[Show abstract][Hide abstract]ABSTRACT: Although non-alcoholic fatty liver disease (NAFLD) is currently the most common form of chronic liver disease there is no pharmacological agent approved for its treatment. Since peroxisome proliferator-activated receptors (PPARs) are closely associated with hepatic lipid metabolism, they seem to play important roles in NAFLD. However, the effects of PPAR agonists on steatosis that is a common pathology associated with NAFLD, remain largely controversial. In this study, the effects of various PPAR agonists, i.e. fenofibrate, bezafibrate, troglitazone, rosiglitazone, muraglitazar and tesaglitazar on oleic acid-induced steatotic HepaRG cells were investigated after a single 24-hour or 2-week repeat treatment. Lipid vesicles stained by Oil-Red O and triglycerides accumulation caused by oleic acid overload, were decreased, by up to 50%, while fatty acid oxidation was induced after 2-week co-treatment with PPAR agonists. The greatest effects on reduction of steatosis were obtained with the dual PPARα/γ agonist muraglitazar. Such improvement of steatosis was associated with up-regulation of genes related to fatty acid oxidation activity and down-regulation of many genes involved in lipogenesis. Moreover, modulation of expression of some nuclear receptor genes, such as FXR, LXRα and CAR, which are potent actors in the control of lipogenesis, was observed and might explain repression of de novo lipogenesis.
Altogether, our in vitro data on steatotic HepaRG cells treated with PPAR agonists correlated well with clinical investigations, bringing a proof of concept that drug-induced reversal of steatosis in human can be evaluated in in vitro before conducting long-term and costly in vivo studies in animals and patients.
Full-text Article · Apr 2014 · Toxicology and Applied Pharmacology
[Show abstract][Hide abstract]ABSTRACT: With the increasing human exposure to nanoparticles (NP), the evaluation of their genotoxic potential is of significant importance.
However, relevance for NP of the routinely used in vitro genotoxicity assays is often questioned, and a nanoparticulate reference positive control would therefore constitute an important
step to a better testing of NP, ensuring that test systems are really appropriate. In this study, we investigated the possibility
of using tungsten carbide-cobalt (WC-Co) NP as reference positive control in in vitro genotoxicity assays, including 2 regulatory assays, the mouse lymphoma assay and the micronucleus assay, and in the Comet
assay, recommended for the toxicological evaluation of nanomedicines by the French Agency of Human Health Products (Afssaps).
Through these assays, we were able to study different genetic endpoints in 2 cell types commonly used in regulatory genotoxicity
assays: the L5178Y mouse lymphoma cell line and primary cultures of human lymphocytes. Our results showed that the use of
WC-Co NP as positive control in in vitro genotoxicity assays was conceivable, but that different parameters have to be considered, such as cell type and treatment
schedule. L5178Y mouse lymphoma cells did not provide satisfactory results in the 3 performed tests. However, human lymphocytes
were more sensitive to genotoxic effects induced by WC-Co NP, particularly after a 24-h treatment in the in vitro micronucleus assay and after a 4-h treatment in the in vitro Comet assay. Under such conditions, WC-Co could be used as a nanoparticulate reference positive control in these assays.
Full-text Article · Oct 2013 · Toxicological Sciences
[Show abstract][Hide abstract]ABSTRACT: Drug hypersensitivity is known to rely on a drug-specific T-cell response. Amplitude of antigen-specific T-cell response is partly controlled by the size of the antigen-specific naïve CD4+ T-cell repertoire, but estimate of this repertoire has never been investigated for allergenic drugs. The purpose of this study was to evaluate the frequency of benzylpenicillin-specific CD4+ T lymphocytes in healthy donors.
Co-cultures were established with CD4+ T lymphocytes from healthy donors and mature autologous dendritic cells loaded with benzylpenicillin coupled to human serum albumin. CD4+ T lymphocytes were stimulated once a week for 4 weeks with benzylpenicillin coupled to human serum albumin. The CD4+ T-cell response was measured using an interferon-γ ELISPOT assay. Frequency of benzylpenicillin-specific naive CD4+ T lymphocytes was then calculated using the Poisson distribution law.
Results showed the presence of benzylpenicillin-specific CD4+ T lymphocytes in 9 of 10 tested healthy donors irrespective of their HLA typing, with a mean frequency of 0.29 cells per million of CD4+ T cells. Experiments performed on naive (CD45RA+) and on memory (CD45RO+) CD4+ T lymphocytes showed that these benzylpenicillin-specific CD4+ T lymphocytes belonged to the naive T-cell subpopulation.
This study showed for the first time the existence of naive CD4+ T lymphocytes specific to benzylpenicillin in healthy donors.
[Show abstract][Hide abstract]ABSTRACT: Interindividual variations in functions other than drug metabolism activity, remain poorly elucidated in human liver. In the present study, the whole transcriptome of several human hepatocyte populations and the differentiated human HepaRG cell line have been analyzed and compared, using oligonucleotide pangenomic microarrays. We show that, although the variation in the percentages of expressed genes did not exceed 14% among the primary human hepatocyte populations, huge interindividual differences in the transcript levels of many genes were observed. These genes were related to various functions; in addition to drug metabolism, they mainly concerned carbohydrate, amino acid, and lipid metabolism. HepaRG cells expressed from 81 to 92% of the genes active in human hepatocytes and, in addition, a specific gene subset mainly related to their transformed status, some chromosomal abnormalities, and the presence of primitive biliary epithelial cells. Of interest, a relationship was evidenced between abnormal basal expression levels of some target genes and their corresponding previously reported fold changes in one of four human hepatocyte populations treated with the hepatotoxic drug troglitazone and not with other nonhepatotoxic peroxisome proliferator-activated receptor agonists (PLoS One 6:e18816, 2011). Taken together, our results support the view that HepaRG cells express most of the genes active in primary human hepatocytes and show that expression of most human hepatic genes can quantitatively greatly vary among individuals, thereby contributing to explain the huge interindividual variability in susceptibility to drugs and other environmental factors.
Article · Jan 2012 · Drug metabolism and disposition: the biological fate of chemicals
[Show abstract][Hide abstract]ABSTRACT: 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 1h 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.
Article · Sep 2011 · Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
[Show abstract][Hide abstract]ABSTRACT: Species-differential toxic effects have been described with PPARα and PPARγ agonists between rodent and human liver. PPARα agonists (fibrates) are potent hypocholesterolemic agents in humans while they induce peroxisome proliferation and tumors in rodent liver. By contrast, PPARγ agonists (glitazones) and even dual PPARα/γ agonists (glitazars) have caused idiosyncratic hepatic and nonhepatic toxicities in human without evidence of any damage in rodent during preclinical studies. The mechanisms involved in such differences remain largely unknown. Several studies have identified the major target genes of PPARα agonists in rodent liver while no comprehensive analysis has been performed on gene expression changes induced by PPARγ and dual PPARα/γ agonists. Here, we investigated transcriptomes of rat hepatocytes after 24h treatment with two PPARγ (troglitazone and rosiglitazone) and two PPARα/γ (muraglitazar and tesaglitazar) agonists. Although, hierarchical clustering revealed a gene expression profile characteristic of each PPAR agonist class, only a limited number of genes was specifically deregulated by glitazars. Functional analyses showed that many genes known as PPARα targets were also modulated by both PPARγ and PPARα/γ agonists and quantitative differences in gene expression profiles were observed between these two classes. Moreover, most major genes modulated in rat hepatocytes were also found to be deregulated in rat liver after tesaglitazar treatment. Taken altogether, these results support the conclusion that differential toxic effects of PPARα and PPARγ agonists in rodent liver do not result from transcriptional deregulation of major PPAR target genes but rather from qualitative and/or quantitative differential responses of a small subset of genes.
Article · Jul 2011 · Toxicology and Applied Pharmacology
[Show abstract][Hide abstract]ABSTRACT: 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.
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.
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.
[Show abstract][Hide abstract]ABSTRACT: Genes involved and fold change after PPAR treatment in main metabolic pathways are listed in this table. The minimum and maximum fold changes obtained with HepaRG cells are indicated in square brackets.
[Show abstract][Hide abstract]ABSTRACT: Genes involved and fold change after PPAR treatment in main metabolic pathways are listed in this table. The minimum and maximum fold changes obtained with primary human hepatocytes are indicated in square brackets.
[Show abstract][Hide abstract]ABSTRACT: 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.