Exposition in vitro de lymphocytes T humains aux hydrocarbures aromatiques polycycliques : étude des effets immunotoxiques

Polycyclic aromatic hydrocarbons (PAHs), such as benzo(a)pyrene (B[a]P), are ubiquitous environmental contaminants generated during organic matter combustion. These compounds have been associated with the development of toxic effects on human health, including carcinogenic and immunotoxic effects, mainly related to Aryl hydrocarbon Receptor (AhR) activation. Among the immune system cells, T lymphocytes appear as major targets of PAHs. Previous results, obtained in the laboratory, have shown that activation of primary human T lymphocytes leads to a functional AhR expression increase, suggesting their ability to respond to PAH exposure. Our specific aims are: (1) to determine the effects of B[a]P on gene expression profiles in human normal lymphocytes by using large-scale approaches such as microarray-based transcriptome analysis, (2) to monitor the genotoxic and immunotoxic effects of B[a]P by measuring DNA damage and immunosuppressive actions, respectively and, (3) to analyze the modulation of these effects by the presence of other PAHs. Our work propose primary cultures of activated human T lymphocytes as a good model for studying both genotoxic and immunotoxic effects of environmental contaminants such as PAHs and predicting human health issues. It also gains a comprehensive insight into the immune response regulation after PAH exposure and provides potential new biomarkers of exposure to these environmental contaminants.Les hydrocarbures aromatiques polycycliques (HAPs), tels que le benzo(a)pyrène (B[a]P), sont des contaminants environnementaux ubiquistes générés lors de la combustion de matière organique. Ces composés ont été associés au développement d'effets toxiques sur la santé humaine, notamment des effets cancérigènes et immunotoxiques, principalement liés à l'activation du récepteur aux hydrocarbures aromatiques (RAh). Parmi les cellules du système immunitaire, les lymphocytes T apparaissent comme des cibles majeures des HAPs. Des résultats antérieurs, obtenus au laboratoire, ont montré que l'activation des lymphocytes T humains en culture primaire conduit à l’augmentation de l'expression et de la fonction du RAh, suggérant la capacité accrue de ces cellules à répondre à une exposition aux HAPs. Nos objectifs sont : (1) de déterminer les effets du B[a]P sur les profils d'expression génique dans les lymphocytes humains normaux en utilisant des approches à haut débit telle que l'analyse transcriptomique sur puce à ADN, (2) d’évaluer les effets génotoxiques et immunotoxiques du B[a]P en mesurant respectivement les dommages à l'ADN induits et leurs actions immunosuppressives et (3) d’analyser la modulation de ces effets en présence d'autres HAPs. Notre travail identifie les lymphocytes T humains normaux comme un bon modèle pour étudier les effets génotoxiques et immunotoxiques des HAPs, et pour prédire les problèmes de santé humaine liés à l’exposition à ces contaminants. Il permet également de mieux comprendre la régulation par les HAPs de la réponse immune et propose de nouveaux biomarqueurs potentiels de l'exposition à ces contaminants environnementaux

In vitro exposure of human T lymphocytes to polycyclic aromatic hydrcarbons : study of immunotoxic effects

Les hydrocarbures aromatiques polycycliques (HAPs), tels que le benzo(a)pyrène (B[a]P), sont des contaminants environnementaux ubiquistes générés lors de la combustion de matière organique. Ces composés ont été associés au développement d'effets toxiques sur la santé humaine, notamment des effets cancérigènes et immunotoxiques, principalement liés à l'activation du récepteur aux hydrocarbures aromatiques (RAh). Parmi les cellules du système immunitaire, les lymphocytes T apparaissent comme des cibles majeures des HAPs. Des résultats antérieurs, obtenus au laboratoire, ont montré que l'activation des lymphocytes T humains en culture primaire conduit à l’augmentation de l'expression et de la fonction du RAh, suggérant la capacité accrue de ces cellules à répondre à une exposition aux HAPs. Nos objectifs sont : (1) de déterminer les effets du B[a]P sur les profils d'expression génique dans les lymphocytes humains normaux en utilisant des approches à haut débit telle que l'analyse transcriptomique sur puce à ADN, (2) d’évaluer les effets génotoxiques et immunotoxiques du B[a]P en mesurant respectivement les dommages à l'ADN induits et leurs actions immunosuppressives et (3) d’analyser la modulation de ces effets en présence d'autres HAPs. Notre travail identifie les lymphocytes T humains normaux comme un bon modèle pour étudier les effets génotoxiques et immunotoxiques des HAPs, et pour prédire les problèmes de santé humaine liés à l’exposition à ces contaminants. Il permet également de mieux comprendre la régulation par les HAPs de la réponse immune et propose de nouveaux biomarqueurs potentiels de l'exposition à ces contaminants environnementaux.Polycyclic aromatic hydrocarbons (PAHs), such as benzo(a)pyrene (B[a]P), are ubiquitous environmental contaminants generated during organic matter combustion. These compounds have been associated with the development of toxic effects on human health, including carcinogenic and immunotoxic effects, mainly related to Aryl hydrocarbon Receptor (AhR) activation. Among the immune system cells, T lymphocytes appear as major targets of PAHs. Previous results, obtained in the laboratory, have shown that activation of primary human T lymphocytes leads to a functional AhR expression increase, suggesting their ability to respond to PAH exposure. Our specific aims are: (1) to determine the effects of B[a]P on gene expression profiles in human normal lymphocytes by using large-scale approaches such as microarray-based transcriptome analysis, (2) to monitor the genotoxic and immunotoxic effects of B[a]P by measuring DNA damage and immunosuppressive actions, respectively and, (3) to analyze the modulation of these effects by the presence of other PAHs. Our work propose primary cultures of activated human T lymphocytes as a good model for studying both genotoxic and immunotoxic effects of environmental contaminants such as PAHs and predicting human health issues. It also gains a comprehensive insight into the immune response regulation after PAH exposure and provides potential new biomarkers of exposure to these environmental contaminants

Genome-Wide Transcriptional and Functional Analysis of Human T Lymphocytes Treated with Benzo[α]pyrene

International audiencePolycyclic aromatic hydrocarbons (PAHs) are widely distributed environmental contaminants, known to affect T lymphocytes. However, the molecular targets and pathways involved in their immunotoxic effects in human T lymphocytes remain unknown. Here, we analyzed the gene expression profile of primary human T lymphocytes treated with the prototypical PAH, benzo[]pyrene (B[]P), using a microarray-based transcriptome analysis. After a 48 h exposure to B[]P, we identified 158 genes differentially expressed in T lymphocytes, including not only genes well-known to be affected by PAHs such as the cytochromes P450 (CYP) 1A1 and 1B1, but also others not previously shown to be targeted by B[]P such as genes encoding the gap junction beta (GJB)-2 and 6 proteins. Functional enrichment analysis revealed that these candidates were significantly associated with the aryl hydrocarbon (AhR) and interferon (IFN) signaling pathways; a marked alteration in T lymphocyte recruitment was also observed. Using functional tests in transwell migration experiments, B[]P was then shown to significantly decrease the chemokine (C-X-C motif) ligand 12-induced chemotaxis and transendothelial migration of T lymphocytes. In total, this study opens the way to unsuspected responsive pathway of interest, i.e., T lymphocyte migration, thus providing a more thorough understanding of the molecular basis of the immunotoxicity of PAHs

Benzo[a]pyrene-induced DNA damage associated with mutagenesis in primary human activated T lymphocytes

International audiencePolycyclic aromatic hydrocarbons (PAHs), such as benzo[a]pyrene (B[a]P), are widely distributed environmental contaminants exerting toxic effects such as genotoxicity and carcinogenicity, mainly associated with aryl hydrocarbon receptor (AhR) activation and the subsequent induction of cytochromes P-450 (CYP) 1-metabolizing enzymes. We previously reported an up-regulation of AhR expression and activity in primary cultures of human T lymphocyte by a physiological activation. Despite the suggested link between exposure to PAHs and the risk of lymphoma, the potential of activated human T lymphocytes to metabolize AhR exogenous ligands such as B[a]P, and produce DNA damage has not been investigated. In the present study, we characterized the genotoxic response of primary activated T lymphocytes to B[a] P. We demonstrated that, following T lymphocyte activation, B[a]P treatment triggers a marked increase in CYP1 expression and activity generating, upon metabolic activation, DNA adducts and double-strand breaks (DSBs) after a 48-h treatment. At this time point, B[a]P also induces a DNA damage response with ataxia telangiectasia mutated kinase activation, thus producing a p53-dependent response and T lymphocyte survival. B[a]P activates DSB repair by mobilizing homologous recombination machinery but also induces gene mutations in activated human T lymphocytes which could consequently drive a cancer process. In conclusion, primary cultures of activated human T lymphocytes represent a good model for studying genotoxic effects of environmental contaminants such as PAHs, and predicting human health issues

Co-exposure to benzo[a]pyrene and ethanol induces a pathological progression of liver steatosis in vitro and in vivo

Abstract Hepatic steatosis (i.e. lipid accumulation) and steatohepatitis have been related to diverse etiologic factors, including alcohol, obesity, environmental pollutants. However, no study has so far analyzed how these different factors might interplay regarding the progression of liver diseases. The impact of the co-exposure to the environmental carcinogen benzo[a]pyrene (B[a]P) and the lifestyle-related hepatotoxicant ethanol, was thus tested on in vitro models of steatosis (human HepaRG cell line; hybrid human/rat WIF-B9 cell line), and on an in vivo model (obese zebrafish larvae). Steatosis was induced prior to chronic treatments (14, 5 or 7 days for HepaRG, WIF-B9 or zebrafish, respectively). Toxicity and inflammation were analyzed in all models; the impact of steatosis and ethanol towards B[a]P metabolism was studied in HepaRG cells. Cytotoxicity and expression of inflammation markers upon co-exposure were increased in all steatotic models, compared to non steatotic counterparts. A change of B[a]P metabolism with a decrease in detoxification was detected in HepaRG cells under these conditions. A prior steatosis therefore enhanced the toxicity of B[a]P/ethanol co-exposure in vitro and in vivo; such a co-exposure might favor the appearance of a steatohepatitis-like state, with the development of inflammation. These deleterious effects could be partly explained by B[a]P metabolism alterations

Mechanisms involved in the death of steatotic WIF-B9 hepatocytes co-exposed to benzo[a]pyrene and ethanol a possible key role for xenobiotic metabolism and nitric oxide

International audienceWe previously demonstrated that co-exposing pre-steatotic hepatocytes to benzo[a]pyrene (B[a]P), a carcinogenic environmental pollutant, and ethanol, favored cell death. Here, the intracellular mechanisms underlying this toxicity were studied. Steatotic WIF-B9 hepatocytes, obtained by a 48h-supplementation with fatty acids, were then exposed to B[a]P/ethanol (10nM/5mM, respectively) for 5 days. Nitric oxide (NO) was demonstrated to be a pivotal player in the cell death caused by the co-exposure in steatotic hepatocytes. Indeed, by scavenging NO, CPTIO treatment of co-exposed steatotic cells prevented not only the increase in DNA damage and cell death, but also the decrease in the activity of CYP1, major cytochrome P450s of B[a]P metabolism. This would then lead to an elevation of B[a]P levels, thus possibly suggesting a long-lasting stimulation of the transcription factor AhR. Besides, as NO can react with superoxide anion to produce peroxynitrite, a highly oxidative compound, the use of FeTPPS to inhibit its formation indicated its participation in DNA damage and cell death, further highlighting the important role of NO. Finally, a possible key role for AhR was pointed out by using its antagonist, CH-223191. Indeed it prevented the elevation of ADH activity, known to participate to the ethanol production of ROS, notably superoxide anion. The transcription factor, NFκB, known to be activated by ROS, was shown to be involved in the increase in iNOS expression. Altogether, these data strongly suggested cooperative mechanistic interactions between B[a]P via AhR and ethanol via ROS production, to favor cell death in the context of prior steatosis