Sewage effluents disposed into the marine environment from De Beers Marine Namibia diamond mining vessels have the potential to cause endocrine disruptive effects in marine organisms. Endocrine disruption refers to the alteration of the normal functioning of the endocrine system and various chemicals have the ability to mimic hormones, effecting endogenous hormone synthesis, transport, receptor interaction and intracellular signaling. The potential endocrine disruptive effects, caused by the release of different types of sewage effluents into the ocean, on fish species is a concern due to the commercial importance of fish species found in the mining area e.g. hake, sole, horse mackerel. Increased awareness of marine environmental degradation due to the presence of chemical contaminants has resulted in research being done on early warning systems, in the form of biomarkers. Cytochrome P450 monooxygenase 1A (CYP1A) and vitellogenin (vtg) are important proteins found in fish liver and blood, that have been used as biomarkers for the detection of pollutants in fish. CYP1A is a subfamily of the P450 superfamily of enzymes and catalyzes the oxidation, hydrolysis and reduction of exogenous and endogenous compounds (phase I reactions) and thus has the capacity to regulate the metabolism of several organic contaminants. CYP1A expression is altered by exposure to planar xenobiotic compounds e.g. polyaromatic hydrocarbons. Vtg is an important precursor for egg yolk proteins and plays a role in the growth and development of an oocyte. Expression of this protein is altered upon exposure to estrogenic compounds. The aim of this project was to isolate CYP1A from fish liver by differential centrifugation and optimize conditions for the CYP1A-mediated ethoxyresorufin-Odeethylase (EROD) assay and western blot analysis (to assess CYP1A expression). Another aim of this study was to evaluate the potential effects of biologically disruptive chemicals from sewage effluents, discharged into the marine environment, on the expression of CYP1A in two species of hake, Merluccius capensis and M. paradoxus (Cape hake). CYP1A in Cape hake is approximately a 60 kDa protein and the highest EROD activity was detected in the microsomal fraction after differential centrifugation. Optimal EROD assay conditions were observed at pH 7.5, a temperature of 25 °C, 10 μl of sample and a reaction time of 30 seconds. Enzyme stability assays indicated a drastic decrease in enzyme activity after 30 seconds. The EROD assay was not NADPH dependent but was limited by NADPH supply, with an increase of 300% in EROD activity being observed with the addition of 0.1 M exogenous NADPH. The addition of dicumarol (40 μM), a phase II enzyme inhibitor, showed a 232% increase in EROD activity. This is because dicumarol inhibited enzymes with the capacity to metabolize the product (resorufin) of the EROD reaction. With regard to western blot analysis, the optimal primary (rabbit antifish CYP1A peptide) and secondary (anti-mouse/rabbit antibody-horseradish peroxidase conjugate (POD)) antibody dilutions were determined to be 1:1000 and 1:5000, respectively. The comparison of CYP1A expression in Cape hake samples from De Beers Marine mining area and reference sites showed higher EROD activity (16.29 ± 0.91 pmol/min) in fish samples from the mining area in comparison to the reference site (10.42 ± 2.65 pmol/min). Western blot analysis was in agreement with the EROD assay results and a higher CYP1A expression was observed in fish from the mining sites. The increased CYP1A expression observed in fish from the mining area is not definitively an indication of a pollutant effect in the environment, as several environmental and biological factors (e.g. photoperiod and age) must also be considered before reaching this conclusion. Another aim of this study was to purify vtg from Cape hake blood samples. Cape hake vtg was purified from fish plasma by selective precipitation with MgCl2 and EDTA. Precipitated sample was subjected to anion exchange chromatography using fast protein liquid chromatography (FPLC). Vtg eluted as two broad peaks and had a molecular weight above 200 kDa. SDS-PAGE analysis also resolved smaller molecular weight proteins below 70 kDa, which were thought to be vitellogenin cleavage proteins, lipovitellin and phosphovitins. Western blot analysis was performed; however, it did not produce any conclusive results. The purification of vtg enables further studies in characterizing this protein and developing assay aimed at detecting estrogenic pollutants in the marine environment

Amphibians develop in aquatic environments where they are very susceptible to the effects of pesticides and other environmental contaminants. Glyphosate-based herbicides are widely used and have been shown to affect survival and development of tadpoles under laboratory conditions. The goal my thesis is to determine if agriculturally relevant exposure to Roundup WeatherMax®, a herbicide formulation containing the potassium salt of glyphosate and an undisclosed surfactant, influences the survival and development of wood frogs tadpoles (Lithobates sylvaticus) under both laboratory and field conditions. In the field, experimental wetlands were divided in half using an impermeable curtain so that each wetland contained a treatment and control side. Tadpoles were exposed to two pulses of this herbicide at environmentally realistic concentration (ERC, 0.21 mg acid equivalent (a.e.)/L) and predicted environmental concentrations (PEC, 2.89 mg a.e./L), after which survival, growth, development, and expression of genes involved in metamorphosis were measured. Results indicate that exposure to the PEC is extremely toxic to tadpoles under laboratory conditions but not under field conditions. Results from both experimental conditions show sublethal effects on growth and development, and demonstrate that ERC of glyphosate-based herbicides have the potential to alter hormonal responses during metamorphosis. My secondary objectives were to compare the effects of Roundup WeatherMax® to the well-studied Vision® formulation (containing the isopropylamine (IPA) salt of glyphosate and POEA), and to determine which ingredient(s) are responsible for the sublethal effects on development. Survival, growth and gene expression results indicate that Roundup WeatherMax® has greater toxicity than Vision® formulation. Contrary to my prediction, results suggest that, under realistic exposure scenarios, POEA is not the sole ingredient responsible for the observed developmental effects. However, my results demonstrate that chronic exposure to the POEA surfactant at the PEC (1.43 mg/L) is extremely toxic to wood frog tadpoles in laboratory. As part of the Long-term Experimental Wetlands Area (LEWA) project, this research contributes to overall knowledge of the impacts of glyphosate-based herbicides on aquatic communities.

Les méthodes actuellement disponibles pour la surveillance de la qualité du milieu se réfèrent au suivi de composés définis par les réglementations et ne permettent pas d’appréhender l’(éco)toxicité réelle de tous les composés présents dans l’environnement (e.g. composés inconnus, produits de transformation, effets de mélange…). Dans ce contexte, une méthode d’analyse pluridisciplinaire intégrative, l’analyse dirigée par l’effet (EDA), a été développée. Cette approche bio-analytique vise à établir un lien de causalité entre une exposition aux contaminants et l’effet biologique observé. La démarche est basée sur la simplification séquentielle d’un échantillon guidée par les bio-essais afin d’individualiser puis identifier, par des techniques analytiques chimiques performantes, les composés biologiquement actifs. Les objectifs de cette thèse ont porté sur (1) le développement et la validation d’une méthodologie, allant du pré-traitement jusqu’au fractionnement, adaptée à différentes matrices environnementales ; (2) la mise en place d’une stratégie d’identification moléculaire par spectrométrie de masse haute résolution ; (3) l’application de la démarche EDA à plusieurs échantillons afin d’illustrer l’adaptabilité et l’apport d’une telle méthode à des problématiques environnementales. Ces travaux de thèse permettent de disposer aujourd’hui d’une démarche bio-analytique complète de la préparation d’échantillon à l’identification de molécules, et adaptée à tous types d’échantillons. Les différentes études environnementales réalisées au cours de ces travaux ont permis, entre autre, d’établir une liste de composés diverses non recherchés pouvant représenter un risque pour l’environnement. / To date, environmental risk assessment is based on targeted chemical analyses. These analyses allow the detection of known active compounds and this preselected set does not often explain the observed toxic effects in complex environmental samples (e.g. unknown compounds, transformation product, cocktail effect...). For an integrative contamination assessment, a multidisciplinary approach, Effect-Directed Analysis (EDA) was developed. This approach combining biological tools and chemical analyses allows determining active compounds and identitying them in such environmental complex mixtures. EDA aims at the establishment of cause-effect relationships by sequential reduction of the complexity of environmental mixtures, eventually to individual toxicants. The aims of this thesis were (1) to develop and to validate the EDA-based strategy on various environmental complex samples; (2) to set up an identification strategy for identifying non targeted or unknown molecules using high resolution mass spectrometry; (3) to prove the usefulness of EDA approach for the identification of active compounds in environnemental complex samples. This thesis work provides to a complet bio-analytical approach from the preparation step of samples to the identification of molecules. The potential of this tool was fully confirmed on various environmental studies. These investigations allowed establishing a list of non-target compounds which can represent a risk for the environment.

Orientador: Maria Aparecida Carvalho de Medeiros / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Tecnologia / Made available in DSpace on 2018-08-27T02:18:56Z (GMT). No. of bitstreams: 1 Oliveira_AndreFelipede_M.pdf: 2184303 bytes, checksum: b46c30ed510d7f32eb7aae82b851f80d (MD5) Previous issue date: 2015 / Resumo: Há uma preocupação crescente na área de Saneamento Ambiental com relação a qualidade das águas dos corpos hídricos, tendo em vista que pesquisas têm revelado que os sistemas de tratamentos convencionais de esgotos e de água para abastecimento público não possuem eficiências adequadas para remoção de compostos orgânicos classificados como Desreguladores Endócrinos (DE). Nesta classe de compostos são destacados os estrógenos naturais 17 ß-estradiol (E2), estriol (E3), estrona (E1) e o sintético 17 ?-etinilestradiol (EE2). Os efeitos destes hormônios sexuais, que são compostos extremamente ativos biologicamente, têm sido citados como agentes etiológicos de feminilização em peixes e também de vários tipos de cânceres. Neste sentido, trabalhos aplicando tecnologias avançadas de tratamento destes DE estão sendo desenvolvidos por vários grupos de pesquisas. Apesar de que as Legislações ambientais relacionadas aos parâmetros de lançamento (RESOLUÇÃO CONAMA no. 430 de 2011) e de parâmetros de potabilidade (Portaria no. 2914 de 2011) não contemplarem estes compostos DE, sendo que diversos trabalhos têm sido publicados com quantificação destes compostos em água da ordem de ng L-1. Neste contexto, o presente trabalho teve como objetivos: (1) adaptação e validação de metodologia analítica para confirmar e quantificar simultaneamente os hormônios E1, E2, E3 e EE2, nas águas bruta e tratada do rio Atibaia, manancial que abastece o município de Campinas, utilizando a técnica de extração em fase sólida (EFS), juntamente com a técnica de Cromatografia Líquida acoplada à espectrometria de massas em tandem (CL-EM/EM), (2) aplicação de tecnologias de degradação destes compostos E1, E2, E3 e EE2 em escala piloto, pelos processos de ozonização e cloroamoniação, visando contribuir com as determinação das dosagens, do tempo de contato e da eficiências de remoção. Os resultados obtidos nas análises via EFS-LC-MS/MS mostraram a ocorrência do hormônio sexual natural E1, na água bruta do rio Atibaia com concentração de 17 ng L-1, sendo que para os demais E2 e E3 os valores obtidos foram inferiores ao limite de quantificação 2 ng L-1 e para o EE2 inferior a 5 ng L-1. As técnicas de oxidação ozonização e cloroamoniação utilizadas nos ensaios demonstraram eficiências de degradação em média, maiores do que 95% dos DE estudados, com exceção de E1, cuja eficiência de remoção ficou entre 87% a 93%, dependendo da faixa de fortificação / Abstract: There is growing concern in the field of Environmental Sanitation regarding the water quality of water bodies, given that research has shown that systems of conventional treatments of sewage and public water supply does not have adequate efficiencies to removal of some organic compounds Endocrine Disruptors (DE). In this class of compounds are included the natural estrogens 17 ß-estradiol (E2), estriol (E3), estrone (E1) and the synthetic 17 ?-ethinylestradiol (EE2). The effects of these sex hormones, which are highly biologically active compounds, have been cited as etiological agents of feminization in fish and various types of cancers. In this regard, several works by applying advanced technologies for treating these DE are being developed by many research groups. Although the environmental legislations related to the parameters of launch (CONAMA RESOLUTION. 430 of 2011) and potability parameters (Ordinance no. 2914 2011) does not address these DE compounds, several studies have been published with quantification of these compounds in the order of ng L-1. In this context, the aimsmof this paper were: (1) adaptation and validation of the analytical methodology to confirm and to quantify simultaneously the E1, E2, E3 and EE2 hormones, raw water and treated in the Atibaia River, a source that supplies the municipality Campinas, using the technique of solid phase extraction (EFS) with the technique of Liquid Chromatography coupled to mass spectrometry in tandem (LC-MS/MS), (2) applying degradation technologies of these compounds E1, E2, E3 and EE2 in pilot scale, by using the processes of ozonation and cloroamoniation, to contribute to the determination of dosage, contact time and degradation efficiencies. The results obtained in this study via EFS-LC-MS / MS showed the occurrence of natural sex hormone E1 in the Atibaia River raw water with a concentration of 17 ng L-1, and for the other E2 and E3 the values were lower the quantification limit of 2 ng L-1 and EE2 less than 5 ng L-1. The ozonation and chloroamoniation oxidation techniques used for the degradation tests were with efficiencies on average more than 95% of the studied DE, except E1, the removal efficiency was between 87% to 93% depending on the fortification range, by using ozonation in pre oxidation and cloroamoniation and the disinfection step / Mestrado / Tecnologia e Inovação / Mestre em Tecnologia

Philosophiae Doctor - PhD / In this work, DNA aptamer biosensor systems were developed for the detection of l7p-estradiol - an estrogeneous endocrine disrupting chemical (EDC). Endocrine disrupting chemicals are group of compounds that impact negatively on the endocrine system of humans and wildlife. High concentrations of l7p-estradiol in water or food chain disrupts the physiology of the endocrine system of various animal species, leading to feminisation in fish and stimulates the proliferation of cancer cells in humans. Aptasensor systems for the determination of l7pestradiol were prepared with three immobilization platforms: (i) poly(3,4- ethylenedioxythiophene) {PEDOT} doped with gold nanoparticles (AuNPs) to form PEDOTIAuNPs polymeric nanocomposite, (ii) generation 1 poly(propylene thiophenoimine)-copoly( 3 ,4-ethy lenedioxythiophene) dendritic star copolymer (G 1PPT -co-PEDOT), and (iii) generation 2 poly (propylene thiophenoimine)-co-poly(3,4-ethylenedioxythiophene) dendritic star copolymer (G2PPT-co-PEDOT). The morphological properties of the sensor platforms were interrogated by scanning emission microscopy (SEM) and atomic force microscopy (AFM), while their spectroscopic characteristics were studied by Fourier transform infra red spectroscopy (FTIR) and fluorescence spectroscopy. The electrochemical behaviour of the platforms and the aptasensors were studied by electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV) and square wave voltammetry (SWV). The DNA aptamer developed for detecting 17~-estradiol and which was used in the fabrication of all aptamer biosensors in this study is a 76-mer biotinylated aptamer (5'-BiotinGCTTCCAGCTTATTGAATTACACGCAGAGG TAGCGGCTCTGCGCATTCAATGCTGCGCGCTGAAGCGCGGAAGC-3'). AulPEDOTIAuNPslAptamer (platform 1) was obtained by covalently attaching streptavidin to the polymeric nanocomposite platform using carbodiimide chemistry and the aptamer immobilized via streptavidin-biotin interaction. The electrochemical signal generated from the aptamer-target molecule interaction was monitored electrochemically using cyclic voltammetry and square wave voltammetry in the presence of [Fe(CN)6J 3-/4- as a redox probe. The signal current observed was inversely proportional to the concentration of 17Bestradiol. The aptasensor demonstrated specificity toward 17~-estradiol. The detectable concentration range of the 17B estradiol was 0.01 nM-O .09 nM with a detection limit of 3.2 pM. The 76-mer biotinylated aptamer for 17~-estradiol was incorporated into a generation 1 poly(propylenethiophenoimine )-co-poly(3 ,4-ethylenedioxythiophene) dendritic star copolymer modified Au electrode via biotin-avidin interaction (platform 2). The Bode plot shows that the charge transfer dynamics of the nanoelectrode can be frequency modulated while the AulG 1PPTco- PEDOT nanoelectrode exhibited greater semi-conductor behavior (higher phase angle value) than AulG 1PPT due to the incorporation of charged functionalized dendrimer at low frequencies (100 mHz). The biosensor response to 17~-estradiol was based on the decrease in the SWV current as the EDC binds to the ssDNA aptamer on the biosensor. The dynamic linear range of the sensor was 0.01-0.07 nM with a detection limit of7.27 pM. Synthesis of electro synthetic generation G2PPT-co-PEDOT (platform 3) was performed by copolymerization of PEDOT with G2PPT dendrimer modified electrode immersed in a solution of 0.1 M LiCI04 containing 0.1 M EDOT monomer and 0.1 M sodium dodecyl sulphate (SDS) for ten (10) cycles. The electrochemical behaviour of the dendritic star copolymer was investigated with CV and EIS in LiCI04 and phosphate buffer solutions. The results show that the electrochemical deposition of G2PPT-co-PEDOT on gold electrode decreased the electrochemical charge transfer resistance when compared to AuiPEDOTILiCI04 and AuiLiCI04 interfaces. Bode impedimetric analysis indicates that G2PPT-co-PEDOT is a semiconductor. The fabrication of two novel aptasensors (based on platforms 2 and 3) simultaneously on a screen printed micro array electrode of 96-well multichannel electrochemical robotic sensor testing system for the detection of endocrine disrupting l7~-estradiol, was also carried out. The aptasensors responses to l7~-estradiol, based on the decrease in the SWV current, were evaluated.

Endocrine disrupting chemicals (EDCs) are ubiquitous in the environment and their presence in water bodies is documented. They discharge into surface water (SW) unmonitored, posing a threat to both aquatic and terrestrial lives. This is a challenge as not all populations have access to treated drinking water (TDW). The EDC contaminated serves as a route of exposure, together with ineffective treatment plants. Given the complexity of the endocrine system, EDCs may mimic or antagonise natural hormones or disrupt their synthesis, metabolism and excretion. The associated health effects include testicular dysgenesis syndrome, metabolic disorders and cancers. Policy and internationally standardised test methods are however sti ll limited. This study therefore aimed to assess the suitability of two assays used for screening estrogenic activity and one for androgenic activity in different water sources. The study consisted of two phases. In phase 1, water sample (tap, surface and treated wastewater) were collected from a catchment area in Pretoria. The samples and a spiked MilliQ laboratory water sample were extracted with solid phase extraction (SPE) and sent to Germany for distribution to participating laboratories. Samples (n=24) from six different countries were received to test for androgenic activity in the MDA-kb2 reporter gene assay. In phase 2, SW and TDW samples were collected from April 2015 until March 2016. The samples were filtered, extracted using SPE and assayed with the YES assay, T47D-KBluc reporter gene assay for estrogenic activity and MDA-kb2 reporter gene assay for androgenic activity. In phase 1, androgenic activity was detected in 4 out of 24 (21%) samples and ranged from 0.23 ± 0.040 ng/L to 0.008 ± 0.001 ng/L DHTEqs. In phase 2, estrogenic activity was detected in 16 out of 24 (67%) SW samples in the T47DKBluc reporter gene assay and ranged from 0.31 ± 0.05 pg/L to 10.51 ± 5.74 pg/L EEqs. It was below the detection limit (dl) in the YES assay. Androgenic activity was detected in 4 out of 24 (17%) SW samples, ranging from 0.0033 ± 0.0050 ng/L to 0.090 ± 0.040 ng/L DHTEqs. Androgenic and estrogenic activity was higher i n pretreatment samples compared to post-treatment in both treatment plants. In phase 1, the MDA-kb2 reporter gene assay was successfully applied to water samples from different sources. Androgenic activity was highest in treated wastewater. In phase 2, treatment plants proved to be effective in removing estrogens detected in the SW samples, as the TDW samples were below the dl. Estrogenic activity is within the ranges reported in other studies. Positive samples were below the 0.7 ng/L proposed trigger value for health risk assessments. Detected androgenic activity was lower in TDW samples compared to the SW samples supplying the two treatment plants indicating that they were both effective in removing the androgenic activity detected. Few studies have reported androgenic activity in tap water. This study strengthens the argument for using a battery of assays when monitoring endocrine activity as EDCs occur at low concentrations in mixtures. / Dissertation (MSc)--University of Pretoria, 2017. / School of Health Systems and Public Health (SHSPH) / MSc / Unrestricted

Endocrine-disrupting chemicals (EDCs) are known to cause endocrine disruption (ED), developmental neurotoxicity (DNT), infertility and impaired embryo development. EDCs do therefore impose a threat to humans, wildlife and the environment. The present study investigated the effects of the reference compounds dihydrotestosterone, estradiol, benzo(a)pyrene, rosiglitazone, as well as the EDCs bisphenol F and perfluorooctanesulfonic acid at various concentrations on zebrafish embryos. The scientific questions of the study were to investigate how these environmental pollutants impact the development of zebrafish, what their molecular mechanisms are and what the link between ED and DNT is. Zebrafish embryos were exposed for 5 days to the compounds and various parameters on development were collected at different time points. The expression of 41 genes (qPCR) related to ED and DNT, and the levels of 23 steroids (LC-MS/MS) were determined. Gene correlations were determined with Pearson’s correlation test and paired t-tests were used to determine significantly altered gene activities. The significant gene expression changes were further related to the pathways of steroids in order to connect how gene activity impacted steroid levels. Exposure to estradiol, dihydrotestosterone and bisphenol F induced cyp19a1b expression which can affect personality traits. Perfluorooctanesulfonic acid interferes with thyroid hormone transport by binding to TTR causing profound effects on neurodevelopmental processes and cognitive functions. The compounds influenced genes that can disrupt endocrine systems which can cause neurodevelopmental impairments.

No description available.

Humans experience ubiquitous exposures to estrogenic environmental chemicals from food, personal care products, and other industrial and consumer goods. Bisphenol A (BPA), a well-studied xenoestrogen, is known to alter development of estrogen-sensitive organs including the brain, reproductive tract, and mammary gland. Bisphenol S (BPS), which has a similar chemical structure to BPA, is also used in many consumer products, but its effects on estrogen-sensitive organs in mammals has not been thoroughly examined. In our study, pregnant CD-1 mice were orally exposed to BPS or ethinyl estradiol (EE2, a positive control for estrogenicity) from gestational day 9 through postnatal day (PND) 2, the period when many estrogen-sensitive organs are developing. After weaning, the offspring were administered either oil (vehicle) or an estrogen challenge (1 μg EE2/kg/day) for ten days starting at PND21 (prior to puberty), PND80 (early adulthood), or PND260 (later adulthood). Timing of puberty was evaluated in females by noting the date on which vaginal opening occurred. After the 10 day estrogen challenge, we evaluated the response of endocrine sensitive organs through measurements of organ weight, tissue morphology, and gene expression in both males and females. We observed dose- and sex-specific effects of BPS and EE2 treatment, as well as alterations in the responses of males and females to the estrogen challenge. This study sheds light on the effects of low dose xenoestrogen exposures on estrogen-sensitive organs including the reproductive tract and mammary gland. Furthermore, it improves our understanding of the influence of environmental chemicals on secular trends of earlier age of puberty in girls reported over the past few decades.

Endocrine disrupting chemicals (EDCs) are ubiquitous and their adverse impact on nature, wildlife and humans is extensively researched. We are constantly exposed to EDCs, such as the widespread and extensively researched Bisphenol A, as well as its substitute Bisphenol F, which is coming into wider use, even though it is much less is researched and limited information is available about its endocrine effects. Realistically, we are exposed to mixtures rather than single substances. In the Swedish Environmental Longitudinal, Mother and Child, Asthma and allergy (SELMA) study, the co-exposure of EDCs was assessed. Based on the SELMA cohort data, a study identified a mixture of EDCs, Mixture N1, which is associated with delayed language development. In recent years, it has been hypothesized that epigenetic alterations are one of the underlying mechanisms for the effect of EDC exposures. For example, EDC induced changes in DNA Methylation of the promoter region of a gene might lead to altered gene expression, which can result in adverse health effects. Several studies already indicate an impact of the formerly introduced chemicals/mixtures on the DNA methylation on genes such as NR3C1 and GRIN2B in animals. However, limited research is available on the impact on NR3C1 and GRIN2B in the early human brain, which is of interest since both genes are crucial for the development of the brain and altered gene expression often leads to adverse effects. This study aimed to investigate the impact of BPA, BPF and Mixture N1 on NR3C1 and GRIN2B in the developing human brain as well as establish a protocol for differentiation of human stem cells into neuroprogenitor cells that express GRIN2B and NR3C1. In the end stem cells were differentiated in vitro into neural progenitor cells (NPCs) using the protocol of Hosseini et al. (2020). During the differentiation, the cells were exposed to different concentrations of the former mentioned chemicals. Afterwards, RNA and DNA were extracted, followed by a qPCR and bisulfite-pyrosequencing to investigate the changes in gene expression and DNA methylation of NR3C1 and GRIN2B. This study established the differentiation protocol but revealed no significant results regarding the chemical exposure. However, some chemical exposures showed a clear tendency towards an impact of the chemicals on the gene expression and the DNA methylation. Furthermore, a negative correlation between DNA methylation at 2 CpG sites and gene expression in NR3C1 could be observed. In conclusion, the DNA methylation at promoter region in NR3C1 is important for the gene expression.