Dioxins such as the potent 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are persistent environmental contaminants, exposure to which results in a number of toxic effects in several species. In mice, TCDD-induced hydronephrosis of the kidney is among the most sensitive of developmental abnormalities. It is widely accepted that the toxic effects of dioxins such as TCDD are mediated by the aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor expressed in virtually all tissues. Though it is known that activation of the AHR during development is likely the first step in TCDD-induced hydronephrosis, genetic events occurring downstream of AHR activation remain to be elucidated. To this end, we investigated the effect of TCDD on gene expression during development of the mouse kidney through use of expression arrays. Our analysis indicates that TCDD alters the expression of genes involved in the developmentally-critical Wnt signaling pathway and genes involved in cell cycle and cell proliferation. Further investigation of the effect of TCDD on Wnt pathway gene expression using an mRNA expression time course and in situ hybridization to characterize localized mRNA changes suggests that TCDD inhibits canonical Wnt signaling in the developing kidney. Consistent with our expression array finding that TCDD downregulates genes involved in cell proliferation, we also report a decrease in cell proliferation that coincides with regions of the kidney where inhibitors of canonical Wnt signaling are induced in response to TCDD. TCDD-induced disruption of Wnt pathway gene expression is not limited to the kidney, and alteration of Wnt gene expression is tissue-specific with varying effects observed in the heart, lung, liver, skin, and brain. Preliminary evidence also indicates that exposure to TCDD during development can alter the expression of microRNAs predicted to target Wnt pathway genes, suggesting a new mechanism for TCDD-induced disruption of gene expression. Taken together, our findings suggest that TCDD may disrupt an important pathway that is critical to the normal development of several organs in numerous species.

Dioxins such as the potent 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are persistent environmental contaminants, exposure to which results in a number of toxic effects in several species. In mice, TCDD-induced hydronephrosis of the kidney is among the most sensitive of developmental abnormalities. It is widely accepted that the toxic effects of dioxins such as TCDD are mediated by the aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor expressed in virtually all tissues. Though it is known that activation of the AHR during development is likely the first step in TCDD-induced hydronephrosis, genetic events occurring downstream of AHR activation remain to be elucidated. To this end, we investigated the effect of TCDD on gene expression during development of the mouse kidney through use of expression arrays. Our analysis indicates that TCDD alters the expression of genes involved in the developmentally-critical Wnt signaling pathway and genes involved in cell cycle and cell proliferation. Further investigation of the effect of TCDD on Wnt pathway gene expression using an mRNA expression time course and in situ hybridization to characterize localized mRNA changes suggests that TCDD inhibits canonical Wnt signaling in the developing kidney. Consistent with our expression array finding that TCDD downregulates genes involved in cell proliferation, we also report a decrease in cell proliferation that coincides with regions of the kidney where inhibitors of canonical Wnt signaling are induced in response to TCDD. TCDD-induced disruption of Wnt pathway gene expression is not limited to the kidney, and alteration of Wnt gene expression is tissue-specific with varying effects observed in the heart, lung, liver, skin, and brain. Preliminary evidence also indicates that exposure to TCDD during development can alter the expression of microRNAs predicted to target Wnt pathway genes, suggesting a new mechanism for TCDD-induced disruption of gene expression. Taken together, our findings suggest that TCDD may disrupt an important pathway that is critical to the normal development of several organs in numerous species.

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Previous experiments have demonstrated that the hepatoxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) involves synergism with iron. It has been postulated that it is storage iron that plays a role in toxic effects observed with TCDD poisoning. It was hypothesized that TCDD induction of the Ah gene locus in some way leads to mobilization of storage iron from the iron storage protein ferritin. Microsomal ferritin iron release was investigated in intact microsomes and in a reconstituted system. Evidence is presented which suggests that NADPH-cytochrome c (P450) reductase is capable of effecting the release of iron from flavin (FMN), and that at the same time, the reduced flavin thus generated is capable of effecting the release of iron from ferritin. While no direct evidence was obtained, the results do lend support to the hypothesis that TCDD toxicity could result from the mobilization of iron from ferritin by the TCDD induced microsomal electron transport system. / Thesis / Master of Science (MSc)

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a highly toxic environmental pollutant and is well known for its immunotoxic effects, particularly on the thymus. The exact mechanism by which TCDD induces thymic atrophy is not known. In the current study, we investigated whether TCDD triggers apoptosis in the thymocytes and whether Fas and Fas ligand play a role in TCDD-mediated immunotoxicity. Administration of a single dose of TCDD at 0.1, 1, 5 or 50 mg/kg body weight intraperitoneally into C57BL/6 +/+ mice caused a significant dose-dependent decrease in the thymic cellularity; whereas, in the C57BL/6 lpr/lpr (lpr) (Fas-deficient) and C57BL/6 gld/gld (gld) (Fas ligand-defective) mice, TCDD failed to induce a decrease in thymic cellularity at doses of 0.1-5 mg/kg body weight. In the lpr and gld mice, thymic atrophy was seen only at 50 mg/kg body weight of TCDD. Significant apoptosis was detected within 8-12 hours after injection in the wild type mice, whereas, in the lpr and gld mice apoptosis could not be detected. Upon culturing the thymocytes from TCDD-treated mice for 24 hours in vitro, the wild-type cells showed increased apoptosis when compared to the control; whereas, similar cells from lpr and gld mice did not show apoptosis. Furthermore, TCDD-treatment caused significant alterations in the expression of surface molecules on the thymocytes in the wild-type mice and minimal changes in the lpr or gld mice. Sera from TCDD-treated wild-type mice also exhibited increased levels of soluble Fas ligand. Also, TCDD-induced apoptosis was inhibited both in vitro and in vivo by caspase inhibitors and other inhibitors of apoptosis. Together, the current study demonstrates that TCDD-induced apoptosis plays an important role in thymic atrophy caused by TCDD in vivo. Furthermore, phenotypic changes in the density of thymocyte surface molecules may serve as a useful biomarker for chemical toxicity involving apoptosis. The current study also demonstrates that Fas-Fas ligand interactions play an important role in the induction of apoptosis and immunotoxicity by TCDD. / Ph. D.

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L’impact de l’environnement dans le développement de certaines pathologies est avéré bien qu’il soit difficile à évaluer. L’Homme est chroniquement exposé à des cocktails de xénobiotiques -des molécules étrangères à l’organisme- dont la nature, la concentration et les interactions sont variables avec des effets néfastes ou bénéfiques pour la santé humaine. Parmi ceux-ci, les polluants environnementaux jouent un rôle non négligeable dans l’apparition de certains cancers. Au contraire, des molécules naturelles comme le resvératrol, possède des propriétés anti-cancéreuses. Au cours de la cancérogenèse, les cellules tumorales acquièrent un phénotype métabolique réversible caractérisé par une glycolyse et une production de lactate élevées en présence ou non d’oxygène (effet Warburg). La flexibilité du métabolisme permet aux cellules cancéreuses d’assurer des niveaux d’énergie et de métabolites et de co-facteurs suffisants pour maintenir leur phénotype tumoral dans un microenvironnement qui fluctue. Dans cette étude, les effets de différents xénobiotiques seuls ou en mélange ont été évalués sur la progression du phénotype tumoral dans des cellules tumorales humaines. Dans une 1ère partie, les effets de 2 polluants organiques persistants agissant via des voies de signalisation différentes, la tétrachlorodibenzo-para-dioxine (TCDD) et l’a-endosulfan, un pesticide organochloré, ont été étudiés, seuls ou en mélange, sur le phénotype tumoral des cellules tumorales coliques humaines (Caco2). Nous avons montré que la TCDD (25 nM) et l’a-endosulfan (10µM) diminuent les capacités oxydatives des cellules tumorales. Cet effet est plus marqué lorsque les cellules sont exposées au mélange des 2 polluants, suggérant un effet synergique. Ces altérations sont associées à une diminution drastique de la respiration mitochondriale, corrélée à une forte réduction de l’activité du complexe I de la chaine respiratoire des mitochondries. Ces observations sont en partie liées à une diminution de l’expression de NDUFS3, l’une des sous unité du complexe I. Parallèlement, nous avons mis en évidence que l’exposition aux polluants entraine l’engagement des cellules tumorales vers une transition épithélio-mésenchymateuse (EMT). Ces données suggèrent que les polluants favorisent la progression tumorale en altérant le métabolisme énergétique des cellules tumorales coliques humaines et en favorisant la mise en place d’une EMT. Le lien entre ces 2 processus ainsi que les voies de signalisation impliquées restent à élucider. Dans une 2ème partie, nous avons évalué les effets du resvératrol (RSV), un composé naturel présent entre autre dans le vin, sur sur la progression du phénotype tumoral. Ce polyphénol a été largement décrit pour ses effets bénéfiques sur le cancer et pour ses capacités à modifier le métabolisme, notamment en mimant la restriction calorique. Nous avons montré que le RSV, à une concentration proche des doses sériques mesurées chez l’Homme (10 µM), diminue la prolifération cellulaire sans affecter la viabilité. Le RSV réoriente le métabolisme énergétique des cellules tumorales en augmentant leurs capacités oxydatives et en diminuant leurs capacités glycolytiques et l’activité de la voie des pentoses phosphates. Nous avons identifié le complexe pyruvate déshydrogénase comme une cible du RSV, mis en évidence que le calcium est impliqué dans cette régulation et que les effets métaboliques induits par le RSV sont relayés en partie par la voie CamKKß/AMPK. L’ensemble de ces résultats démontre que l’environnement, via les xénobiotiques, peut moduler le phénotype tumoral, et que le métabolisme tumoral, en raison de son extrême flexibilité, est une cible majeure de ces modulations. / The impact of the environment in the development of several human diseases is well established but difficult to evaluate. Humans are chronically exposed to xenobiotics mixture - foreign chemicals substances which are not normally present within the organism – with different nature, concentration and interactions leading to deleterious or beneficial effects on human health. Among these xenobiotics, environmental pollutants play a significant role in the development of some cancers. On the contrary, a natural molecule like resveratrol has anti-cancer properties. During carcinogenesis, tumor cells acquire a reversible metabolic phenotype characterized by a high glycolysis and a massive lactate production with or without oxygen (Warburg effect). The flexibility of the metabolism allows cancer cells to provide sufficient levels of energy, metabolites and cofactors to maintain their tumor phenotype in a fluctuating microenvironment. In this study, the effects of several xenobiotics alone or in a mixture were assessed on human cancer cell progression. In the first part, the effects of 2 persistent organic pollutants acting by different signaling pathways, tetrachlorodibenzo-para-dioxin (TCDD) and a-endosulfan, an organochlorine pesticide, were studied alone or in a mixture, on human colonic cancer cells (Caco2) progression. We have shown that TCDD (25 nM) and a-endosulfan (10µM) decrease the oxidative capacity of tumor cells. This effect is more pronounced when cells are exposed to the mixture, suggesting a synergistic effect. These alterations are associated with a drastic decrease in mitochondrial respiration, correlated with a strong reduction in the activity of the mitochondrial respiratory chain complex I. These observations are in part linked to a decrease of NDUFS3 gene expression, one of the subunit of the complex I. We have also found that the dysregulation of tumor cell metabolism was associated with an epithelial-mesenchymal transition (EMT). Our data show that pollutants strengthen the Warburg effect associated with an EMT, which suggests that the pollutants affect the progression of the tumor phenotype. The signaling pathways involved in these observations are under investigation. In the second part, we assessed the effects of resveratrol (RSV), a natural compound present among other in wine, on cancer cell progression. This polyphenol has been widely described for its benefits on cancer and its ability to mimic caloric restriction. We have shown that the RSV, with a close concentration of serum doses measured in humans (10 µM), decreases cell proliferation without modulate cell viability. RSV redirects the energy metabolism of tumor cells by increasing their oxidative capacity, decreasing their glycolytic capacity and reducing the activity of the pentose phosphate pathway. We have identified the complex pyruvate dehydrogenase as a target of the RSV and highlighted that the calcium is involved in the regulation of PDH activity. We have also shown that the RSV induces its metabolic effects in part through CamKKß/AMPK signaling pathway. These results demonstrate that the xenobiotics can modulate tumor phenotype, and tumor metabolism, because of its extreme flexibility, is a major target of these modulations.