View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by HAL-Rennes 1 Two persistent organic pollutants which act through different xenosensors (alpha-endosulfan and 2,3,7,8 tetrachlorodibenzo-p-dioxin) interact in a mixture and downregulate multiple genes involved in human hepatocyte lipid and glucose metabolism. Ariane Ambolet-Camoit, Chris Ottolenghi, Alix Leblanc, Min Ji Kim, Franck Letourneur, S´ebastien Jacques, Nicolas Cagnard, Christiane Guguen-Guillouzo, Robert Barouki, Martine Aggerbeck To cite this version: Ariane Ambolet-Camoit, Chris Ottolenghi, Alix Leblanc, Min Ji Kim, Franck Letourneur, et al.. Two persistent organic pollutants which act through different xenosensors (alpha-endosulfan and 2,3,7,8 tetrachlorodibenzo-p-dioxin) interact in a mixture and downregulate multiple genes involved in human hepatocyte lipid and glucose metabolism.. Biochimie, Elsevier, 2015, 116, pp.79-91. <10.1016/j.biochi.2015.07.003>. <hal-01174215> HAL Id: hal-01174215 https://hal-univ-rennes1.archives-ouvertes.fr/hal-01174215 Submitted on 16 Sep 2015 HAL is a multi-disciplinary open access lished or not. The documents may come from archive for the deposit and dissemination of sci- teaching and research institutions in France or entific research documents, whether they are pub- abroad, or from public or private research centers. L'archive ouverte pluridisciplinaire HAL, est ´emanant des ´etablissements d'enseignement et de destin´eeau d´ep^otet `ala diffusion de documents recherche fran¸caisou ´etrangers,des laboratoires scientifiques de niveau recherche, publi´esou non, publics ou priv´es. Two persistent organic pollutants which act through different xenosensors ( alpha- endosulfan and 2,3,7,8 tetrachlorodibenzo-p-dioxin) interact in a mixture and downregulate multiple genes involved in human hepatocyte lipid and glucose metabolism Ariane Ambolet-Camoit 1, 2, ¶, Chris Ottolenghi 1, 2, 3, ¶, Alix Leblanc 1, 2, Min Ji Kim 1, 2 , Franck Letourneur 2, 4, Sébastien Jacques 2, 4, Nicolas Cagnard 2, 4, Christiane Guguen- Guillouzo 5, 6, Robert Barouki 1, 2, 3 and Martine Aggerbeck 1, 2, * 1 Inserm UMR-S 1124, Paris, France 2 Université Paris Descartes, Sorbonne Paris Cité, Paris, France 3 APHP, Hôpital Necker Enfants Malades, Service de Biochimie Métabolique, Paris, France 4 Plateforme de Génomique, Institut Cochin, Paris, France 5 Université de Rennes 1, Rennes, France 6 Inserm, UMR991, Rennes, France * Corresponding author. UMR-S 1124 INSERM, Centre universitaire des Saints-Pères, Université Paris Descartes, 45 rue des Saints-Pères 75006 Paris, France. Tel : 33 (0)142862093. E-mail address : [email protected] ¶ Both authors contributed equally to the manuscript 1 Abstract Individuals, typically, are exposed to mixtures of environmental xenobiotics affecting multiple organs and acting through different xenosensors and pathways in species and cell- type specific manners. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and α-endosulfan are Persistent Organic Pollutants (POPs) and endocrine disruptors which act through different xenosensors and accumulate in the liver. Our objective in this HEALS study was to investigate the effects of the mixture of these POPs on gene expression in a human-derived hepatocyte cell line, HepaRG. We found that, in spite of having largely uncorrelated effects, TCDD and α-endosulfan, when mixed, alter the expression of genes. The combined effects of the mixture of the POPs significantly altered the expression of 100 genes (42 up- and 58 down-regulated) whereas the same concentration of either POP alone did not alter significantly the expression of these genes. For 32 other genes, selective inhibitory crosstalk between TCDD and α-endosulfan was observed. One of the POPs inhibited the effect, on gene expression, of the other in the mixture although, when used alone, that POP did not affect expression. The expression of another 82 genes was significantly altered (up- or down- regulated) by a single POP. The addition of the second POP either increased, in the same direction, the effect on gene expression or had no further effect. At low concentrations (0.2nM TCDD and 1µM α-endosulfan), the POPs still had significant effects and the levels of expression of the corresponding proteins were found to be affected for some genes. Particularly striking was the 80-90% inhibition, by the mixture, of the expression of a number of genes of several hepatic intermediary metabolic pathways (glycerolipid metabolism, FXR/RXR activation, glycolysis/gluconeogenesis, retinoid and bile acid biosynthesis), whereas each pollutant alone had only a moderate effect. Keywords: dioxin, pesticides, HepaRG, mixture, metabolism, microarray 2 1. Introduction Environmental exposure to toxic chemicals for most individuals involves mixtures of compounds and extended periods of exposure. Humans are exposed not only to pesticides (estimated use exceeding 3 billion tons per year worldwide), mainly through food consumption, but also to a variety of other environmental xenobiotics, many of them being endocrine disrupters, which may have a variety of detrimental effects on human health [1, 2]. Epidemiological studies often associate occupational exposure to these compounds with an increase in the incidence of various pathologies, including cancers, neuro-degenerative disorders, reduced fertility and the metabolic syndrome which can lead to type 2 diabetes [3- 10]. Although concerns about the adverse effects of mixtures of chemicals and their potential interactions are increasing, especially for vulnerable populations, most studies on the effects of pollutants to date have focused on exposure to a single compound or to a mixture of compounds that act through the same signaling pathway. Only a few studies [11-19] have addressed the toxicity of mixtures of contaminants which act through different xenosensors, even though combinations of different chemicals, especially at low levels of exposure, probably have significant effects on human health [20, 21]. In this study, part of the HEALS exposome European project, we investigated the effects of a mixture of two endocrine disruptors, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and α-endosulfan, which act via different signalling pathways, accumulate in the liver and may have non-cancerogenic toxic hepatic effects in humans [22-25]. TCDD is one of the most potent ligands of the aryl hydrocarbon receptor (AhR) and a classical one with little metabolism and a half-life of about 7 years in humans, which triggers several biological responses [1]. Alpha-endosulfan is the major isomer of an organochlorine insecticide known to act through two different signalling pathways, the pregnane X receptor (PXR) [26, 27] or the estrogen receptor α (ER α) [12, 28]. Although endosulfan use has been banned in many 3 countries, it is still in use in China and India and decades of agricultural use have built up soil reservoirs [17]. Endosulfan sulfate, which is a major metabolite of α-endosulfan is as toxic as the parent compound and more persistent with a half-life of months to years in soils, sediments and water [22, 23]. Moreover, since dioxins are by-products of chlorinated compounds, both pollutants can be associated during pesticide manufacturing. In vivo , pollutant induced toxic effects on the liver arise from direct effects on hepatocytes and from extra-hepatic factors. Among extra-hepatic factors that may alter hepatocyte function, the disruption of the endocrine system [29, 30] or pollutant induced modification of the environment of the organ may affect hepatic physiology. This may be due to the release of pro-inflammatory cytokines into the systemic circulation, the activation of hormonal or oxidative stress responses and hypoxia [31-37]. The objective of this study was to investigate the effects, related specifically to hepatocyte cell functions, of the mixture of TCDD and α-endosulfan, by studying global gene expression in a human-derived hepatocyte cell line, HepaRG. To our knowledge, few studies have explored the effects on global gene expression of a combination of two Persistent Organic Pollutants (POPs), which act through different xenosensors, using a human liver cell model. HepaRG cells were chosen for the study because, after differentiation, they express high levels of several xenobiotic metabolizing enzymes and xenosensors [38] and, to date, it is the human cell line that most closely resembles human hepatocytes [39, 40]. A recent transcriptomic study, using five carcinogens, revealed that the HepaRG model was better suited for understanding the biological effects of exposure to the chemicals as compared to the HepG2 hepatocarcinoma cell line which has a low metabolic capacity and reduced PXR level [41-43]. Several other studies also have concluded that the HepaRG cell line is an excellent in vitro model to study human drug metabolism [39, 44-46] as well as being a relevant model for studying glucose, lipid and 4 lipoprotein metabolism [47, 48]. The use of the HepaRG cell line, which also exhibits stable and inducible enzyme expression over long periods (weeks), and has reduced variability, thus avoids many of the difficulties associated with the use of human hepatocytes such as scarce availability, complicated isolation procedures, variability, rapid dedifferentiation precluding long term use and cost [49-52]. This study was designed to provide information on the combined effects of TCDD and the pesticide α-endosulfan on genome-wide gene expression in one of the