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. 2020 Jun 3;21(11):3998.
doi: 10.3390/ijms21113998.

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

Madalena Cipriano  1   2 , Pedro F Pinheiro  2   3 , Catarina O Sequeira  4 , Joana S Rodrigues  2 , Nuno G Oliveira  2 , Alexandra M M Antunes  3 , Matilde Castro  2 , M Matilde Marques  3 , Sofia A Pereira  4 , Joana P Miranda  2
Affiliations

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

Madalena Cipriano et al. Int J Mol Sci. .

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.

Keywords: 3D culture; glutathione; hepatocytes; metabolism; nevirapine; stem cells.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Schematic representation of Nevirapine (NVP) biotransformation and toxic metabolites formation.
Figure 2
Figure 2
Gene expression analyses of Phase I, II and III related genes of 3D- and 2D-HLCs after 3 (D27) and 10 days (D34) of NVP treatment. (A) Gene expression of Phase I enzymes comprising CYP3A4, CYP2B6 and CYP2D6 in NVP-treated cells relative to non-treated cells. (B) Gene expression of Phase II enzymes comprising SULT1A1, UGT1A1 and GSTA1-A2 in NVP-treated cells relative to non-treated cells. (C) Gene expression of the MRP7 transporter in NVP-treated cells relative to non-treated cells. (A–C) Data for hnMSC exposed to NVP for 3 days and used as negative controls, are also shown. (D) Phase I, II and III gene expression in 3D-HLCs relative to 2D-HLCs in NVP-treated cells at D27 and D34. The data (Mean ± SD, n = 3) are normalized to the reference gene β-ACTIN and expressed as the log10 of the ratios.
Figure 3
Figure 3
Phase I and Phase II enzymatic induction in 3D- and 2D-HLCs after 3 (D27) and 10 days (D34) of NVP treatment. The data for hnMSC, cultured in monolayer and exposed to NVP for 3 days, are presented as negative control. * p < 0.05 and *** p < 0.001 relative to 2D-HLCs, $ p < 0.05 and $$$ p < 0.001 relative to non-treated and ### p < 0.001 relative to D27 of culture. The data (Mean ± SD, n = 3) are represented as fold induction in NVP-treated cells relative to non-treated cells.
Figure 4
Figure 4
(A) Levels of NVP Phase I and II metabolites in 3D- and 2D-HLCs after 3 (D27) and 10 days (D34) of NVP treatment. hnMSC cultured in monolayer and exposed to NVP for 3 days are also shown and (B) relative proportions of total NVP metabolites (free metabolites + sulfate conjugates + glucuronic acid conjugates) at D27 and D34. The data (Mean ± SD, n = 3) are normalized to total cell number.
Figure 5
Figure 5
Glutathiolomic analysis of 3D- and 2D-HLCs at D27 and D34. (A) Total intracellular availability for cysteine (Cys), glutamylcysteine (GluCys) and glutathione and total extracellular levels of cysteinylglycine (CysGly) and glutathione; (B) Intracellular levels of protein-bound glutathione (GSSP) and free glutathione; * p < 0.05, ** p < 0.01 and *** p < 0.001 relative to 2D-HLCs. The data are represented as Mean ± SD, n = 3.
Figure 6
Figure 6
Glutathiolomic analysis of 3D- and 2D-HLCs treated or non-treated with NVP. (A) Effect of NVP on glutathione synthesis and degradation ratios, after 3 (D27) and 10 days (D34) of NVP treatment; (B) Effect of NVP on total intracellular availability for cysteine (Cys), glutamylcysteine (GluCys) and glutathione and total extracellular levels of cysteinylglicine (CysGly) and glutathione, in 3D-HLCs, after 3 (D27) and 10 days (D34) of NVP treatment; (C) NVP effect on intracellular levels of protein-bound glutathione (GSSP) and free glutathione, after 3 (D27) and 10 days (D34) of NVP treatment; (D) NVP effect on glutathione oxidation (intracellular GSH/GSSG ratio) after 10 days of NVP treatment (D34). * p < 0.05, ** p < 0.01 and *** p < 0.001 relative to 2D-HLCs. The data are represented as Mean ± SD, n = 3.
Figure 7
Figure 7
Schematic representation of the study design.
See this image and copyright information in PMC

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