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Rogue A.,French Institute of Health and Medical Research | Rogue A.,University of Rennes 1 | Lambert C.,French Institute of Health and Medical Research | Lambert C.,University of Rennes 1 | And 8 more authors.
PLoS ONE | Year: 2011

Background: Several glitazones (PPARγ agonists) and glitazars (dual PPARα/γ agonists) have been developed to treat hyperglycemia and, simultaneously, hyperglycemia and dyslipidemia, respectively. However, most have caused idiosyncratic hepatic or extrahepatic toxicities through mechanisms that remain largely unknown. Since the liver plays a key role in lipid metabolism, we analyzed changes in gene expression profiles induced by these two types of PPAR agonists in human hepatocytes. Methodology/Principal Findings: Primary human hepatocytes and the well-differentiated human hepatoma HepaRG cells were exposed to different concentrations of two PPARγ (troglitazone and rosiglitazone) and two PPARα/γ (muraglitazar and tesaglitazar) agonists for 24 h and their transcriptomes were analyzed using human pangenomic Agilent microarrays. Principal Component Analysis, hierarchical clustering and Ingenuity Pathway Analysis® revealed large inter-individual variability in the response of the human hepatocyte populations to the different compounds. Many genes involved in lipid, carbohydrate, xenobiotic and cholesterol metabolism, as well as inflammation and immunity, were regulated by both PPARγ and PPARα/γ agonists in at least a number of human hepatocyte populations and/or HepaRG cells. Only a few genes were selectively deregulated by glitazars when compared to glitazones, indicating that PPARγ and PPARα/γ agonists share most of their target genes. Moreover, some target genes thought to be regulated only in mouse or to be expressed in Kupffer cells were also found to be responsive in human hepatocytes and HepaRG cells. Conclusions/Significance: This first comprehensive analysis of gene regulation by PPARγ and PPARα/γ agonists favor the conclusion that glitazones and glitazars share most of their target genes and induce large differential changes in gene profiles in human hepatocytes depending on hepatocyte donor, the compound class and/or individual compound, thereby supporting the occurrence of idiosyncratic toxicity in some patients. © 2011 Rogue et al.

Menard N.,Technology Servier | Menard N.,University Paris - Sud | Menard N.,CNRS Galen Institute | Tsapis N.,University Paris - Sud | And 10 more authors.
Pharmaceutical Research | Year: 2012

Purpose: Novel surfactants made of diglutamic acid (DG) polar head linked to lithocholic, arachidonic, linoleic or stearic acids were designed for drug solubilization. Methods: Surfactants 3-D conformer and packing parameter were determined by molecular modelling and self-assembling properties by pyrene fluorescence measurements. Cytotoxicity was assessed on Human Umbilical Vein Endothelial Cells (HUVEC) and haemolyitic activity on rat red blood cells. Drug solubilization was quantified and its interaction with hydrophobic moieties was characterized using differential scanning calorimetry and X-ray diffraction. Self organisation of stearoyl-DG was observed by cryogenic transmission electron microscopy. Toxicity after repeated injections of stearoyl-DG was investigated in Wistar rats. Results: DG-based surfactants self-assemble into water and their critical micellar concentrations are comprised between 200 and 920 μg/mL. Cytotoxicity and haemolysis were lower than for polysorbate 80. At best, stearoyl-DG solubilized the drug up to 22% (w/w). Solid-state characterization evidenced drug/lipid interactions leading to the formation of a new complex. Stearoyl-DG formed spherical micelles of 20 nm, as predicted by packing parameter calculation. However, it induced a possible liver toxicity after intravenous administration in rats. Conclusions: Among the surfactants tested, stearoyl-DG is the more efficient for drug solubilization but its use is limited by its possible liver toxicity. © 2012 Springer Science+Business Media, LLC.

Spire C.,Biologie Servier | Rogue A.,French Institute of Health and Medical Research | Rogue A.,University of Rennes 1 | Brun M.,Institute Of Recherches Servier | And 3 more authors.
PPAR Research | Year: 2010

Thiazolidinediones are a class of Peroxisome Proliferator Activated Receptor γ (PPARγ) agonists that reduce insulin resistance in type 2 diabetic patients. Although no detectable hepatic toxicity has been evidenced in animal studies during preclinical trials, these molecules have nevertheless induced hepatic adverse effects in some treated patients. The mechanism(s) of hepatotoxicity remains equivocal. Several studies have been conducted using PCR analysis and microarray technology to identify possible target genes and here we review the data obtained from various in vivo and in vitro experimental models. Although PPARγ is expressed at a much lower level in liver than in adipose tissue, PPARγ agonists exert various PPARγ-dependent effects in liver in addition to PPARγ-independent effects. Differences in effects are dependent on the choice of agonist and experimental conditions in rodent animal studies and in rodent and human liver cell cultures. These effects are much more pronounced in obese and diabetic liver. Moreover, our own recent studies have shown major interindividual variability in the response of primary human hepatocyte populations to troglitazone treatment, supporting the occurrence of hepatotoxicity in only some individuals. Copyright © 2010 Alexandra Rogue et al.

Josse R.,French Institute of Health and Medical Research | Josse R.,University of Rennes 1 | Rogue A.,French Institute of Health and Medical Research | Rogue A.,University of Rennes 1 | And 3 more authors.
Mutagenesis | Year: 2012

The in vitro micronucleus test is considered as an attractive tool for genotoxicity testing of chemicals because of its simplicity of scoring and wide applicability in different cell types. However, most of the cells currently in use are devoid of the enzyme equipment required for activation of promutagens in the genotoxic metabolites. We postulated that the human HepaRG cell line, which can express xenobiotic metabolising enzymes at levels close to those found in primary human hepatocytes and has retained the indefinite growth capacity of transformed cells, could represent a more suitable model for genotoxicity testing of chemicals requiring metabolic activation. Based on the recommendations of the Organisation for Economic Co-operation and Development test guideline TG 487 for testing of chemicals, HepaRG cell cultures containing >80% mature hepatocytes were treated in situ with various chemicals for 24 h followed by a 3-day mitogenic stimulation with epidermal growth factor without cytokinesis block. In such culture conditions, HepaRG cells underwent >1.5 cell cycle per cell during the mitogenic stimulation. While non-genotoxic compounds (mannitol and staurosporine) did not increase the rate of micronucleated mononucleated cells, all aneugens (colchicine, nocodazole and dichlorodiphenyldichloroethylene) as well as the direct acting clastogen methyl methanesulfonate and clastogens requiring metabolic activation (aflatoxin B1, benzo(a)pyrene and 2-nitrofluorene) induced a statistically significant concentration-related increase in the number of mono-micronucleated cells. The micronucleus test was also performed after 7-day repeat exposure of HepaRG cells to the chemicals. Noticeably, a time-dependent effect was obtained with the three clastogens requiring metabolic activation. In conclusion, our results obtained with HepaRG hepatocytes exposed to various genotoxic compounds requiring or not bioactivation, compared favorably with those reported in various other cell types. They support the view that metabolically competent HepaRG cells have unique potential benefits for testing genotoxic compounds using the in vitro micronucleus assay. © The Author 2011. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved.

Platel A.,University of Lille Nord de France | Nesslany F.,University of Lille Nord de France | Gervais V.,Biologie Servier | Claude N.,Biologie Servier | Marzin D.,University of Lille Nord de France
Mutation Research - Genetic Toxicology and Environmental Mutagenesis | Year: 2011

Nowadays, there is clear progress in using the threshold concept in genetic toxicology, but its demonstration and acceptance in risk assessment is still under debate. Although it has been accepted for some non-DNA-reactive agents for which mechanisms of action were demonstrated, there is a growing weight of evidence to also support the existence of thresholded dose-responses for DNA-reactive agents. In this context, we have recently shown in human TK6 lymphoblastoid cells, that DNA-oxidizing agents [potassium bromate, bleomycin and hydrogen peroxide (via glucose oxidase)] produced non-linear dose-responses in the in vitro micronucleus test, thus allowing the determination of No-Observed-Genotoxic-Effect-Levels (NOGELs). Therefore, the aim of the present study was to focus on the analysis of thresholded dose-response curves in order to further investigate the existence of NOGELs for these same directly DNA-damaging agents, by use of other genotoxicity endpoints. Mutation frequency was determined after a 1-h treatment in the thymidine kinase (TK) gene-mutation assay. Primary DNA damage, especially oxidative DNA damage, was also assessed after 1 h of treatment, followed - or not - by a 23-h recovery period, with the modified version of the comet assay (i.e. with the glycosylases Fpg and hOgg1). Overall, our analysis demonstrates that there is convincing evidence to support the existence of thresholded dose-responses for DNA-oxidizing agents. The determination of NOGELs depends on the genotoxic endpoint studied and consequently requires different genotoxicity assays performed concurrently. NOGELs could only be defined for the induction of chromosomal aberrations and gene mutations, i.e. for an effect-endpoint but not for primary DNA damage, i.e. for an exposure-endpoint. Further statistical analyses of these data are now required in order to draw conclusions on the exact level of the thresholds. © 2011 Elsevier B.V.

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