Assessment of human exposure to environmental chemicals is inherently subject to uncertainty and variability. There are data gaps concerning the inventory, source, duration, and intensity of exposure as well as knowledge gaps regarding pharmacokinetics in general. These gaps result in uncertainties in exposure assessment. The uncertainties compound further with variabilities due to population variations regarding stage of life, life style, and susceptibility, etc. Use of physiologically-based pharmacokinetic (PBPK) models promises to reduce the uncertainties and enhance extrapolation between species, between routes, from high to low dose, and from acute to chronic exposure. However, fitting PBPK models is challenging because of a large number of biochemical and physiological parameters to be estimated. Many of these model parameters are non-identifiable in that their estimates cannot be uniquely determined using statistical criteria. In practice some parameters are fixed in value and some determined through mathematical calibration or computer simulation. These estimated values are subject to substantial uncertainties. The first part of this paper illustrates the use of iteratively-reweighted-nonlinear-least-squares for fitting pharmacokinetic (PK) models, highlighting some common difficulties in obtaining statistical estimates of non-identifiable parameters and use bootstrap confidence interval to quantify uncertainties. Statistical estimation of parameters in physiologically based pharmacokinetic (PBPK) models is a relatively new area of research. Over the past decade or so PBPK models have become important and valuable tools in risk assessment as these models are used to describe the absorption, distribution, metabolism, and excretion of xenobiotics in a biological system such as the human or rat. Because these models incorporate information on biological processes, they are well equipped to describe the kinetic behaviors of chemicals and are useful for extrapolation across dose routes, between species, from high-to-low-doses, and across exposure scenarios. A PBPK model has been developed based on published models in the literature to describe the absorption, distribution, metabolism, and excretion of Dioxin and dioxin like compounds (DLCs) in the rat. Data from the National Toxicology Program (NTP) two year experiment TR-526 is used to illustrate model fitting and statistical estimation of the parameters. Integrating statistical methods into risk assessments is the most efficient way to characterize the variation in parameter values. In this dissertation a Markov Chain Monte Carlo (MCMC) method is used to estimate select parameters of the system and to describe the variation of the select parameters.

Notre laboratoire s'intéresse aux mécanismes impliqués dans la régulation de l'expression génique et plus particulièrement au rôle de la chromatine dans cette régulation. En effet, chez les eucaryotes l'ADN est compactée autour de protéines appelées histones créant ainsi des nucléosomes lesquels forment une structure plus complexe, la chromatine. Cette dernière est une barrière aux processus cellulaires touchant l'ADN dont la transcription. La compréhension de la régulation de la structure de la chromatine est essentielle pour saisir les variations de l'expression génique. Mon projet de doctorat a porté sur l'étude de la régulation des gènes cibles du récepteur aryl hydrocarbone (AhR), CYP1A1 et CYP1B1, et plus particulièrement sur le rôle du variant d'histone H2A.Z dans l'expression de ces gènes. AhR est un senseur moléculaire auquel va [i.e. vont] se lier de nombreux polluants appartenant principalement à ces deux grandes familles : les hydrocarbones aromatiques halogènes (HAH) et les hydrocarbones aromatiques polycycliques (PAH). En réponse à la liaison de ces polluants, AhR va induire l'expression de ses gènes cibles. CYP1A1 et CYP1B1 sont impliquées dans le métabolisme de l'estradiol (E2) en 2hydroxyestradiol et 4-hydroxyestradiol respectivement. Il a été proposé qu'une diminution du ratio CYP1A1/CYP1B1 soit importante pour l'initiation du cancer du sein. Au cours de mon doctorat, j'ai pu mettre en évidence un rôle du variant H2A.Z dans la régulation de l'expression de CYP1A1 et CYP1B1. J'ai aussi pu montrer que le statut de ER[alpha] déterminait l'importance de H2A.Z lors de l'induction de CYP1A1. De plus, nous avons observé que la déplétion de H2A.Z induit une augmentation de la méthylation de l'ADN au promoteur de CYP1A1. En parallèle, nous avons confirmé que ER[alpha] réprime spécifiquement l'induction de CYP1A1 sans affecter celle de CYP1B1. Nos résultats montrent qu'en présence de TCDD et d'E2, ER[alpha] et DNMT3B sont recrutés au promoteur de CYP1A1, ce qui conduit à une augmentation de la méthylation du promoteur de CYP1A1 et conséquemment à une diminution de son induction. AhR possède de nombreux ligands d'origine très variée qui peuvent être aussi bien toxiques que bénéfiques. Nous avons choisi de comparer deux de ces ligands : le TCDD et le DIM. Au cours de ces travaux, nous avons montré que le DIM utilisé à forte concentration (>50[mu]M) induit les gènes cibles de AhR (CYP1A1 et CYP1B1) mais aussi un arrêt de la croissance et la mort des cellules. À l'opposé, le traitement avec des concentrations plus faible [i.e. faibles] de DIM (10[mu]M) induit principalement les gènes cibles de ER[alpha] (TFF1 et GREB1) et la prolifération des cellules. Nous avons aussi montré que l'activation de ER[alpha] par le DIM est due à l'action de la protéine kinase A (PKA). En effet, l'inhibition de la PKA ainsi que la déplétion de ER[alpha] abolissent les effets du DIM sur l'expression de GREB1 et CYPIA1 ainsi que sur la prolifération cellulaire. En conclusion, nous avons dans un premier temps mis en évidence le rôle de deux protéines, DNMT3B et H2A.Z, dans la régulation de CYP1A1 dans les cellules MCF7. Nous avons ainsi découvert un nouveau corépresseur partenaire de ER[alpha] en DNMT3B et nous avons proposé une nouvelle façon pour ER[alpha] de promouvoir la carcinogenèse en dérégulant le ratio CYP1A1/CYP1B1. Dans un deuxième temps, nous avons montré que la concentration de DIM utilisée dans les expériences peut conduire à des résultats diamétralement opposés sur la croissance cellulaire.

Orientador: Wellerson Rodrigo Scarano / Resumo: A fertilidade masculina e a espermatogênese estão diretamente ligadas à capacidade das células de Sertoli em produzir fatores associados ao desenvolvimento das células germinativas. As células de Sertoli expressam receptores para FSH e testosterona e são os principais reguladores da espermatogênese. Aproximadamente 60-70% dos casos de infertilidade masculina são considerados idiopáticos, devido aos mecanismos moleculares envolvidos na espermatogênese ainda serem desconhecidos. Estudos recentes relatam que os microRNAs (miRNAs), são capazes de modular a função testicular durante a espermatogênese e sua expressão alterada pode estar envolvida na infertilidade masculina. miRNAs podem desempenhar papel importante na resposta aos xenobióticos que têm todas as consequências adversas para a saúde. Um grupo importante de compostos orgânicos com potencial tóxico são as dioxinas, como o 2,3,7,8-tetraclorodibenzo-p-dioxina (TCDD). Modelos experimentais de exposição ao TCDD, em camundongos, demonstraram que sua exposição provoca baixa contagem de espermatozóides e atraso na puberdade. Neste estudo, analisamos o efeito do TCDD nas células de Sertoli humanas in vitro após 72h a uma dose de 10nM. Nossos resultados mostraram que as enzimas antioxidantes catalase, superóxido dismutase e glutationa peroxidase diminuíram sua atividade e confirmaram o estresse oxidativo causado pelo TCDD nesse tipo celular. 78 miRNAs apresentaram expressão alterada, com regulação positiva de 73 e regulação negat... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Male fertility and spermatogenesis are directly linked to the ability of Sertoli cells to produce factors associated with the development of germ cells. Sertoli cells express receptors for FSH and testosterone, and are the major regulators of spermatogenesis. Approximately 60-70% of male infertility cases are considered idiopathic, due to the molecular mechanisms involved in spermatogenesis are still unknown. Recent studies report that microRNAs (miRNAs) are capable of modulating spermatogenesis in testicular function and its altered expression may be involved in male infertility. miRNAs may play a role in response to xenobiotics that have all the adverse consequences for health. An important group of organic compounds that are potentially toxic are the dioxins such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Experimental models of exposure to TCDD in mice showed that its exposure causes low sperm count and delayed puberty. In this study, we analyzed the effect of TCDD on human Sertoli cells after a exposure of 72h in vitro at a dose of 10nM. Our results showed that the antioxidant enzymes catalase, superoxide dismutase and glutathione peroxidase decreased their activity and confirmed the oxidative stress caused by TCDD in this cell type. 78 miRNAs showed altered expression with upregulation of 73 miRNAs and downregulation of 5 miRNAs compared to the control group. Regarding the gene expression profile, 51 genes showed deregulated, of which 46 genes with upregulation and d... (Complete abstract click electronic access below) / Doutor

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Developmental 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) exposure can permanently alter immune system ontogeny, resulting in the dysregulation of a number of vital immune pathways. We hypothesized that developmental exposure to TCDD may also impair the establishment of self-tolerance, resulting in an increased risk of autoimmunity. For example, we observed that a single prenatal TCDD exposure given to non-autoimmune-prone high affinity aryl hydrocarbon receptor (AhR) C57BL/6 mice resulted in an immune complex-mediated autoimmune disease during the adult stage. Further using a similar TCDD exposure protocol, autoimmune-prone low affinity AhR SNF1 mice exhibited acceleration and exacerbation of lupus-like nephritis in adulthood. Examination of these mice showed that perinatal TCDD exposure adversely affected both primary immune organs of the adaptive immune system. In the thymic compartment, prenatal TCDD affected thymocyte cellularity, differentiation and maturation as well as central tolerance as indicated by high levels of autoreactive Vβ TCR T cells in the periphery. Prenatal TCDD also altered bone marrow B lymphopoiesis and B cell maturation and differentiation in the spleen. Functionally, these B cell changes resulted in high serum autoantibodies titers to dsDNA, ssDNA and cardiolipin suggesting a loss in central B cell tolerance. The functional assessment of T cells, via cytokine production showed that prenatal TCDD mice altered Th1/Th2 levels. As a result, significant changes were detected in the kidney characterized by increased immune complex deposition in the glomeruli, lymphocytic infiltration and general pathologic changes. This would suggest that multiple immune pathways are affected by prenatal TCDD and work either independently or synergistically to display immune-mediated disease during aging. Importantly, this study has also shown that the sex of an individual appears to influence both the type of immune pathways affected by TCDD as well as the progression and severity of the autoimmunity. In summary, these studies clearly demonstrate that postnatal immune system impairment due to prenatal TCDD exposure is not limited to immunosuppression but also can include inappropriate immune activation manifested as a hypersensitivity that can lead to the onset of autoimmune disease. / Ph. D.

2, 3, 7, 8-Tetrachlorodibenzo-p-dioxin is well known for its immunotoxic effects on the thymus, as well as on B and T lymphocyte functions. Previous studies suggested that TCDD exerted immunotoxic effects only on cells differentiating in response to antigenic challenge. To this date, no work has been done to characterize the long-term effects of TCDD on the activated cells. Additionally, no studies have been done to determine whether TCDD has any effect on resting T cells. In the current study, therefore, we investigated the effects of TCDD on activated and resting cells within the same animal. T cells in the popliteal lymph node cells were activated by rear footpad immunizations with anti-CD3 antibodies. Distally-located axillary lymph nodes were chosen as a source of naive and resting T cells. Our results demonstrate that TCDD acted at the time of cell differentiation to suppress the immune responses of activated T cells, but failed to suppress, and at times, enhanced the immune responses of resting T cells. The TCDD-induced immunomodulations were temporary; responsiveness of both activated and resting T cells from TCDD-treated animals returned to normal by two weeks post-treatment, suggesting that TCDD does not affect memory cells. Futhermore, we provide direct evidence that the TCDD-induced immunosuppression in activated cells is due to increased apoptosis of CD3+ T cells. TCDD also induced significant changes in cell surface markers expressed by naive and activated T cells. Together our data suggested that TCDD suppresses the proliferative responsiveness of only the activated, but not naive, T cells and that this is accomplished by induction of increased apoptosis of activated T cells. These studies shed new light on the mechanism through which TCDD induces increased susceptibility to infections and cancer in the vertebrate host. / Master of Science

During the US – Vietnam War (1961 – 1972), Vietnam was subjected to widespread spraying of the chemical herbicide that is also called Agent Orange containing the most toxic dioxin congener, of 2,3,7,8-Tetrachlorodibenzo(p)dioxin (2,3,7,8-TCDD). A Luoi district belongs to Thua Thien- Hue province, located in the western part of the North Central coast region of Vietnam. During the Ranch Hand campaign (1965 -1970), A Luoi was heavily sprayed with this herbicide. In order to assess potential human health risks for people due to 2,3,7,8-TCDD exposure from contaminated soil, more than 50 soil samples were collected in A Luoi district area in 2013 and 2014 to determine dioxin concentrations by HRGC/HRMS. Human health risk assessment was applied using internationally recognized approaches. Hazard Quotient (HQ) values, assuming 2,3,7,8-TCDD to be a threshold contaminant, were calculated to be 13.2 and 6.1; and Incremental Lifetime Cancer Risk (ILCTR) values, assuming 2,3,7,8-TCDD to be carcinogenic non threshold, were 0.00314 and 0.00627 for adults and children, respectively. These results from exposures in A Luoi show risk values, which are several hundred times higher than acceptable TRVs. The results of this study indicate that, although the war ended nearly 50 years ago, communities living in A Luoi are still at risk of residual dioxin exposure from soils contaminated. Therefore, risk management and mitigation measures are needed, including targeted soil remediation and provision of improved medical and health systems. To our knowledge, this is the first human health risk assessment (HRRA) study in areas sprayed by herbicides during the war in Vietnam. / Trong thời kỳ chiến tranh giữa Mỹ và Việt Nam (1961 - 1972), Việt Nam phải hứng chịu một lượng lớn chất diệt cỏ còn gọi là chất Da cam, trong đó chứa chất hóa học siêu độc 2,3,7,8- Tetrachlorodibenzo (p) dioxin (2,3,7,8-TCDD). Huyện A Lưới thuộc tỉnh Thừa Thiên-Huế, nằm ở phía tây của vùng duyên hải Bắc Trung Bộ Việt Nam. Trong chiến dịch Ranch Hand (1965-1970), huyện A Lưới đã nhiều lần bị phun rải chất diệt cỏ này. Trong 2 năm 2013 và 2014, hơn 50 mẫu đất và thực phẩm đã được thu thập ở khu vực huyện A Lưới và phân tích xác định nồng độ dioxin nhằm đánh giá rủi ro về sức khỏe đối với người dân sống trong vùng bị phun rải chất diệt cỏ trong chiến tranh. Nếu giả định chất 2,3,7,8-TCDD là chất độc có ngưỡng, giá trị HQ (hệ số rủi ro) tính được là 13,2 và 6,1; và nếu giả định 2,3,7,8-TCDD là chất độc gây ung thư không ngưỡng, các giá trị ILCR (nguy cơ ung thư tăng dần suốt đời) tìm được là 0,00314 và 0,00627, tương ứng đối với người lớn và trẻ em sống ở A Lưới. Khi so sánh với các giá trị TRVs (rủi ro chấp nhận được) cho thấy các giá trị rủi ro ở A Lưới cao hơn vài trăm lần. Từ kết quả này chỉ ra mặc dù chiến tranh đã kết thúc gần 50 năm trước, cộng đồng ở A Lưới vẫn có nguy cơ phơi nhiễm dioxin. Cần thiết phải sớm có các biện pháp quản lý rủi ro và giảm thiểu phơi nhiễm dioxin cho người dân, bao gồm việc xử lý đất và cung cấp các hệ thống bảo vệ môi trường, y tế và cải thiện sức khỏe. Đây là bài báo đầu tiên về đánh giá rủi ro sức khỏe cộng đồng dân cư do phơi nhiễm dioxin ở những vùng bị phun rải chất diệt cỏ trong chiến tranh.