Studying drug-induced cholestasis in human hepatocytes spheroid cultures

  • Dina Zu Alghina ((PhD) Student)
  • Vânia Vilas-Boas (Co-promotor)
  • Mathieu Vinken (Promotor)

Student thesis: Master's Thesis

Abstract

Background: Cholestasis induced by drugs is a major concern for pharmaceutical industry during drug development and marketing. Therefore, understanding its mechanisms and identifying potential hepatotoxins will allow an on-time prediction and control with benefits for both industry and society.
Aim: This study aimed at studying in vitro the cytotoxicity and the cholestatic liability of a well-known cholestatic drug, cyclosporine A (CsA), in relevant concentrations upon long-term repeated exposure. Additionally, the observed synergistic toxicity of CsA and a concentrated bile acids (BA) mixture was further investigated by detecting the transcriptional deregulation of some genes involved in BA homeostasis.
Methods: Primary human hepatocytes from one donor were seeded in spheroid configuration and exposed to different concentrations of CsA for 28 days. The viability of treated cells was assessed by measuring ATP content at 5 time-points. The cholestatic riskwas assessed upon spheroids co-exposure to different concentrations of CsA and BA mixture. The cholestatic index was calculated by measuring ATP content on days 1, 7, 14, 21 and 28. The deregulation of BSEP, MRP2, NTCP and CYP7A1 was evaluated only on day 1 and day 7 using RT-qPCR analysis.
Results: Based on the dose-response curve, CsA 0.01µM, 0.05µM, 0.5µM, and 5µM were chosen to study their cholestatic liability. Only 5µM CsA exhibited cholestatic liability from day 14 on. BA mixture combined with CsA seemed to potentiate the downregulation of the mRNA expression of most of the studied targets compared to exposure to CsA alone. Downregulation of BSEP and MRP2 mRNA expression by CsA may result in the accumulation of intracellular BA to toxic concentrations that initiate the cholestatic insult. In turn, downregulation of mRNA expression of CYP7A1 and NTCP may aim at preventing further intracellular accumulation of BA.
Conclusion: This study confirms the cholestatic liability of relevant CsA concentrations, as well as the delayed onset of its cholestatic insult. This is reinforced by the likely downregulation of the mRNA expression of target genes responsible for the maintenance of intracellular concentrations of BA. Further research is needed to confirm these results and to determine the interdonor variability that may underlie unequal responses towards the investigated drug.
Date of Award31 Jan 2020
Original languageEnglish
Awarding Institution
  • Vrije Universiteit Brussel
SupervisorVânia Filipa Esteves Vilas Boas (Co-promotor)

Keywords

  • drug-induced cholestasis
  • cyclosporine A
  • primary human hepatocytes spheroids
  • cholestatic index

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