Nevirapine Biotransformation Insights: An Integrated In Vitro Approach Unveils the Biocompetence and Glutathiolomic Profile of a Human Hepatocyte-Like Cell 3D Model

Abstract

The need for competent in vitro liver models for toxicological assessment persists. The differentiation of stem cells into hepatocyte-like cells (HLC) has been adopted due to its human origin and availability. Our aim was to study the usefulness of an in vitro 3D model of mesenchymal stem cell-derived HLCs. 3D spheroids (3D-HLC) or monolayer (2D-HLC) cultures of HLCs were treated with the hepatotoxic drug nevirapine (NVP) for 3 and 10 days followed by analyses of Phase I and II metabolites, biotransformation enzymes and drug transporters involved in NVP disposition. To ascertain the toxic effects of NVP and its major metabolites, the changes in the glutathione net flux were also investigated. Phase I enzymes were induced in both systems yielding all known correspondent NVP metabolites. However, 3D-HLCs showed higher biocompetence in producing Phase II NVP metabolites and upregulating Phase II enzymes and MRP7. Accordingly, NVP-exposure led to decreased glutathione availability and alterations in the intracellular dynamics disfavoring free reduced glutathione and glutathionylated protein pools. Overall, these results demonstrate the adequacy of the 3D-HLC model for studying the bioactivation/metabolism of NVP representing a further step to unveil toxicity mechanisms associated with glutathione net flux changes.

A Critical Perspective on 3D Liver Models for Drug Metabolism and Toxicology Studies

“…More recently, Cipriano et al (2017a) generated hnMSC-derived HLCs with more partial hepatic phenotype, sharing expression of gene groups with hpHep that was not observed between HepG2 and hnMSCs, as shown by genome-wide analysis ( Cipriano et al, 2017a ). Importantly, when resorting to the 3D culture technology, MSC-derived HLCs demonstrate an improvement in phase I biotransformation activity, urea and ALB production, as well as relevant diclofenac and nevirapine biotransformation capacity, which supports its potential usefulness for toxicological studies ( Cipriano et al, 2017b , 2020 ). Nevertheless, despite the growing efforts made in this research field a complete mature hepatocyte phenotype of HLCs derived from MSCs has not yet been achieved.…”

“…To overcome the limitations of the above mentioned cell sources, SC-derived human hepatocyte-like cells (HLCs) have been suggested as a reliable alternative ( Szkolnicka et al, 2014 ; Takayama et al, 2014 ; Freyer et al, 2016 ; Cipriano et al, 2017a , b , 2020 ; Figure 2 ). SCs represent normal primary cells with a mostly stable genotype than hepatoma cell lines.…”

“…However, this culture approach negatively impacts cell expression profiles ( Engler et al, 2006 ) and causes primary hepatocytes to rapidly lose their differentiation markers ( Treyer and Müsch, 2013 ), compromising long-term and repeated dose studies. On the other hand, 3D cell culture systems have been shown to improve the biotransformation capacities in primary hepatocytes ( Tibbitt and Anseth, 2009 ; Miranda et al, 2010 ; Mandenius et al, 2011 ; Mueller et al, 2011 ; Zeilinger et al, 2011 ; Schyschka et al, 2013 ), hepatoma cell lines ( Fey and Wrzesinski, 2012 ; Molina-Jimenez et al, 2012 ; Wrzesinski et al, 2014 ) and SC-derived HLCs ( Gieseck et al, 2014 ; Freyer et al, 2016 ; Cipriano et al, 2017b , 2020 ) over time in culture.…”

“…Non-adhesive surfaces, hanging-drop method, hydrogels, and nanoimprinted structures are some examples of small-scale 3D systems that allow the formation of organoids or multicellular spheroids of hepatocytes ( Messner et al, 2013 , 2018 ; Bell et al, 2016 ; Koyama et al, 2018 ; Peng et al, 2018 ), hepatic cell lines ( Ramaiahgari et al, 2014 ; Leite et al, 2016 ) or other cell types ( Huch et al, 2015 ; Asai et al, 2017 ; Cipriano et al, 2017b , 2020 ; Takebe et al, 2017 ; Wang S. et al, 2019 ; Wang Z. et al, 2019 ; Ramli et al, 2020 ; Table 2 ).…”

Oxidative Stress in Drug-Induced Liver Injury (DILI): From Mechanisms to Biomarkers for Use in Clinical Practice

“…ROS production and antioxidant compounds depletion (e.g., GSH) are considered two of the most sensitive parameters of drug-induced hepatotoxicity [ 300 , 301 , 302 ]. Thus, future preclinical DILI studies will probably include high content screening assays in which oxidative stress would be an essential endpoint to measure [ 303 , 304 , 305 , 306 ].…”