • Caspase 3