Predictive toxicology using systemic biology and liver microfluidic "on chip" approaches: Application to acetaminophen injury

International audienceWe have analyzed transcriptomic, proteomic and metabolomic profiles of hepatoma cells cultivated inside a microfluidic biochip with or without acetaminophen (APAP). Without APAP, the results show an adaptive cellular response to the microfluidic environment, leading to the induction of anti-oxidative stress and cytoprotective pathways. In presence of APAP, calcium homeostasis perturbation, lipid peroxidation and cell death are observed. These effects can be attributed to APAP metabolism into its highly reactive metabolite. N-acetyl-p-benzoquinone imine (NAPQI). That toxicity pathway was confirmed by the detection of GSH-APAP, the large production of 2-hydroxybutyrate and 3-hydroxybutyrate, and methionine, cystine, and histidine consumption in the treated biochips. Those metabolites have been reported as specific biomarkers of hepatotoxicity and glutathione depletion in the literature. In addition, the integration of the metabolomic, transcriptomic and proteomic collected profiles allowed a more complete reconstruction of the APAP injury pathways. To our knowledge, this work is the first example of a global integration of microfluidic biochip data in toxicity assessment. Our results demonstrate the potential of that new approach to predictive toxicology

Transcriptional and Epigenetic Consequences of DMSO Treatment on HepaRG Cells

Dimethyl sulfoxide (DMSO) is used to sustain or favor hepatocyte differentiation in vitro. Thus, DMSO is used in the differentiation protocol of the HepaRG cells that present the closest drug-metabolizing enzyme activities to primary human hepatocytes in culture. The aim of our study is to clarify its influence on liver-specific gene expression. For that purpose, we performed a large-scale analysis (gene expression and histone modification) to determine the global role of DMSO exposure during the differentiation process of the HepaRG cells. The addition of DMSO drives the upregulation of genes mainly regulated by PXR and PPARα whereas genes not affected by this addition are regulated by HNF1α, HNF4α, and PPARα. DMSO-differentiated-HepaRG cells show a differential expression for genes regulated by histone acetylation, while differentiated-HepaRG cells without DMSO show gene signatures associated with histone deacetylases. In addition, we observed an interplay between cytoskeleton organization and EMC remodeling with hepatocyte maturation

Effet inhibiteur de la roquefortine sur les cytochromes P450 hépatiques

TOULOUSE3-BU Santé-Centrale (315552105) / SudocSudocFranceF

The glutathione transferase kappa family.

International audienceGlutathione transferase (GST) kappa, also named mitochondrial GST, is a very ancient protein family with orthologs in bacteria and eukaryotes. Both the structure and the subcellular localization of GSTK1-1, in mitochondria and peroxisomes, make this enzyme distinct from cytosolic GSTs. Rodent and human GSTK1 exhibit activity towards a number of model GST substrates and, in Caenorhabditis elegans, this enzyme may be involved in energy and lipid metabolism, two functions related to mitochondria and peroxisomes. Interestingly, GST kappa is also a key regulator of adiponectin biosynthesis and multimerization suggesting that it might function as a chaperone to facilitate correct folding and assembly of proteins. Since adiponectin expression has been correlated with insulin resistance, obesity and diabetes, GSTK1 expression level which is negatively correlated with obesity in mice and human adipose tissues may be an important factor in these metabolic disorders. Furthermore, a polymorphism in the hGSTK1 promoter has been associated with insulin secretion and fat deposition

Métabolisme et pharmacocinétique de composés peptidiques et cyclopeptidiques (utilisation de radioéléments et d'isotopes stables)

Actuellement plus d'une centaine de peptides et protéines sont commercialisés ou en cours de développement. Les études de pharmacocinétique et de métabolisme de ces composés reposent sur l'utilisation de 3 techniques principales : l'utilisation de composés marqués " ponctuellement " sur un acide aminé avec des radioéléments, de détections immunologiques et depuis quelque temps de la spectrométrie de masse.La première partie de ce travail a consisté à évaluer à l'aide de peptides modèles (insuline et peptide C) l'intérêt du marquage uniforme avec des isotopes stables ( 13C, 15N et 13C/15N) ou de 14C dans ce type d'étude. Ces travaux se basent principalement sur un dosage par dilution isotopique couplé à la spectrométrie de masse.Dans un second temps, nous nous sommes intéressés à une famille de cyclopeptides composés de deux acides aminés, les dicétopipérazines. Ces produits sont retrouvés à la fois chez les mammifères et chez les microorganismes. Non reconnus par les enzymes protéolytiques des mammifères, nous avons évalué leur prise en charge, à l'aide de deux composés modèles la roquefortine et la phenylahistin, par les principales enzymes intervenant dans le métabolisme des xénobiotiques, les monooxygénases à cytochrome P450s.More and more peptides and proteins are used in therapeutic. Three mainly technics are used for pharmacokinetic and metabolism studies: immunoassay, radioactively labeled molecules and mass spectrometry.In the first part of this work, we've used uniformely labelled peptides (C-peptide and insulin) with stables (13C, 15N, and 13C/15N) or radioactive (14C) isotopes to investigated these kind of studies. These works are based on isotope dilution mass spectrometry assay.In a second time we've investigated the metabolism of a particular cyclopeptides families composed of two amino acids : the diketopiperazine. These compounds are found in mammals and in microorganisms. There are not recognized by proteolytic enzymes. We've estimated if the main enzymes implicated in the metabolism of xenobiotics, the P450 cytochrome monooxygenases, were able to recognized them.PARIS-BIUP (751062107) / SudocSudocFranceF

Vente de médicaments sur internet (encadrement, dérives possibles et adaptation de l'exercice officinal)

Les chiffres en témoignent, de plus en plus nombreuses sont les personnes à avoir déjà effectué un achat de médicaments par le biais d'internet. La France présente un statut particulier en étant l'un des derniers états européens à ne pas avoir légiférer sur la question entraînant un flou juridique. En effet, depuis l'arrêt doc Morris rendu en décembre 2003, l'interdiction de vente par correspondance des médicaments sans ordonnance constitue une entrave à la liberté de circulation des marchandises par jurisprudence européenne. Tout en apprenant des autres systèmes européens et en tenant compte des particularités françaises, ce travail a pour but de réfléchir à une éventuelle évolution du système, celle qui sera la plus adaptée, en pesant notamment les risques et les intérêts.All the statistics testify for it, more and more people have already been buying medecines through internet. France presents a peculiar status, being one of the last european contries that hasn't been working on the legislation of this practice, which implies a legal desert . Indeed, since the doc Morris judgement of december 2003, the prohibition of medecines online selling without prescription constitutes a restriction to the merchandises free trade by european justice. Learning from other european systems and taking into account the french specificities, this work aims at thinking about a potential evolution of the actual system, balancing the risks and interests to propose the most adapted solution.RENNES1-BU Santé (352382103) / SudocLYON1-BU Santé (693882101) / SudocSudocFranceF

Les compléments alimentaires du sportif et le risque de dopage par inadvertance

Les compléments alimentaires sont de plus en plus présents dans notre quotidien mais que sont réellement ces produits? Quelle différence existe-t-il entre eux et les médicaments ou l'alimentation? Quelle est leur sécurité d'emploi et les effets supposés de ces produits sont-ils appuyés par des études validées? En prenant l'exemple des compléments alimentaires destinés aux sportifs, nous allons essayer de répondre à ces questions et également mentionner le problème de la pureté de ces compléments, pouvant parfois mener à des cas de dopage à l'insu du consommateur. Enfin, nous évaluerons par un petit questionnaire l'opinion des sportifs envers les compléments alimentaires.The dietary supplements are more and more present in our everyday life, but what about these products? What are the differences between these and medicines or the basic food? What is their really safety use and the supposed effects are they argued by scientifically studies? By takng the example of the dietary supplements intended for the sportsmen, we would expected to answer these questions and also mention the problem of high-purity of these supplements, which can sometimes lead to positive doping cases without the knowledge of the consumer. At least, we will try to know by some question what the sportsmen think about the dietary supplements.LYON1-BU Santé (693882101) / SudocRENNES1-BU Santé (352382103) / SudocSudocFranceF

Protective role of glutathione transferases Pi class against stress in Caenorhabditis elegans and identify novel protein partners of glutathione transferases M1 in human

Les glutathion transférases (GST) représentent une superfamille de gènes constituée de sept familles multigéniques. Certaines de ces GST, notamment les GST de la famille Pi, sont surexprimées dans les tumeurs et peuvent être responsables de phénomènes de résistance aux traitements chimiothérapeutiques. Le premier objectif de ce travail a été d utiliser Caenorhabditis elegans pour l étude d un agent anticancéreux, le 5-fluorouracil, et pour évaluer le rôle des GST pi dans la résistance à des agents anticancéreux, mais également à d autres types de stress. Nous avons démontré que le 5-FU entraîne une perturbation du développement de la vulve chez 30 à 40% des vers entraîne l impossibilité pour les œufs formés de sortir de l utérus. Nous avons également démontré que les taux d ARNm de lin-29, un gène impliqué dans le développement de la vulve, est réprimé par le 5-FU. Par la suite, nous avons démontré que l expression des cinq gènes gst-pi présents dans le génome de C. elegans est régulée différemment dans par des stress induits par des molécules chimiques, un choc thermique ou des agents anticancéreux. L extinction de ces différents gènes gst-pi entraine une diminution de la durée de vie des vers et une sensibilité plus importante aux molécules chimiques, au choc thermique ou aux agents anticancéreux. Le deuxième objectif de mon étude a été recherché de nouveaux partenaires protéiques de la GST Mu1 à partir de cellules d hépatome stressées ou non. L utilisation d un approche protéomique, le Seldi-Tof, nous a permis d identifier 7 partenaires potentiels qui devront être maintenant caractérisés afin de déterminer dans quels processus biologiques.The glutathione transferases (GST) are a superfamily divided in seven families. GSTs, and more particularly GST pi, have retained much attention because their overexpression has been demonstrated in various tumors and, in certain cases, are responsible of chemotherapeutic resistance. For this purpose, we have used the C. elegans model to study the effects of anticancer drugs and the role of pi class GSTs in the resistance to these drugs compared to other stress-generating conditions. We have demonstrated that 5-FU affects vulva development, some animals being vulvaless, as well as dysfunction of vulval and egg laying muscles leading to an 8 10 days delay in reproductive time. Interestingly, 5-FU represses levels of mRNA encoding LIN-29, a transcription factor that affects vulva development and egg laying system. Thereafter, we have investigated the role of the five GST pi class enzymes in protection against various conditions of stress in C. elegans. Expression of these genes is regulated by oxidative-stress generating chemicals, heat shock and three anticancer drugs. By using specific silencing of the gst-pi genes, we observe a shorter lifespan for gst-pi RNAi worms when compared to control strain. Specific repression of each gene led to increased susceptibility to stress conditions. The second objective of my thesis was to identify new protein partners of GST Mu1. By using a proteomic approach, Seldi-Tof, we found 7 putative GSTM1 protein partners. These proteins will be further characterized in order to determine their roles and whether they could represent some new therapeutic targets.RENNES1-BU Santé (352382103) / SudocSudocFranceF

Anticancer drug 5-fluorouracil induces reproductive and developmental defects in Caenorhabditis elegans.

International audienceIn order to examine the chronic effects of anticancer drug 5-fluorouracil (5-FU) on reproduction and development, we exploited Caenorhabditis elegans as a model system. We demonstrate that 5-FU induces cell-cycle arrest and apoptosis of germline cells and reduces by approximately 30-40% the number of mitotic nuclei per gonad arm when compared to untreated worms. This drug also affects vulva development, some animals being vulvaless, as well as dysfunction of vulval and egg laying muscles leading to an 8-10 days delay in reproductive time. Interestingly, 5-FU represses levels of mRNA encoding LIN-29, a transcription factor that affects vulva development and egg laying system. Finally, we demonstrate that RNAi-dependent repression of ung-1 gene, which encodes a uracil-DNA glycosylase, partially abolishes 5-FU effects on embryo hatching. Thus, we proposed that C. elegans could be a useful model system for studying the mechanisms by which 5-FU might affect either embryo, adult or organ development