Elsevier

Toxicology in Vitro

Volume 23, Issue 3, April 2009, Pages 466-475
Toxicology in Vitro

Reproducible chemical-induced changes in gene expression profiles in human hepatoma HepaRG cells under various experimental conditions

https://doi.org/10.1016/j.tiv.2008.12.018Get rights and content

Abstract

The use of in vitro human liver cell models is an attractive approach in toxicogenomic studies designed to analyze gene expression changes induced by a toxic chemical. However, in such studies, reliability, reproducibility and interlaboratory concordance of microarrays, as well as the choice of the most suitable cell model, remain a matter of debate. This work was aimed at evaluating the robustness of microarray technologies and the suitability of the highly differentiated human HepaRG cell line in the investigation of gene expression changes induced by a toxic compound in human liver. The influence of various experimental conditions including cell cultures grown at different test sites, different generations of microarrays, RNA analysis platforms and softwares, was tested on gene expression profiles induced by a 20 h treatment with an 8 mM concentration of phenobarbital as the toxic compound. As many as 1099 genes (p-value < 0.01 and 1.5-fold-change), representing 74% and 30% of the signature genes detected with Agilent 22 and 44K pangenomic microarrays, respectively, were shown to be modulated in common in six independently performed experiments. The most modulated genes included both those known to be regulated by phenobarbital, such as cytochromes P450 and membrane transporters, and those involved in oxidative stress, inflammation and apoptosis, typifying a toxic insult. These data provide strong support for the use of a toxicogenomic approach for the in vitro prediction of chemical toxicity, and for the choice of human HepaRG cells as a promising model system for human hepatotoxicity testing.

Introduction

Toxicogenomics is becoming a valuable approach to predict toxicity of a new compound and/or its mechanism of toxicity. Microarray technologies are the most frequently used tools in such studies, as demonstrated by several recent publications. However, some problems have arisen regarding the reliability, reproducibility and cross-platform comparability of results from such studies, even when the same RNA samples and the same commercial microarrays are used (Irizarry et al., 2005a, Irizarry et al., 2005b, Larkin et al., 2005, Tan et al., 2003, Waring et al., 2004, Zhang et al., 2005). Indeed, when using different platforms, the number of up- and down-regulated genes has been shown to vary greatly between experiments. Nevertheless, continuous improvements are being made to the quality and conditions of use of microarrays and recent data have shown an increase in reproducibility of interlaboratory data, thus establishing the robustness of microarray technology (Dobbin et al., 2005, Guo et al., 2006, Kuo et al., 2006, Patterson et al., 2006, Petersen et al., 2005, Piper et al., 2002, Shi et al., 2006).

Even though most studies to date have been performed with reference RNA samples, a recent report has shown that a comparison of gene expression profiles from primary cultures of rat hepatocytes prepared in different laboratories is also very difficult (Beekman et al., 2006). These findings are of major concern since the liver is the main target organ for potentially toxic compounds. Moreover, data obtained in animals cannot be extrapolated with certainty to the human situation and therefore in vitro human cell models would appear to be the most appropriate alternatives for investigating xenobiotic hepatotoxicity in humans. Primary human hepatocytes and immortalized hepatocytes are widely used; however, both model systems have limitations. Primary human hepatocytes are considered to be the most pertinent model in spite of their scarcity and unpredictable availability, limited growth activity and life-span and early and variable phenotypic alterations in vitro (Guillouzo, 1998, Hewitt et al., 2007). Hepatocyte cell lines lack a variable and substantial set of liver-specific functions, especially the major cytochromes P450 (CYP) activities and consequently, are unsuitable for mimicking in vivo normal parenchymal cells.

A new human hepatoma cell line, named HepaRG, was recently shown to be capable of expressing most of the liver-specific functions, including the major CYPs involved in drug metabolism at levels comparable to those found in primary human hepatocytes (Aninat et al., 2006, Gripon et al., 2002). In this study, we attempted to determine whether HepaRG cells could represent a robust model for evaluating the toxic effects of chemicals by a transcriptomic approach. For this purpose, we used cells prepared and drug-exposed at different laboratory test sites and analyzed mRNA samples on two platforms using different generations of Agilent oligonucleotide microarrays.

As suggested by others (Beekman et al., 2006, McMillian et al., 2004, Reymann and Borlak, 2006, van Delft et al., 2005), a high chemical concentration causing limited cytotoxicity, if at all, has to be investigated in order to evaluate toxic effects together with an active transcriptional activity. We show here that an 8 mM phenobarbital (PB) concentration caused reproducible effects, characterized by more than one thousand constantly modulated genes, in spite of the use of widely different experimental conditions.

Section snippets

HepaRG cell cultures

The HepaRG cell line, derived from a liver tumor of a female patient suffering from hepatocarcinoma, was cultured as previously described (Gripon et al., 2002). These cells reach maximum differentiation when maintained for two weeks at confluence in the presence of 2% dimethyl sulfoxide (DMSO) and 5 × 10−5 M hydrocortisone hemisuccinate. They are composed of both hepatocyte-like and biliary-like cells (about 50% of each type) (Cerec et al., 2007). For these studies, HepaRG cells were initially

Cytotoxic-induced effects

Intracellular ATP content was comparable following a 20 h treatment with 8 mM PB, representing around 40–50% of control values, in all experiments with the exception of one in which the content dropped to around 22% (experiment B) (specific data not shown). Examination of PB-exposed HepaRG cell cultures under phase-contrast microscopy revealed some alterations in a fraction of hepatocyte-like and biliary epithelial-like cells; these morphological alterations were more pronounced in cultures from

Discussion

The emerging toxicogenomic technologies offer new opportunities in pharmaceutical drug discovery and development since they can represent a novel way to predict toxicity and to determine the mode of toxicity of new drugs. However, these technologies create many challenges, due in part to problems related to reproducibility, interpretation, understanding and sharing of data. Various parameters must be considered when comparing different sets of data, including the biological models, RNA

Acknowledgments

Carine Lambert was a recipient of a CIFRE contract. We thank the Ouest-Genopole transcriptomic platform (Rennes) for help with some experiments. This work was supported by Servier group and in part by an EEC contract (LIINTOP-STREP-037499). We are grateful to Drs Lydie Sparfel, Françoise Goldfain-Blanc, Nicolas Sajot and Anne Licznar for helpful comments; and Miss Anne Platel for technical help. We also wish to thank Dr. Wynne Ellis and Dr. Delphine Allorge for careful reading of the manuscript.

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