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Investigation of acetaminophen toxicity in HepG2/C3a microscale cultures using a system biology model of glutathione depletion

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Abstract

We have integrated in vitro and in silico information to investigate acetaminophen (APAP) and its metabolite N-acetyl-p-benzoquinone imine (NAPQI) toxicity in liver biochip. In previous works, we observed higher cytotoxicity of HepG2/C3a cultivated in biochips when exposed to 1 mM of APAP for 72 h as compared to Petri cultures. We complete our investigation with the present in silico approach to extend the mechanistic interpretation of the intracellular kinetics of the toxicity process. For that purpose, we propose a mathematical model based on the coupling of a drug pharmacokinetic model (PK) with a systemic biology model (SB) describing the reactive oxygen species (ROS) production by NAPQI and the subsequent glutathione (GSH) depletion. The SB model was parameterized using (i) transcriptomic data, (ii) qualitative results of time lapses ROS fluorescent curves for both control and 1-mM APAP-treated experiments, and (iii) additional GSH literature data. The PK model was parameterized (i) using the in vitro kinetic data (at 160 μM, 1 mM, 10 mM), (ii) using the parameters resulting from a physiologically based pharmacokinetic (PBPK) literature model for APAP, and (iii) by literature data describing NAPQI formation. The PK-SB model predicted a ROS increase and GSH depletion due to the NAPQI formation. The transition from a detoxification phase and NAPQI and ROS accumulation was predicted for a NAPQI concentration ranging between 0.025 and 0.25 μM in the cytosol. In parallel, we performed a dose response analysis in biochips that shows a reduction of the final hepatic cell number appeared in agreement with the time and doses associated with the switch of the NAPQI detoxification/accumulation. As a result, we were able to correlate in vitro extracellular APAP exposures with an intracellular in silico ROS accumulation using an integration of a coupled mathematical and experimental liver on chip approach.

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Abbreviations

Ahr:

Aryl hydrocarbon receptor

APAP:

Acetaminophen

CYP:

Cytochrome P450 gene

DCFDA:

2′,7′-Dichlorofluorescin diacetate

GCLC:

Glutamate-cysteine ligase catalityc subunit gene

GCLM:

Glutamate-cysteine ligase regulatory subunit gene

GPX:

Glutathione peroxidase gene

GSH:

Glutathione

GS:

Glutathione synthetase gene

GST:

Glutathione S transferase gene

Keap-1:

Kelch-like ECH-associated protein 1

MRP:

Multidrug resistance-associated protein gene

NAPQI:

N-Acetyl-p-benzoquinone imine

NFE2L2:

Nuclear factor (erythroid-derived 2)-like 2 gene

Nrf2:

Protein code by NFE2L2 gene

PBPK:

Physiologically based pharmacokinetic

PK:

Pharmacokinetic

ROS:

Reactive oxygen species

SB:

Systems biology

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Acknowledgments

This work was carried out in the framework of the Labex MS2T, which was funded by the French Government, through the program “Investments for the future” managed by the National Agency for Research (ANR-11-IDEX-0004-02). The work was supported by the “fondation UTC pour la recherche” via the chaire in predictive toxicology and the project “Puces à cellules 3D”

Conflict of interest

We declare no conflict of interest.

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Authors and Affiliations

  1. CNRS UMR 7338, Laboratoire de Biomécanique et Bio ingénierie, Université de Technologie de Compiègne, Compiègne, France

    Eric Leclerc, Jeremy Hamon, Isabelle Claude, Rachid Jellali, Marie Naudot & Frederic Bois

  2. Chaire de Toxicologie Prédictive, Université de Technologie de Compiègne, Compiègne, France

    Frederic Bois

  3. Institut National de l’Environnement Industriel et des Risques (INERIS), Unité Modèles pour l’Ecotoxicoclogie et la Toxicologie, Parc ALATA, BP2, 60550, Verneuil en Halatte, France

    Frederic Bois

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  1. Eric Leclerc
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  2. Jeremy Hamon
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  3. Isabelle Claude
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Correspondence to Eric Leclerc.

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Leclerc, E., Hamon, J., Claude, I. et al. Investigation of acetaminophen toxicity in HepG2/C3a microscale cultures using a system biology model of glutathione depletion. Cell Biol Toxicol 31, 173–185 (2015). https://doi.org/10.1007/s10565-015-9302-0

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