Phthalate esters (PEs) and organophosphate esters (OPEs) are common indoor pollutants frequently detected in environmental (dust, air), personal (hand wipes, diet) and human matrices (urine, serum etc.). In this thesis, mathematical models were used to establish links between intake and body burden for a comprehensive dataset based on a Norwegian study population. Also, the relative importance of different PE uptake pathways was assessed and discussed. Furthermore, the suitability of human nails as an alternative, non-invasive biomonitoring matrix for PEs was investigated. Additionally, information regarding alternative plasticizers to PEs was collected and presented extensively. Results showed that for PEs (paper II), daily intakes based on external exposure media agree with back-calculations using urinary metabolite concentrations, leading to the conclusion that human exposure for the general adult population is well understood and that the most important uptake routes were captured. Overall intake levels are comparable or lower than level presented in recent comprehensive studies and hazard quotients were well below 1 (low risk). As expected, diet was found to be the most important uptake route for all PEs. For lower molecular weight PEs, inhalation becomes a strong contributing pathway whereas for higher molecular weight PEs, dust ingestion was also important. Daily intake based on hand wipes was found to be much lower than the estimated total dermal intake based on air, dust and personal care products, questioning the relevance of hand wipes to represent total dermal exposure. Human nails were found to be unsuitable for replacing urine as a biomonitoring matrix for PEs as internal intake (from blood) cannot explain measured nail concentrations and uptake from air is too slow to reach observed concentrations within a realistic time frame (paper III). Hence, the kinetic links between intake and nail concentrations could not be established. Although exposure to traditional PEs is decreasing, use and body burden of some alternatives are increasing (paper I). Fortunately, most alternative plasticizers have favorable toxicological properties, resulting in low risk for humans. In contrast to PEs, OPEs still remain a group of poorly studied substances in terms of human exposure (paper IV). Due to lack of information regarding human metabolism, reliable links between intake and concentrations in serum and urine could not be established. Modelling results showed that concentrations in serum, and to some extent, urine, were underestimated for 2 compounds. It is likely that a combination of missing intake and suboptimal biomarkers were the cause for this under-prediction. Because of this, further studies regarding human metabolism should be performed for OPEs and potentially more specific biomarkers identified in the future. For PEs, there is a need for more comprehensive datasets to study exposure for high risk groups such as infants and children. Furthermore, dermal uptake remains poorly understood and the uptake of PEs into human nails should be studied in more detail to establish the kinetic links between exposure and body burden. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 4: Manuscript.</p>

Les phtalates et les alkylphénols sont des composés organiques générés par l'industrie chimique, qui entrent dans la composition des matières plastiques, pour les phtalates, et des détergents, pour les alkylphénols. Leurs productions mondiales avoisinent les 6 millions de tonnes pour les phtalates et 500 000 tonnes pour les alkylphénols. Ces composés sont considérés comme ubiquitaires dans l'environnement, c'est-à-dire qu'ils sont retrouvés dans tous les compartiments environnementaux (de l'atmosphère au milieu naturel). Par ailleurs, ils sont considérés comme des perturbateurs endocriniens et peuvent donc causer des dommages aux faunes et flores présentes dans le milieu naturel. Ce travail de thèse a permis de déterminer les niveaux de contamination des eaux usées en phtalates et alkylphénols dans plusieurs compartiments environnementaux situés en zone urbaine. Ainsi, des prélèvements effectués, par temps sec, ont permis de déterminer la qualité des eaux brutes vis-à-vis des paramètres globaux, des phtalates et des alkylphénols dans les rejets industriels, domestiques, les émissaires parisiens ainsi que dans les eaux usées transitant au sein d'une station d'épuration majeure d'Ile-de-France (Seine Centre, Colombes, 92). Ces résultats reflètent certaines spécificités et disparités du réseau d'assainissement, notamment marqués par la forte variabilité qui entoure, à la fois, le prélèvement et les niveaux de contamination par ces micropolluants. Au niveau des rejets industriels, de forts contrastes ont été observés entre les différents secteurs répertoriés (traitement des textiles, traitement de surface, métallurgie, industries pharmaceutiques, etc.). Globalement, les résultats des flux par équivalent-habitant soulignent que les apports domestiques sont la première source de contamination pour ces polluants en région parisienne. En effet, la contribution des apports industriels est inférieure à 5 %, pour tous les composés, quelque soit le secteur industriel considéré. L'étude des émissaires parisiens a permis de montrer une certaine stabilité temporelle de la qualité des effluents dans la partie avale du réseau d'assainissement. En revanche, cette étude a également mis en exergue des divergences et des similitudes entre les sous-bassins versants drainés par les différents émissaires. Ainsi, une étude statistique a montré que les émissaires Saint Denis Achères et Clichy Achères Argenteuil apparaissent significativement plus contaminés que les émissaires Sèvres Achères Reuil et Clichy Achères Bezons. Au niveau de l'efficacité des ouvrages de traitement, les résultats obtenus pour la station d'épuration de Seine Centre ont confirmé son efficacité pour les paramètres globaux communément utilisés pour déterminer la qualité des eaux usées. D'autre part, les résultats obtenus sur l'abattement des phtalates et des alkylphénols (rendements supérieurs à 83 % pour tous les composés) soulignent que même si les stations d'épuration ne sont, initialement, pas construites pour réduire et/ou éliminer les micropolluants, elles y parviennent de façon significative. A l'échelle des ouvrages, le traitement primaire par décantation physico-chimique lamellaire a permis de mettre en évidence l'élimination des polluants hydrophobes tels que le DEHP, le NP, le DnBP et le BBP. Le traitement secondaire par biofiltration a mis en exergue son efficacité à abattre significativement tous les polluants, aussi bien les composés légers (DEP) que les autres composés / Phthalates and alkylphenols are organic molecules generated by the chemical industry, entering the composition of plastics, for phthalates, and detergents for alkylphenols. Their worldwide productions are around 6 million tons and 500,000 tons, respectively. It is now obvious from the concentrations and contents measured in the various environmental compartments that phthalates and alkylphenols are ubiquitous environmental contaminants. Indeed, they are found in all environmental compartments (from the atmosphere to the natural environment) at significant levels. These contaminants are also recognized as endocrine disrupting compounds and can cause damages to fauna and flora present in the natural environment. This work has identified levels of wastewater contamination of phthalates and alkylphenols in various environmental compartments in urban areas. Thus, samples taken, during dry weather periods, were used to determine the quality of raw water for global parameters, phthalates and alkylphenols within industrial and domestical discharges as well as the sewer network and a major Parisian sewage treatment plant (Seine Centre, Colombes, 92). These results reflect certain characteristics and disparities of the sewer network, including both high variability of the sampling and the contamination levels by these micropollutants. For industrial discharges, significant disparities have been observed between the different sectors listed (textile manufacturing, surface treatment, metallurgy, pharmaceutical industries, etc.). Overall, the results confirm that household inputs correspond to the first source of contamination in Parisian conurbation. Indeed, the contribution of industrial inputs is less than 5%, for all compounds, whatever the industrial sector. The study of the Parisian sewer network showed some temporal stability of wastewaters in the downstream part. This work however also highlighted disparities and similarities between the sub-watersheds drained by the sewer network. Thus, the northern part of the Parisian network (SDA and CAA) appears to be more contaminated than the Southern part (CAB and SAR).The results obtained for Seine Centre wastewater treatment plant (WWTP) confirms the effectiveness of the treatment processes on the removal on the routine water quality parameters. Additionally, the results on the elimination of phthalates and alkylphenols in sewage treatment plant (yields above 83% for all compounds) confirm that even if WWTP are initially not designed for reducing and/or eliminating micropollutants, they proceed significantly. During primary treatment, physicochemical lamellar clarification underlined the removal of hydrophobic pollutants such as DEHP, NP, DnBP and BBP. During the secondary treatment, biofiltration highlighted its effectiveness in eliminating all pollutants, as well as volatile compounds (DEP) and other contaminants

Les phtalates (PAEs) sont un groupe de contaminants organiques émergents qui sont devenus une préoccupation majeure en raison de leur omniprésence dans le milieu marin et de leurs effets perturbateurs sur le système endocrinien. Cependant, leur répartition en Méditerranée est assez mal connue, principalement en raison des difficultés analytiques et du risque élevé de contamination des échantillons lors du processus analytique. En outre, les débris de plastique en milieu marin contiennent des additifs comme les PAEs, qui sont ajoutés lors de l’étape de fabrication du plastique. Dans la première partie de cette thèse, une méthode analytique a été optimisée et validée pour la détermination de 8 PAEs à l'état de traces en milieu marin. Brièvement, 1L d'échantillon filtré est passé à travers des cartouches de verre contenant 200 mg d'Oasis HLB, puis élué à l’aide de 6mL d'acétate d'éthyle. Notre méthode donne des rendements d’extraction compris entre 95 et 115 % et des blancs acceptables. Dans un second temps, un suivi annuel de la contamination de la Baie de Marseille a été effectué de décembre 2013 à novembre 2014. Les résultats ont montré la présence de 7 PAEs, avec des concentrations totales variant de 130 à 1330 ng L-1. Dans la dernière partie de cette thèse, deux échantillons de plastique communs (sac de poubelle et gaine de câble électrique) ont été incubés dans de l'eau de mer naturelle dans des conditions contrôlées de laboratoire, de manière à évaluer la migration de PAEs depuis ces matériaux en fonction de différentes conditions environnementales comme la température, la présence de lumière, ou encore la présence de bactéries. / Phthalic Acid Esters (PAEs) are a group of emerging organic contaminants that have become a serious issue because of their ubiquitous presence on the marine environment worldwide and for the endocrine disrupting effects in animals and humans. However, little is known about their distribution in the Ocean, mainly because of analytical difficulties and the high possibility of ambient sample contamination. Plastic debris in marine environment includes resin pellets, macro- and microplastic fragments, and contains additives such as PAEs that might be released in the aquatic environment in unknown proportion. In our work, we improved an analytical method for the determination of 8 PAEs, at trace levels in marine and fresh waters. In the first part of the thesis, the method was validated for different seawater salinities and wastewater treatment plant outlet. By passing 1 L of sample through glass cartridges packed with 200 mg of Oasis HLB and eluted with 6 mL of ethyl acetate, the recoveries of 8 PAEs ranged from 95 to 115 % with acceptable blank values (below 0.4-4.0 % of the masses measured in different seawater samples). In a second part, PAE distribution in the Marseille Bay has been evaluated though a one-year monitoring from December 2013 to November 2014. The results showed the occurrence of six PAEs with total concentrations ranging from 130 to 1330 ng L-1. In the last part of this thesis, two common plastic samples as trash plastic bags and insulation layer of electric cables were incubated in natural seawater in laboratory conditions and studied for the release of PAE. Release ws observed for the different PAEs with kinetics varying according to the plastic type.

Obesity is a public health problem. The global obesity problem has been attributed to unhealthy diet, inactivity, and hereditary factors. However, phthalates may also contribute to the obesity epidemic. The purpose of this cross-sectional study was to examine whether there was a relationship between exposure to phthalates and obesity among adults 18 years and older in the United States. The theoretical framework was based on the socioecological model. The quantitative, cross-sectional design, and data analysis were based on the National Health and Nutrition Examination Survey, 2011-2012 database. Descriptive statistics and multiple regression analysis were used to analyze the association between phthalates, obesity, and other independent variables (phthalates exposure, socioeconomic status, gender, age, race/ethnicity, physical activity and place of residence) According to study results, exposure to phthalates does not have a statistically significant effect on the odds of obesity. In terms of the influence of the sociodemographic variables such as race, age, and gender on the relationship between total phthalate exposure and obesity, only race showed statistical significance at p < 0.05. In addition, physical activity did not have influence on relationship between total phthalate exposure and obesity among adults 18 years and older in the United States. Despite the lack of significant relationships, the results of this study are useful in addressing the concerns about the exacerbation of obesity. The results of this study could enhance academic research by showing the effect of phthalate chemical exposure, age, gender, race/ethnicity, physical activity, income, and educational level on obesity.

A number of investigations during the last two decades describe adverse trends in male reproductive health, which have been proposed to be caused by environmental factors with endocrine disrupting properties. In contrast to many other toxicants, endocrine disruptors often do not show linear dose-response relationships typical of those found in traditional toxicological studies. For many compounds, low-dose exposure causes effects opposite to the ones seen after high-dose exposure. In addition, the timing of exposure has been found to be critical. Hence, to correctly assess the impact of endocrine disruptors on reproductive health requires in-depth knowledge of their mechanisms of action. This thesis aimed at identifying the mechanisms underlying the effects of cadmium (Cd), phthalates and phytoestrogens on testicular steroidogenesis. For this purpose, in vitro as well as in vivo models were used. Cd was found to inhibit testosterone synthesis in vivo by down-regulating LH receptor gene expression and reducing the testicular levels of cAMP and StAR protein. In addition, Cd caused a pronounced increase in testicular prostaglandin F2ɑ (PGF2ɑ), suggesting that Cd exerts its suppressive effect on steroidogenesis also by inducing the inhibitory PKC pathway. Pre-treatment with zinc (Zn) protected completely against Cd-induced effects on testosterone and PGF2ɑ. Furthermore, we observed that Cd exposure increased glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression in the testis. GAPDH is a potent coactivator of androgen receptor-mediated transcription and the up-regulation found in our study is probably a compensatory response to reduced testosterone concentrations. This finding is interesting since GAPDH has been proposed to have an important role in the regulation of apoptosis as well as sperm motility. We discovered that mono-(2-ethylhexyl) phthalate (MEHP), the active metabolite of the frequently used phthalate di-(2-ethylhexyl) phthalate (DEHP), stimulates Leydig cell steroidogenesis in vitro, by a cAMP- and StAR-independent mechanism. MEHP exposure caused a similar effect in granulosa cells. Gene expression analysis revealed that MEHP is likely to stimulate steroidogenesis by increasing the amount of cholesterol available for steroid synthesis. In the last investigation, we examined the effects of low-dose phytoestrogen exposure on testosterone synthesis during puberty in male goats. Isoflavones present in clover increased plasma concentrations of testosterone and free as well as total triiodothyronine (T3). T3 has previously been shown to induce testosterone synthesis and it is possible that an elevated T3 secretion underlies the increased plasma testosterone levels. Reduced fertility and reproductive tract malformations affect both the individual and the society. Hence, a sound knowledge of reproductive toxicants is of crucial importance. The findings presented in this thesis provide new insights into the reproductive toxicology of endocrine disruptors and may be valuable for risk assessment purposes.

Phthalates are industrial plasticizers with a wide range of applications. Di-(2-ethylhexyl) phthalate (DEHP) is one of the most highly produced and frequently studied phthalates. Its metabolite, mono-(2-ethylhexyl) phthalate (MEHP) is known as a testicular toxicant. The objective of this study was to examine expression of the genes of interest in testicular germ cells exposed to MEHP in a dose- and time-dependent manner at concentrations of 1µM, 10µM, and 100µM at 24, 48, 72 and 96hr time points. The genes consisted of Testisin, GSPT1, and MGMT genes which are a tumor suppressors, phase II xenobiotic metabolizing enzyme and DNA repair gene respectively. These genes were analyzed by Quantitative Real Time PCR (RT-PCR). The results revealed an overall down-regulation for each gene as the concentration and/or time increased. Testisin was the focus of the gene expression analysis. Testisin is epigenetically silenced in testicular germ cell tumors (TGCT) by DNA methylation at the 5’CpG island of the gene. To investigate if MEHP is capable of DNA hypermethylation, a co-exposure with 5-azacytidine (demethylating agent) was conducted. Compared with the 5-azacytidine treatment alone, there was a significant down-regulation of the Testisin gene in the co-exposure. This suggests that MEHP may down-regulate Testisin gene expression by DNA methylation. These findings provide evidence that MEHP can alter the expression of Testisin, GSTP1 and MGMT, genes that are associated in the risk of developing testicular germ cell tumors. In addition, results indicated that MEHP may cause DNA methylation leading to the down-regulation/silencing of genes such as Testisin.

Ambient ionization mass spectrometry, which has witnessed a flurry of recent developments, is a set of useful techniques for the analysis of samples under open-air conditions. It allows direct, rapid, real-time, high-throughput analysis with little or no sample pretreatment for the chemicals in solids or liquids. In this study, thermal desorption electrospray ionization mass spectrometry ( TD-ESI/MS ) involving direct insertion probe ( DIP ), thermal desorption ( TD ) and electrospray ionization ( ESI ) was used for the rapid screening of various types of samples. The source mainly consists of the sampling probe device, thermal desorption heating device, electrospray ionization device, ion source and temperature controller. A novel strategy involved in TD-ESI/MS processes where sampling, desorption, and ionization are separated as three independent events. The sampling probe is first used for the sampling of analytes and then inserted into a heat unit for thermal desorption. The desorbed analytes are finally carried into a reaction region with a stream of nitrogen gas, where charged methanol droplets were generated continuously by electrospray for post-ionization. Total analysis time is less than 10 seconds. Traditionally, three standard methods are used for the analysis for pesticide residues, biochemical, immunoassay and instrument. And, the instrument analysis is the most widely used because it provides lots of advantages in particularly accurate quantitative approach. However, its complicated steps take a long period of time for preparation. Here, we used TD-ESI/MS to rapidly screen the pesticide residues on the surface of fruits and vegetables. The MS/MS analysis was also performed to confirm those detected compounds. The experimental results of the standard deviation for reproducibility is 13.2% (n = 10), and the detection limit is approximately 10 ppb. Furthermore, several fruits and vegetables purchased from local market were used as test samples and pesticide residues on the surface of samples can be successfully detected via TD-ESI/MS. In addition, the TD-ESI/MS technique was also applied to the analysis of illegal additives or phthalates in food. In this study, the TD-ESI/MS technique emerges lots of advantages such as direct, rapid, real-time analysis of sample surface and sample pretreatment is not necessary, and shows highly potential for rapid screening of chemicals in food safety.

Phthalates are a class of ubiquitous environmental contaminants that cause health problems including reproductive disorders, asthma, and obesity. Advocacy against phthalates has been taking place in the US since the mid-1990s, and eight in-depth interviews were conducted with advocates and scientists in order to construct a history of this advocacy. There have been a variety of campaigns and victories; those around medical devices, children’s products, and personal care products are examined in detail. Phthalate exposure data for a representative sample of the US population indicates that exposure to DEP, DEHP, DnBP, and BBzP went down between 1999 and 2010. As these were the phthalates that had the largest volume of advocacy during the period researched, this decrease suggests that advocacy around specific phthalates is effective in reducing exposure and that more advocacy around phthalates, and potentially other harmful chemicals, could result in further decreased exposure and improved health in the US population. Additional research using more finely graded biomonitoring data would help deepen understanding about correlations between advocacy and phthalate exposure. In reviewing the health effects of phthalates, it was found that a disproportionate amount of the research is on male reproductive health effects, which is partially responsible for the fact that a disproportionate amount of phthalate advocacy is on heath effects relating to men, particularly male babies. Both phthalate science and advocacy sometimes treat women instrumentally, objectifying them or regarding them as incubators. To combat this, scientists could do more research on the health effects of phthalates on women and advocates could take more care not to neglect or instrumentalize women in their efforts to reduce phthalate exposure for all people.

Abstract Peroxisome proliferator-activated receptors (PPARs) are transcription factors that belong to the family of nuclear hormone receptors and exist as three isoforms namely PPARα, PPARβ and PPARγ. PPARs function as key regulators of glucose and lipid metabolism and are potential targets for drugs used in the treatment of glucose and lipid metabolism dysregulation. PPARs also regulate the expression of genes involved in the process of cellular proliferation and differentiation. Since it was discovered that PPAR ligands cause liver tumourigenesis in rodents, PPARs and their modulators have been investigated widely in in vitro and in vivo studies of carcinogenesis of the liver, colon, prostate, lung and skin. PPARα and PPARγ are the most studied PPAR isoforms in relation to cancer, while the association of PPARβ with cancer is increasingly being investigated. Some studies suggest that PPARβ and its ligands may have anticancer activity, while other studies identify a role for PPARβ in tumour promotion and progression. Breast cancer is one of the most common types of cancer in women with the majority caused by non-hereditary mechanisms. The activation of PPARα in breast cancer cells is associated with an increase in proliferation, while PPARγ activation in breast cancer cells is related to differentiation and an inhibition of cell proliferation. The role of PPARβ and its modulators in breast cancer is uncertain, as there have been limited studies addressing the effects of PPARβ modulation in breast cancer cell lines. Environmental contaminants such as the phthalate plasticizers and alcohol are putative risk factors for breast cancer. The phthalates di(2-ethylhexyl)phthalate (DEHP) and di-n-butyl phthalate (DBP) are plasticizers that are used in a range of common household, medical and beauty products and as a consequence humans are exposed to significant levels of these compounds. DEHP and DBP are known teratogens in rodents and DEHP induces hepatocarcinogenesis in a process thought to be mediated via PPARα. DEHP and DBP are metabolized in vivo by esterases to the monoesters, mono-(2-ethylhexyl) phthalate (MEHP) and mono-n-butyl phthalate (MBP), and these compounds have been identified in human biological samples. MEHP and MBP modulate PPARs in various tissues and cell types, but their ability to modulate PPARs in human breast cancer cells is not known. Like phthalates, ethanol is another modulator of PPARs and alcohol consumption is associated positively with breast cancer development, but the molecular mechanisms involved are unknown and there are no studies that examine the effects of ethanol and its metabolite acetaldehyde on PPARs in breast cancer cell lines. This thesis describes studies establishing and validating a breast cancer cell line that conditionally expresses human PPARβ under the control of a tetracycline regulator. Using this model, the ability of PPARβ over-expression and/or activation by the PPARβ specific ligand GW0742 to promote breast cancer cell proliferation was studied. Furthermore, putative PPARβ regulated genes were examined for alterations in expression in the presence of the PPARβ ligand. This work determined that over-expression of PPARβ and/or its activation by GW0742 does not promote proliferation in MCF-7 breast cancer cells. This thesis also investigated the effects of the phthalate monoesters MEHP and MBP on PPARs in MCF-7 breast cancer cells. It was found that MEHP activated both PPARα and PPARγ but was unable to activate PPARβ, whereas MBP could not activate any of the PPAR isoforms. MBP was an antagonist for both PPARγ and PPARβ. Using breast cancer cell lines, studies were conducted addressing the effects of an increasing concentration of ethanol (0-300 mM) on the transcription and transactivation of PPARα and PPARβ isoforms. Estrogen receptor positive MCF-7 breast cancer cells were more sensitive to the effects of ethanol than estrogen receptor negative MDA-MB-231 cells, with changes in PPARα mRNA more pronounced than PPARβ mRNA. Studies in MCF-7 cells conditionally expressing either PPARα or PPARβ in the presence of their respective specific ligands, GW7647 and GW0742, revealed that ethanol concentrations of 20 mM and 100 mM suppressed the maximal response to ligand-mediated activation for PPARα. Studies using the ethanol metabolism enzyme inhibitors 4-methylpyrazole and cyanamide, suggested that while ethanol was responsible for the modulation of PPARβ transactivation, the primary metabolite acetaldehyde was responsible for the effects on PPARα transactivation. Lastly, it was determined that ethanol and/or GW0742 did not increase the proliferation of MCF-7 Tet-off cells. The findings in this thesis suggest that given the different consequences of MEHP, MBP and ethanol on PPARs, PPAR expression and activation by ligands may have tissue specific consequences and that PPARβ may have a complex role in mammary gland tumourigenesis.

Made available in DSpace on 2014-10-09T12:27:18Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T13:59:13Z (GMT). No. of bitstreams: 0 / Tese (Doutoramento) / IPEN/T / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP