Integrated Proteomic and Transcriptomic Investigation of the Acetaminophen Toxicity in Liver Microfluidic Biochip

Jean Matthieu Prot,Franck Merlier,Yves Grandvalet,Franck Letourneur,Eric Leclerc,Anne-Sophie Briffaut,Philippe Chafey,Cécile Legallais,Christophe Egles

doi:10.1371/journal.pone.0021268

Abstract

Microfluidic bioartificial organs allow the reproduction of in vivo-like properties such as cell culture in a 3D dynamical micro environment. In this work, we established a method and a protocol for performing a toxicogenomic analysis of HepG2/C3A cultivated in a microfluidic biochip. Transcriptomic and proteomic analyses have shown the induction of the NRF2 pathway and the related drug metabolism pathways when the HepG2/C3A cells were cultivated in the biochip. The induction of those pathways in the biochip enhanced the metabolism of the N-acetyl-p-aminophenol drug (acetaminophen-APAP) when compared to Petri cultures. Thus, we observed 50% growth inhibition of cell proliferation at 1 mM in the biochip, which appeared similar to human plasmatic toxic concentrations reported at 2 mM. The metabolic signature of APAP toxicity in the biochip showed similar biomarkers as those reported in vivo, such as the calcium homeostasis, lipid metabolism and reorganization of the cytoskeleton, at the transcriptome and proteome levels (which was not the case in Petri dishes). These results demonstrate a specific molecular signature for acetaminophen at transcriptomic and proteomic levels closed to situations found in vivo. Interestingly, a common component of the signature of the APAP molecule was identified in Petri and biochip cultures via the perturbations of the DNA replication and cell cycle. These findings provide an important insight into the use of microfluidic biochips as new tools in biomarker research in pharmaceutical drug studies and predictive toxicity investigations.

Highlights

In drug development and toxicity studies, there is increased demand from pharmaceutical companies to develop new approaches that make it possible to focus on optimum drugs at the preclinical stage
To determine the real impact of the microscale models, we developed a microfluidic biochip applied to mammalian cell cultures [12,13]
Ingenuity pathway analysis of the resulting combination markers for genes and proteins highlighted the NRF2 pathway and fatty acid metabolism. This NRF2 pathway is related to oxidative stress and xenobiotic response. It led to the over-expression of genes and proteins involved in glutathione metabolism, protein ubiquitination, phase 1 (CYP P450) and phase 2 (SULT, GST, UGT) enzymes and phase 3 transporters (ABCC)

Summary

Introduction

In drug development and toxicity studies, there is increased demand from pharmaceutical companies to develop new approaches that make it possible to focus on optimum drugs at the preclinical stage. Recent progress in proteomic analysis has led to identify biomarkers at the protein level providing new insights into drug development and toxicological science [2,3]. Hepatoxicity is one of the primary causes of late drug withdrawal, mainly because of the lack of a pertinent model for both reproducing functional liver tissue and addressing the systemic toxicity of molecules [5]. Microscale models have been built to cultivate liver cells in order to refine the investigations with hepatocytes [6]. By controlling the microfluidic flow conditions inside these environments, the microscale model makes it possible to propose successful co-cultures based on various organ cell types (such as liver, lung etc...), reproducing systemic interactions [9]. Complex and structured liver microscale models (using human primary hepatocytes and liver non parenchymal cells) can be successfully applied to pharmaceutical drug screening [10,11]

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