Abstract
The development of metabolically competent in vitro models is of utmost importance for predicting adverse drug reactions, thereby preventing attrition-related economical and clinical burdens. Using the antiretroviral drug nevirapine (NVP) as a model, this work aimed to validate rat hepatocyte 3D spheroid cultures as competent in vitro systems to assess drug metabolism and bioactivation. Hepatocyte spheroids were cultured for 12 days in a stirred tank system (3D cultures) and exposed to equimolar dosages of NVP and its two major Phase I metabolites, 12-OH-NVP and 2-OH-NVP. Phase I NVP metabolites were detected in the 3D cultures during the whole culture time in the same relative proportions reported in in vivo studies. Moreover, the modulation of SULT1A1 activity by NVP and 2-OH-NVP was observed for the first time, pointing their synergistic effect as a key factor in the formation of the toxic metabolite (12-sulfoxy-NVP). Covalent adducts formed by reactive NVP metabolites with N-acetyl-l-cysteine and bovine serum albumin were also detected by high-resolution mass spectrometry, providing new evidence on the relative role of the reactive NVP metabolites, 12-sulfoxy-NVP, and NVP quinone methide, in toxicity versus excretion pathways. In conclusion, these results demonstrate the validity of the 3D culture system to evaluate drug bioactivation, enabling the identification of potential biomarkers of bioactivation/toxicity, and providing new evidence to the mechanisms underlying NVP-induced toxic events. This model, integrated with the analytical strategies described herein, is of anticipated usefulness to the pharmaceutical industry, as an upstream methodology for flagging drug safety alerts in early stages of drug development.
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Abbreviations
- 2D:
-
Two-dimensional
- 3D:
-
Three-dimensional
- Alb:
-
Albumin
- BSA:
-
Bovine serum albumin
- CK-18:
-
Cytokeratin-18
- CYP:
-
Cytochrome P450
- DAPI:
-
4′,6-Diamidino-2-phenylindole
- DILI:
-
Drug-induced liver injury
- DMSO:
-
Dimethyl sulfoxide
- ECOD:
-
Ethoxycoumarin-O-deethylation
- EROD:
-
Ethoxyresorufin-O-deethylation
- HIV:
-
Human immunodeficiency virus
- HNF4-α:
-
Hepatocyte nuclear factor 4 alpha
- HSA:
-
Human serum albumin
- MeOH:
-
Methanol
- MRP2:
-
Multidrug resistance-associated protein 2
- NAC:
-
N-acetyl-l-cysteine
- NVP:
-
Nevirapine
- OATP-C:
-
Liver-specific organic anion-transporting polypeptide
- PAPS:
-
3′-Phosphoadenosine-5′ phosphosulfate
- PFA:
-
Paraformaldehyde
- PBS:
-
Phosphate-buffered saline
- SFSC:
-
Spinner flask suspension culture
- SEM:
-
Standard error of the mean
- SULT:
-
Sulfotransferase
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Acknowledgments
The authors thank Dr. F. A. Beland (National Center for Toxicological Research, Jefferson, AR, USA) for the kind gift of nevirapine, and the Portuguese MS Network (IST-ULisboa Node) for providing access to the facility. A preliminary account of these results was presented and selected for oral communication at the EUROTOX 2013 meeting, Interlaken, Switzerland.
Financial support
This work was supported by Fundação para a Ciência e a Tecnologia (FCT), through Grants PTDC/QUI-QUI/113910/2009, RECI/QEQ-MED/0330/2012, RECI/QEQ-QIN/0189/2012, UID/DTP/04138/2013, UID/QUI/00100/2013, PTDC/SAU-TOX/110457/2009, and PEst-OE/SAU/UI4013/2011, and fellowships SFRH/BPD/96719/2013 (to JPM), SFRH/BD/80690/2011 (to SGH), SFRH/BD/92191/2013 (to ATM) and SFRH/BD/75426/2010 (to ILM). AMMA would also like to acknowledge FCT, “Programa Operacional Potencial Humano” and the European Social Fund for the IF Program (IF/01091/2013).
Authors’ contributions
JM, AMMA, SAP, and MMM contributed to the study design. JM, AMMA, and PFP contributed to data interpretation. CCJ contributed to the mass spectrometry data acquisition and interpretation. AMMA, PFP, SGH, and ILM contributed to the synthesis of metabolite and adduct standards. PFP and JM contributed to the cell isolation and culture. JM and MC performed the immunofluorescence assays. PFP, ATM, and SAP contributed to the quantification of Phase I and Phase II metabolites. PFP, JM, AMMA, CCJ, NGO, MFC, and MMM contributed to the writing and editing of the manuscript.
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Alexandra M. M. Antunes and Joana P. Miranda have contributed equally to the study.
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Pinheiro, P.F., Pereira, S.A., Harjivan, S.G. et al. Hepatocyte spheroids as a competent in vitro system for drug biotransformation studies: nevirapine as a bioactivation case study. Arch Toxicol 91, 1199–1211 (2017). https://doi.org/10.1007/s00204-016-1792-x
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DOI: https://doi.org/10.1007/s00204-016-1792-x