Octylphenol and genistein are two of the growing list of endocrine disrupting chemicals found in the environment that mimic estrogen in reproductive tissue both in vitro and in vivo. It is well established that endogenous estrogens modulate not only the reproductive system, but also the immune system. However, the effects of many endocrine disrupting chemicals, such as octylphenol and genistein, on the immune system have yet to be determined. Preliminary studies on short-term treatment with genistein (0.6 mg) and octylphenol (10 mg) showed that the thymus of orchiectomized (NZBxNZW)F1 males is sensitive to these agents. Further studies focused on the effects of short-term treatment of octylphenol on the morphology and function of the thymus in adult, reproductively intact non-autoimmune C57BL/6 and pre-autoimmune (NZBxNZW)F1 males. Oral dosing of 0.1 mg, 1 mg, or 10 mg of octylphenol 3 times a week for 3 weeks did not affect the morphology or function of the thymus as assessed by its weight, thymocyte cellularity, proportion of immature and mature thymocytes, level of apoptosis, apoptotic rates of stimulated thymocytes, and proportion of mature T cells in the spleen. Furthermore, oral dosing of 0.1 mg, 1 mg, or 10 mg of octylphenol did not result in estrogenic changes in the reproductive tract in our model. Subcutaneous injection of 10 mg of octylphenol resulted in skin lesions that confounded the assessment of its affects on the thymus. Further studies are needed to definitively determine the effects of octylphenol on the immune system of both males and females of various ages and to determine the effect of long-term exposure. / Master of Science

Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: During the last two and a half decades a large body of research has accumulated indicating the presence of various natural and synthetic chemical compounds within the environment capable of inducing hormone-like responses in humans and animals. Such compounds, termed endocrine disruptors, have been implicated in a variety of developmental, reproductive and physiological abnormalities which have been shown to converge on the endocrine system. Given that endocrine disrupters are comprised of a diverse group of molecules with dissimilar chemical structures, general screening techniques are not feasible for effective environmental monitoring. A primary method of action by which these exogenous molecules affect the homeostatic regulation of the endocrine system is believed to be via the modulation of gene transcription. It is now well established that many endocrine disrupting compounds act upon a principal group of transcription factors, the nuclear receptors, by chance interaction with the ligand binding domains of these proteins. With a view to ultimately design a portable kit for the detection of endocrine disrupting compounds in water based on the bio-specific immobilisation of nuclear receptor ligand binding domains to a stationary membrane matrix, this study specifically describes: 1. The effects on recombinant protein expression by the addition of small molecules to the cultivation media of bacteria. 2. The optimisation of conditions for the lysis of bacterial cells to increase the solubility of heterologously expressed proteins. 3. The purification of recombinant proteins from bacterial cell lysates by means of a two-step chromatographic methodology. 4. The cloning of the genes for the human androgen and estrogen receptors’ ligand binding domains into baculovirus transfer plasmids. 5. Transfer of genetic material from the created baculovirus transfer plasmids to a linearised baculovirus genome for the generation of recombinant viruses. 6. The cultivation, and baculoviral infection, of Spodoptera frugiperda and Trichoplusia ni cell lines. 7. Expression and purification of N-terminal hexahistidine-tagged human nuclear receptor LBDs from insect cell lysates by means of immobilised metal affinity chromatography. / AFRIKAANSE OPSOMMING: Die teenwoordigheid van natuurlike en sintetiese chemiese middels wat oor die vermoë beskik om die aksies van hormone in die mens en dier na te boots het toenemend aftrek gekry in navorsing gedurende die laaste twee en ’n halwe dekades. 'n Verskeidenheid van ontwikkelings-, reproduktiewe- en fisiologiese abnormaliteite ontstaan as gevolg van die aksies van hierdie molekule, genaamd endokriene-ontwrigters, op die natuurlike funksionering van die endokriene-sisteem. Gegewe dat die groep chemiese middels waaruit endokriene-ontwrigters bestaan van diverse oorsprong afkomstig is lei dit daartoe dat algemene analitiese tegnieke nie in alle gevalle geskik is vir effektiewe omgewingsmonitering is nie. Die modulasie van geentranskripsie is een van die metodes wat voorgestel word as ’n metode waarop hierdie eksogene molekule die homeostatiese regulering deur die endokriene-sisteem omverwerp. ’n Algemene metode waarop vele endokrien-ontwrigtende stowwe geentranskripsie beïnvloed, is deur interaksie met die hormoon-bindende gedeeltes van ’n belangrike groep transkripsiefaktore, die nukluêre reseptore. Hierdie studie, met die uiteindelike ontwikkeling van ’n draagbare toetsstelsel vir die opsporing van endokrien-ontwrigtende-stowwe in water, gebasseer op die bio-spesifieke immobilisering van nukluêre reseptor ligand bindingsdomeins op ’n stasionêre membraanmatriks, het ten doel om die volgende te beskryf: 1. Die effek wat die byvoeging van klein molekule tot die groeimedium van bakteriëe het op die uitdrukking van rekombinante proteïene. 2. Die optimisering van bakteriese sel-lisering in terme van verhoging in die oplosbaarheid van heteroloë proteïene. 3. Die suiwering van rekombinante proteïen vanuit bakteriese sellisate deur middel van ’n twee-stap chromatografiese sisteem. 4. Die klonering van die gene vir die menslike androgeen en estrogeen reseptore se ligand bindingsdomeine in bakulovirus oordragplasmiede. 5. Die oordrag van genetiese materiaal vanaf hierdie bakulovirus oordragplasmiede na ’n gelineariseerde bakulovirus genoom deur middel van homoloë rekombinasie vir die produksie van rekombinante virusse. 6. Die groei en infeksie van Spodoptera frugiperda en Trichoplusia ni sellyne wat lei tot die uitdrukking van menssoortgelyke nukluêre reseptor ligandbindingsdomains. 7. Suiwering van N-terminaal heksahistidien-etiket-gekoppelde menslike nukluêre reseptor ligandbindingsdomeins vanuit inseksellisate deur middel van geïmmobiliseerde metaal affiniteitschromatografie.

Submitted in fulfillment of the requirements for the degree of Doctorate of Technology: Biotechnology, Durban University of Technology, Durban, South Africa, 2014. / All over the world concerns have been raised over the possible adverse effects that may occur when exposed to chemicals that have the potential to interfere and affect the endocrine system. The concern is directed at both humans and wildlife. There is still a lack of public awareness regarding Endocrine Disrupting Chemicals (EDCs) and the harmful effects on humans and wildlife. It has only been within the last decade that South Africa began the actual task for proper management and control for water and wastewater quality. There are many ways to detect these EDCs all of which are very laborious and most of the cases these EDCs are either in the pico or nano gram per litre range, too minute for many methods to detect effectively; so therefore the research project aimed to use rapid and sensitive techniques to determine the quickest means to detect the very low concentrations of theses EDCs. Two techniques were researched, i.e., Enzyme Linked immunoassays (ELISAs) and Radio-immunoassays (RIAs). The research study thus assessed the solid phase extraction (SPE) technique for total recovery of hormones; the ELISA and RIA techniques for rapid detection of natural (estrone (E1), estradiol (E2) and estriol (E3) and synthetic ethinylestradiol (EE2) by validating the precision and reproducibility . These techniques were then applied to determine hormone EDC removal first at laboratory scale investigations and then applied to full scale wastewater treatment plants (WWTP) with different configurations in order to deduce removal efficiency of each type of plant. The next phase assessed the toxicity of individual and combined estrogen standards as well as the toxicity in the WWTPs and classify and to determine if there was a correlation between hormone concentration and toxicity in final effluents. The assessment of the SPE and the immunoassay procedures (ELISA AND RIA) using standards and controls found that both these assays can be utilised to quantify hormone estrogens in wastewater. The small sample volume required reduced the labour time and application of the procedure made it cost effective and reliable techniques. The intra-assay and inter-assay validation procedures as well as the standard recoveries confirmed reproducibility and precision of the immunoassays. The % CV were <10% for both the intra-assay and inter-assay validations. The laboratory scale investigations included the operation of a modified Ludzak-Ettinger (MLE) process which enabled control and manipulation over the operational parameters in order to establish how certain parameters influenced the removal of hormone EDCs. One such parameter that was manipulated was the sludge retention time (SRT). The MLE tests showed that the SRTs definitely have an effect on the removal of hormones from the influent as well as the overall performance of sewage treatment. The 10 day SRT proved that longer SRTs will definitely aid in the removal of hormones and possibly other EDCs in raw sewage. During the 10 day SRT the influent hormone concentrations (E1: 59.11 ng/L, E2: 61.40 ng/L) were almost double than the influent hormone concentrations (E1: 26.46 ng/L, E2: 27.60 ng/L) during the 5 day SRT, which impacted on the removal efficiency. The 5 day SRT had an overall average E2 and E1 removal of 78.11% and 81.71% respectively while the 10 day SRT had average E2 and E1 removal of 91.24 % and 80.56% respectively. The 24 hour batch test provided evidence of the reversible metabolism of the E2 hormone. This was seen by the rapid decrease of E2 and the rapid increase of E1 in less than 3 hours, which proved that E2 can be metabolized in to E1. An average reduction of 94.44% of E2 was seen after 5 hours and after 10 hours was no longer detected. After 13 hours E1 could no longer be detected. This finding also provided clarity as to the lower percentage removal of E1 during the 10 day SRT of the MLE process. The Vibrio fischeri biotox method implemented was the most economic and easiest way to conduct the toxicity tests. The validation of the test used a 52.9 mg/L K2Cr2O7 standard which provided a Cr (VI) concentration of 18.7 mg/L in the final test suspension which is the theoretical effective concentration causing 50% inhibition (EC50). This specific concentration of the Cr (VI) exhibited an EC50 at 20.08 mg/L. The toxicity investigations of the individual and mixed hormone standards revealed that at the 10 ng/L concentration the individual E2 standard had the highest percentage inhibition (%INH) of 45.99% after the 30 minute contact time (T30), and when this standard was further diluted to 5 and 1 ng/L also showed higher % INH (26.04 and 23.66 %INH, respectively) than the individual EE2 standard (21.92 %INH) at 10 ng/L. . According to the toxicity classification system and after interpretation of the data, all the hormone standards were classified as Class II as they all exhibited slight acute toxicity. The 10 ng/L E2 standard had Toxicity Units (TU) of 0.8 which was close to the Class III level; however when it was in a mixture with E1 and E3, the TU was much lower (0.6 TU). The synthetic EE2 hormone also showed slight acute toxicity and had the lowest TU of 0.4. The application of the above mentioned techniques to full scale WWTPs with different configurations showed different removal efficiencies. The WWTPs ranged from the most primary consisting of just oxidation ponds to biological trickling filters, to biological nutrient removal (BNR) to conventional activated sludge (AS) plants. Removal rates ranged from 29% to 96% for E2, 0% to 89% for E1 and 0% to 100% for EE2. The overall ranking of the WWTPs from the most efficient to least efficient in terms of hormone removal were as follows: Plant E (91%) = Plant D (before UF) (91%) > Plant B (east side) (88%) > Plant B (west side) (77%) > Plant C (east side) (71%) > Plant D (after UF) (57%) > Plant A (56%) > Plant C (west side) (12%). Using the Vibrio fischeri method to evaluate the reduction of toxicity in WWTPs C, D and E proved effective. It was seen immediately after secondary biological treatment in the clarifier effluent the toxicity was reduced. Plants C, D and E had reduced the toxicities by 100, 80 and 97 % immediately after secondary biological treatment, while after the addition of the Chlorine disinfectant in the final stage of treatment the toxicity increased having %INH of 99.9, 15.7 and 99.9 respectively. In conclusion the SPE can be used as an extraction procedure for hormones in wastewater and the immunoassays can be used as rapid techniques for quantification of hormone EDCs in wastewater. The ELISA technique proved to be the slightly superior to the RIA in terms of facilities required. The laboratory scale procedures proved that some hormones can be oxidised to other hormones and therefore longer sludge retention times may be required to improve the removal. The study of the different WWTPs configuration showed that plant configuration and operational parameters impact the removal of hormone EDCs. The composition of the influent received by the plant also has an effect on the removal, i.e., whether it’s industrial, domestic or a mixture of both. Results concluded that plants which have either mixing and/or aeration with activated sludge and longer SRTs of more than 10 days have a higher rate of hormone removal than those plants with shorter SRTs and that the activated sludge processes were capable of reducing the toxicity of the influent. Overall results indicated that hormone EDCs are indeed being discharged with the effluents from WWTPs in South Africa. However whether the concentrations left in the final effluents will still have an adverse effect on the aquatic life is a question that still remains unanswered. The aquatic ecosystems are inevitably being polluted with these EDCs and their breakdown products. / D

<p>There is a critical need to target tumor hypoxia as patients with hypoxic tumors have worse prognosis due to aggressive phenotypes and resistance to radiotherapy and chemotherapy. The overall goal of this work is to improve response to conventional cancer therapies by targeting tumor hypoxia. This has been carried out and evaluated through the use of polymersome-encapsulated myoglobin (PEMs) with the hypothesis that O2-releasing PEMs will increase tumor oxygenation, and thereby improve response to radiotherapy. Mb was chosen as an O2 carrying protein to deliver to tumors because it has a strong association to O2, providing a mechanism to deliver O2 only within the hypoxic regions of the tumor. Mb was loaded within nanoscale polymeric vesicles that were expected to accumulate within solid tumors due to the enhanced permeability and retention (EPR) effect. This hypothesis has been tested through the following aims:</p><p>1. Develop NIR imaging techniques for studying the biodistribution and pharmacokinetics of polymersomes</p><p>2. Establish the effects of Mb-containing polymersomes on tumor physiology</p><p>3. Modify tumor growth through delivery of Mb polymersomes in combination with a cytotoxic therapy specific to aerobic tumors</p><p>These aims have been evaluated through numerous in vivo studies. First, polymersomes of various polymer formulations and diameters ranging from 110-550 nm were prepared with a near-infrared (NIR) -emissive fluorophore. Using live animal fluorescence imaging, I was able to study the biodistribution of the polymersomes following i.v. administration, demonstrating significant polymersome accumulation in orthotopic 4T1 mammary carcinomas. In addition, a novel method for measuring pharmacokinetics was developed, using serial small volume blood draws from individual mice. The plasma fluorescence in microcapillary tubes was used to quantify polymersome concentrations, demonstrating long circulation half-lives that varied from 6-23 h. Toxicity of various polymersome formulations were also studied in vitro and in vivo, revealing negligible toxicities.</p><p>For the second aim, PEMs were administered i.v. in tumor-bearing mice. Unexpectedly, we observed a dramatic gross tumor effect within hours of treatment in both orthotopic 4T1 tumors and flank Renca renal cell carcinomas. Histological analysis revealed endothelial cell apoptosis as early as 1 h following treatment, with scattered tumor cell death throughout the tumor by 4 h. Hematoxylin and eosin staining showed significant necrosis 24 h following PEM treatment. Vascular effects and polymersome distribution were studied in 4T1 window chamber tumors. Following i.v. treatment with PEMs, intravital microscopy was used to image polymersome fluorescence, brightfield transmission was imaged for vessel morphology and blood flow, and a tunable filter was used for determining hemoglobin (Hb) oxygen saturation. Tumor hemorrhaging was observed within hours of PEM treatment, which was not seen with empty polymersomes. This was consistent with the gross tumor effects observed initially. Hb saturation decreased in both the PEM and empty polymersome groups, but not in saline-treated mice. While we expected to observe an increase in tumor oxygenation by using Mb as an oxygen carrier, we actually observed hemorrhage, decreased oxygenation, and central tumor necrosis. In vitro studies using human endothelial cells demonstrated dramatic changes in cell morphology and increased permeability due to Mb and PEM treatments, which appear to be enhanced in an oxidative environment. These in vitro and in vivo observations are similar to what is seen with tumor vascular disrupting agents.</p><p>For the third aim, I combined radiotherapy (RT) and PEM treatment with a new hypothesis. I originally expected the PEMs to increase tumor oxygenation, thus making the tumor more susceptible to RT. However, considering the results from the second aim, this hypothesis was modified: the PEMs would result in necrosis of the tumor core, while RT would target the more oxygenated rim of the tumor, thus leading to improved tumor growth delay compared with PEM or RT alone. This hypothesis was tested in both orthotopic, syngeneic 4T1 tumors as well as flank FaDu xenografts. 4T1 tumor cells were surgically implanted within the dorsal mammary fat pad of mice and grown until ~200 mm3. A CT microirradiator with a square collimator was used in order to locate and specifically irradiate the tumor. Within 1 h following RT, the PEMs were administered i.v.. Mice receiving PEMs with no RT showed a significant decrease in tumor growth compared with saline-treated mice (p = 0.0001 for time to 3x original tumor volume). In addition, the combination of RT plus PEMs reduced tumor growth compared with RT alone (p = 0.0144 for time to 3x original tumor volume). However, this effect was not seen with FaDu tumors. This may have been due to excessive radiation dose or other compounding factors: the timing between RT and PEM treatment was not optimized, and the number of mice per group was small (3-4). </p><p>Thus, the conclusions for each aim are as follows:</p><p>1. Develop NIR imaging techniques for studying the biodistribution and pharmacokinetics of polymersomes</p><p>NIR imaging techniques were optimized for studying polymersomes, demonstrating long plasma circulation times and accumulation within tumors.</p><p>2. Establish the effects of Mb-containing polymersomes on tumor physiology</p><p>While the hypothesis was that PEMs would accumulate within hypoxic tumors and subsequently increase O2 tension, we observed a rapid decrease in tumor oxygenation followed by a dramatic hemorrhagic effect of Mb polymersomes, which appear to be due to both endothelial cell apoptosis and morphological changes, resulting in central tumor necrosis.</p><p>3. Modify tumor growth through delivery of Mb polymersomes in combination with a cytotoxic therapy specific to aerobic tumors</p><p>Combination therapy of PEMs with RT results in enhanced tumor growth delay in aggressive 4T1 mammary carcinomas compared with RT or PEMs alone.</p><p>These studies have led to a proposed mechanism for the PEM anti-tumor effect in combination with RT. Prior to PEM administration, RT is administered, resulting in tumor cell kill of the well-oxygenated tumor periphery. Mb polymersomes are then injected i.v. and begin to accumulate within tumors due to the EPR effect. As shown in Aim 1, this accumulation occurs over a short time scale. Within 30 min of PEM treatment, the Mb is believed to act on tumor vessels, resulting in morphological changes and apoptosis of endothelial cells. These effects are expected to increase permeability of the vessels and expose the basement membrane, which leads to clotting and decreased blood flow. Both decreased perfusion and increased permeability are believed to have a catastrophic effect on interior tumor vessels. Hemorrhage results as the endothelial cells die, resulting in tumor core necrosis. Therefore, the result is tumor cell kill at the periphery due to RT and central tumor necrosis due to PEM treatment.</p><p>PEMs have potential in cancer therapy as a new class of VDAs. While the mechanism requires further investigation, this work has demonstrated that PEM treatment results in tumor vessel destruction and central necrosis. PEMs accumulate within tumors, thus minimizing the systemic toxicity of treatment commonly seen with VDAs. By combining PEMs with a therapy that kills the better perfused tumor periphery, PEMs show promise in improving tumor response. Future mechanistic studies will be needed in order to maximize vessel damage and optimize combination dosing schedules to improve outcome.</p> / Dissertation

Persistent, anthropogenic contaminants collectively known as endocrine-disrupting chemicals (EDCs), are considered to be potential threats to wildlife reproductive fitness (Wingfield, 2002). The modes by which EDCs act on the reproductive system, and establishment of steroid profiles in wildlife, are important areas of study. Two means of performing an endocrine snapshot profile from dolphin tissues (serum and urine) were tested on samples, comparing a novel high performance liquid chromatography method to newly-validated enzyme-linked immunoassays (ELISAs). Serum and urine samples were obtained from two marine parks, and from free-ranging dolphins in the Indian River Lagoon (IRL) and Charleston Harbor (CHS) systems. Relative binding affinities of steroids versus putative EDCs to steroid receptors were compared in dolphin reproductive tissues, obtained by the Georgia Aquarium's Dolphin Conservation Field Station (GADCFS) from strained bottlenose dolphins (four male, three female) during 2010 and 20ll.........I conclude that endocrine disruptors are tissue and receptor specific, and both legacy and emerging contaminants are of concern to dolphin stocks, particularly those facing other significant environmental stressors. / by Sharon M. Homer-Drummond. / Vita. / Thesis (Ph.D.)--Florida Atlantic University, 2012. / Includes bibliography. / Mode of access: World Wide Web. / System requirements: Adobe Reader.

Nas últimas décadas, a ocorrência de atividade estrogênica e interferentes endócrinos (IEs) no ambiente aquático têm se tornado uma crescente preocupação. Dentre as diversas substâncias classificadas como IEs, destacam-se os fármacos, produtos de higiene e cuidados pessoais, hormônios naturais e sintéticos, produtos químicos industriais, praguicidas e muitos outros compostos que atingem o ambiente aquático por meio de descargas de esgoto doméstico, industrial ou de escoamento agrícola. Os objetivos deste estudo foram determinar a atividade estrogênica em amostras de águas superficiais, e avaliar seus efeitos biológicos no desenvolvimento de embriões de Danio rerio, a fim de propor faixas baseadas em valores de desencadeamento de efeitos para categorizar a atividade estrogênica. As amostras ambientais também foram analisadas por cromatografia líquida acoplada com a espectrometria de massas para identificar as substâncias que são suspeitas de causar alteração endócrina. Os compostos analisados foram: praguicidas, hormônios, triclosan, bisfenol A, octilfenol, nonilfenol, e a cafeína como indicador de atividade antrópica. A atividade estrogênica foi medida pelo ensaio Bioluminescent Yeast Estrogen (BLYES), que fornece os resultados em equivalente de 17&beta;-estradiol (EEQ). No entanto, este ensaio não é capaz de prover informações sobre os efeitos adversos em organismos aquáticos. Para observação de possíveis efeitos na biota, os embriões foram expostos a amostras de águas superficiais com resultados acima de 0,1 EEQ no BLYES. Os ensaios foram realizados de acordo com a OECD No. 236 (2013), verificando efeitos agudos como: ausência de batimento cardíaco, não formação de somitos, não desprendimento da cauda, e embrião coagulado. Malformações embrionárias tais como: redução do tamanho do organismo, edema cardíaco e vitelínico, curvatura da coluna vertebral, também foram avaliadas. As informações obtidas pelo ensaio com embriões de Danio rerio foram adequadas para mostrar os efeitos da mistura de contaminantes em organismos não-alvo. A atividade estrogênica medida pelo BLYES ficou abaixo do limite de quantificação (0,1 EEQ) em 44,8% do total de 116 amostras analisadas, e a faixa de atividade estrogênica variou de 0,11 a 14,6 EEQ. Além disso, a presença de contaminantes mesmo que em concentrações baixas ressalta a necessidade de mais estudos para entender os efeitos dessas substâncias nos organismos aquáticos. / Over the last few decades, the occurrence of estrogenic activity and endocrine disrupting chemicals (EDCs) in aquatic environment has become a worldwide issue of increasing environmental concern. The EDCs have the ability to alter the endocrine system of organisms, and includes pharmaceuticals, personal care products, steroid hormones, industrial chemicals, pesticides and many other compounds. Such compounds are present in several industrial and domestic activities and reach the aquatic environment via wastewater discharges or agricultural runoff. The aim of this study was to determine the overall estrogenic activity of surface water, evaluate biological effects on fish embryos development, in order to propose concentrations range based on trigger value to categorize estrogenic activity. Environmental samples were also analyzed by liquid chromatography tandem mass spectrometry to identify substances that are suspected to be an endocrine disruptor. The analyzed compounds were: pesticides, hormones, triclosan, bisphenol A, octylphenol, nonylphenol, and caffeine as an indicative of anthropic activity. The estrogenic activity was measured by Bioluminescent Yeast Estrogen assay (BLYES), with the results expressed in 17&beta;-estradiol equivalent quotient (EEQ). However, this assay is not able to provide information about adverse effects to aquatic organisms. In order to observe effects on aquatic organisms, organic extracts of surface water with results &ge; 0.1 EEQ in BLYES were tested in a bioassay using Danio rerio embryos. The methodology was conducted according OECD No. 236 and verified effects such as: lack of heart beat, lack of somites formation, non-detachment tail and coagulated embryo. Embryonic malformations were also evaluated, such as: reduction of organism size, edema and spine curvature, which are chronic effects. These effects probably are associated with contaminants mixtures. The obtained information by embryonic assay with Danio rerio was suitable to show the effects of contaminants mixture and was used to a categorization proposal of estrogenic activity. Estrogenic activity was below the limit of quantification (0.1 EEQ) in 44.8% of 116 analyzed samples, and range of estrogenic activity was from 0.11 to 14.6 EEQ. The tested samples in FET test were analyzed for acute or chronic toxicity in Danio rerio embryos. Based on the obtained results, even when estrogenic activity is present in surface water, the contaminants mixture can cause toxic effects in non-target organisms. Besides this, the widespread presence of these chemicals highlight the need for further studies in order to understand the harmfulness of these contaminants to aquatic organisms.

Nas últimas décadas, a ocorrência de atividade estrogênica e interferentes endócrinos (IEs) no ambiente aquático têm se tornado uma crescente preocupação. Dentre as diversas substâncias classificadas como IEs, destacam-se os fármacos, produtos de higiene e cuidados pessoais, hormônios naturais e sintéticos, produtos químicos industriais, praguicidas e muitos outros compostos que atingem o ambiente aquático por meio de descargas de esgoto doméstico, industrial ou de escoamento agrícola. Os objetivos deste estudo foram determinar a atividade estrogênica em amostras de águas superficiais, e avaliar seus efeitos biológicos no desenvolvimento de embriões de Danio rerio, a fim de propor faixas baseadas em valores de desencadeamento de efeitos para categorizar a atividade estrogênica. As amostras ambientais também foram analisadas por cromatografia líquida acoplada com a espectrometria de massas para identificar as substâncias que são suspeitas de causar alteração endócrina. Os compostos analisados foram: praguicidas, hormônios, triclosan, bisfenol A, octilfenol, nonilfenol, e a cafeína como indicador de atividade antrópica. A atividade estrogênica foi medida pelo ensaio Bioluminescent Yeast Estrogen (BLYES), que fornece os resultados em equivalente de 17&beta;-estradiol (EEQ). No entanto, este ensaio não é capaz de prover informações sobre os efeitos adversos em organismos aquáticos. Para observação de possíveis efeitos na biota, os embriões foram expostos a amostras de águas superficiais com resultados acima de 0,1 EEQ no BLYES. Os ensaios foram realizados de acordo com a OECD No. 236 (2013), verificando efeitos agudos como: ausência de batimento cardíaco, não formação de somitos, não desprendimento da cauda, e embrião coagulado. Malformações embrionárias tais como: redução do tamanho do organismo, edema cardíaco e vitelínico, curvatura da coluna vertebral, também foram avaliadas. As informações obtidas pelo ensaio com embriões de Danio rerio foram adequadas para mostrar os efeitos da mistura de contaminantes em organismos não-alvo. A atividade estrogênica medida pelo BLYES ficou abaixo do limite de quantificação (0,1 EEQ) em 44,8% do total de 116 amostras analisadas, e a faixa de atividade estrogênica variou de 0,11 a 14,6 EEQ. Além disso, a presença de contaminantes mesmo que em concentrações baixas ressalta a necessidade de mais estudos para entender os efeitos dessas substâncias nos organismos aquáticos. / Over the last few decades, the occurrence of estrogenic activity and endocrine disrupting chemicals (EDCs) in aquatic environment has become a worldwide issue of increasing environmental concern. The EDCs have the ability to alter the endocrine system of organisms, and includes pharmaceuticals, personal care products, steroid hormones, industrial chemicals, pesticides and many other compounds. Such compounds are present in several industrial and domestic activities and reach the aquatic environment via wastewater discharges or agricultural runoff. The aim of this study was to determine the overall estrogenic activity of surface water, evaluate biological effects on fish embryos development, in order to propose concentrations range based on trigger value to categorize estrogenic activity. Environmental samples were also analyzed by liquid chromatography tandem mass spectrometry to identify substances that are suspected to be an endocrine disruptor. The analyzed compounds were: pesticides, hormones, triclosan, bisphenol A, octylphenol, nonylphenol, and caffeine as an indicative of anthropic activity. The estrogenic activity was measured by Bioluminescent Yeast Estrogen assay (BLYES), with the results expressed in 17&beta;-estradiol equivalent quotient (EEQ). However, this assay is not able to provide information about adverse effects to aquatic organisms. In order to observe effects on aquatic organisms, organic extracts of surface water with results &ge; 0.1 EEQ in BLYES were tested in a bioassay using Danio rerio embryos. The methodology was conducted according OECD No. 236 and verified effects such as: lack of heart beat, lack of somites formation, non-detachment tail and coagulated embryo. Embryonic malformations were also evaluated, such as: reduction of organism size, edema and spine curvature, which are chronic effects. These effects probably are associated with contaminants mixtures. The obtained information by embryonic assay with Danio rerio was suitable to show the effects of contaminants mixture and was used to a categorization proposal of estrogenic activity. Estrogenic activity was below the limit of quantification (0.1 EEQ) in 44.8% of 116 analyzed samples, and range of estrogenic activity was from 0.11 to 14.6 EEQ. The tested samples in FET test were analyzed for acute or chronic toxicity in Danio rerio embryos. Based on the obtained results, even when estrogenic activity is present in surface water, the contaminants mixture can cause toxic effects in non-target organisms. Besides this, the widespread presence of these chemicals highlight the need for further studies in order to understand the harmfulness of these contaminants to aquatic organisms.

Background: Evidence from experimental studies suggests that atrazine and its analytes alter the timing of puberty in laboratory animals. Such associations have not been investigated in humans. Objective: To determine the association between in utero exposure to atrazine analytes and earlier menarche attainment in a nested case-control study of the population-based Avon Longitudinal Study of Parents and Children. Methods: Cases were girls who reported menarche before 11.5 years while controls were girls who reported menarche at or after 11.5 years. Seven atrazine analyte concentrations were measured in maternal gestational urine samples (sample gestation week median (IQR): 12 (8–17)) during the period 1991–1992, for 174 cases and 195 controls using high performance liquid chromatography-tandem mass spectrometry. We evaluated the study association using multivariate logistic regression, adjusting for potential confounders. We used multiple imputation to impute missing confounder data for 29% of the study participants. Results: Diaminochlorotriazine (DACT) was the most frequently detected analyte (58%>limit of detection [LOD]) followed by desethyl atrazine (6%), desethyl atrazine mercapturate (3%), atrazine mercapturate (1%), hydroxyl atrazine (1%), atrazine (1%) and desisopropyl atrazine (0.5%). Because of low detection of other analytes, only DACT was included in the exposure–outcome analyses. The adjusted odds of early menarche for girls with DACT exposures≥median was 1.13 (95% Confidence Interval [95% CI]:0.82, 1.55) and exposure Conclusions: This study is the first to examine the association between timing of menarche and atrazine analytes. We found a weak, non-significant association between in-utero exposure to atrazine metabolite DACT and early menarche, though the association was significant in the subset of girls with complete confounder information. Further exploration of the role of these exposures in female reproduction in other cohorts is needed.

In recent years exposure to endocrine-disrupting chemicals (EDCs) in humans and wildlife has become an increasing concern. These compounds have been found ubiquitously in the environment and are suspected to induce adverse effects on the health of aquatic organisms. The results of health effects due to EDCs are clearly presented in many aquatic organisms, such as the feminization of male fish and a near extinction of some species. No clear effects on human health have been documented at this time. The major sources of these contaminants in the environment are discharges from wastewater treatment plants (WWTP) and diffuse pollution. Conventional wastewater treatment processes are not designed to remove such emerging pollutants and removal efficiency depends on many factors, including treatment technology and pollutant species. Passage through WWTPs and changes due to treatment technologies lead to detection of minute concentrations of EDCs in water downstream from discharge points. In New Orleans, Louisiana, discharge from its East Bank WWTP is being considered for potential reuse for wetland restoration. Therefore, effluents must be treated adequately to prevent adverse effects on the natural biota. Since effluents from wastewater treatment plants using conventional technologies may prove potentially unsafe for the environment due to the presence of EDCs, improved and/or new treatment processes for removal of these contaminants are needed. Ferrate (Fe+6) is known as an alternative oxidant for the treatment of wastewater that can be used as an oxidizing, disinfectant, and/or coagulating agent. Because of its redox potential, ferrate has been used as a disinfection agent and has been reported as a tool for enhanced treatment to remove many micropollutants without producing undesirable disinfection byproducts in contrast to other disinfection processes. Recent research has noted the ability of ferrate to deactivate a wide range of EDCs present in wastewater effluents. The negative effect of effluent's soluble organic matter on ferrate has been reported and higher doses of ferrate may be needed to obtain desired effluent quality. This study found that aerobic biological treatment processes reduce more than half of EDCs in wastewater and that free chlorine disinfection increases estrogenic activity in discharged effluent. Higher organic content in wastewater results in increased ferrate demand. The optimum ferrate dosage to deactivate EDCs in lab scale is 6 ppm, and a dosage of 8 ppm is possibly the operational optimum dose. pH neutralization by concentrated sulfuric acid was not found to affect EDCs deactivation efficiency by ferrate when added at the end of designed contact time. Ferrate was observed to have a high oxidation rate in the first10 minutes after application into wastewater and then degraded to other iron states, such as iron III. Higher oxidation rates can be obtained when more organics are present in wastewater as TOC. Higher dosages required longer oxidation reaction times. Ferrate was observed to generate fewer disinfection byproducts as compared to chlorine. Haloacetic acids in ferrate-treated effluent are generated from organics in wastewater and reactions with residual hypochlorite from the incomplete ferrate synthesis process. The reduction of trihalomethanes may be related to EDCs deactivation by ferrate. Because this study was performed on a lab scale, assessment of onsite production and application of ferrate is required to determine the feasibility of the ferrate treatment process at a full-scale treatment plant and to optimize required dosage. / acase@tulane.edu

Les substances perturbatrices du système endocrinien sont des substances qui, de par leur capacité à induire des changements hormonaux chez les organismes vivants, génèrent des préoccupations dans le domaine de la qualité des eaux et, par extension, dans le domaine du traitement des effluents aqueux. Particulièrement, ce projet de recherche s’est attardé sur l’élimination des perturbateurs endocriniens phénoliques nonylphénol (NP), bisphénol A (BPA) et triclosan (TCS) en solution aqueuse à l’aide de la laccase (E.C. 1.10.3.2) sécrétée par la souche fongique Coriolopsis polyzona. Cette oxydase est une métalloprotéine pouvant catalyser l’oxydation d’une vaste gamme de substances phénoliques. En premier lieu, l’impact du pH et de la température sur l’élimination de ces composés à l’aide de la laccase libre en utilisant un design factoriel. L’oxydation de ces composés produit des oligomères (dimère à pentamère) via le couplage des radicaux phénoxy produits par l’action de la laccase. Il s’avère que les substances produites suite à l’oxydation du NP et du BPA par la laccase ont perdu leurs similitudes structurales avec l’estrogène. Ainsi, l’élimination de l’activité estrogénique de ces substances est directement liée à la transformation des composés. Finalement, l’utilisation d’ABTS comme médiateur a permis d’augmenter le taux d’oxydation enzymatique de ces composés chimiques. Puis, de façon à augmenter la possibilité d’utilisation de la laccase dans des biotechnologies environnementales, cette enzyme a été immobilisée sur un support siliceux et via la réticulation d’agrégats. L’impact des conditions d’immobilisation sur l’activité enzymatique, la stabilité du catalyseur et les propriété biocatalytiques apparentes a été déterminé pour différentes stratégies d’immobilisation. Globalement, l’immobilisation génère un biocatalyseur stable vis-à-vis les dénaturations chimique, physique et biologique. Particulièrement, l’immobilisation sur un support solide produit un biocatalyseur facile à utiliser ayant une faible activité massique et des propriétés cinétiques moindres que celle de l’enzyme libre. La formation de CLEAs de laccase a permis d’obtenir une activité massique élevée et des propriétés cinétiques supérieures à celle de l’enzyme soluble. Ces biocatalyseurs solides ont étés utilisés pour éliminer en continu le NP, BPA et TCS dans différents types de bioréacteur. Le biocatalyseur sur silice a été utilisé pour éliminer ces substances dans un réacteur garni, tandis que les CLEAs ont été utilisés dans un réacteur à lit fluidisé et un réacteur à perfusion développé au cours de ce projet. Ces différentes configurations de bioréacteur ont permis d’éliminer efficacement ces différents perturbateurs endocriniens. Globalement, les différents résultats obtenus, à l’échelle de laboratoire, au cours de ce projet de recherche démontrent que la laccase et particulièrement les biocatalyseurs formés via les différentes stratégies d’immobilisation testées représentent des approches extrêmement prometteuses pour le développement de biotechnologies environnementales vouées à l’élimination des perturbateurs endocriniens phénoliques.