Consumption of whole grains has been linked to reduced risk of colon cancer. This study determined estrogenic activity of sorghum phenolic extracts of different phenolic profiles and identified possible estrogenic compounds in sorghum in vitro, as well as evaluated the potential of estrogenic sorghum phenolic extracts to prevent colon carcinogenesis in vivo. The thermal stability of sorghum 3-deoxyanthocyanins was also studied, to determine their suitability as functional food colorants. White and TX430 (black) sorghum extracts showed estrogenic activity in cell models predominantly expressing estrogen receptor-α (ERα) or ERβ at 5 and 10 µg/mL, respectively. The same treatments led to induction of apoptosis in cells expressing ERβ. The red TX2911 sorghum did not possess these activities. Compositional analysis revealed differences in flavones and flavanones. Flavones with estrogen-like properties, i.e. luteolin and apigenin, were detected in White and TX430 (black) sorghum extracts, but not in red TX2911 extract. Naringenin, a flavanone known to antagonize ERα signalling, was only detected in the red TX2911 extract. Additional experiments with sorghum extracts of distinct flavones/flavanone ratio, as well as with pure apigenin and naringenin, suggested that flavones are the more potent ERβ agonists in sorghum. On the other hand, 3-deoxyanthocyanins were probably not estrogenic. Estrogenic white and black sorghum phenolic extracts (fed at 1% level in the diet) reduced the number of azoxymethane induced colon premalignant lesion (aberrant crypt foci) by 39.3% and 14.7%, respectively, in ovariectomized mice. Further studies are needed to elucidate the protective mechanisms induced by these sorghum extracts. Sorghum 3-deoxyanthocyanins retained good color stability after 30 minutes of heat treatment at 121 °C under pressure: More than 80% of color retained in pH 1 and 2 HCl and citric acid solutions, and 39-84% retained from pHs 3-7. Formic acid negatively affected the color stability at pH 1 and pH 2 due to its reducing capacity. Methoxylation decreased the thermal stability of 3-deoxyanthocyanins. The heat stability of 3-deoxyanthocyanins indicates good potential for food use. Overall, the inherent estrogenic activity of specific sorghum phenolic extracts is a likely mechanism for colon cancer prevention. Further studies are needed to assess physiologically relevant dietary level of sorghum phenolics for prevention of colon cancer, and effect of food processing on the activity and bioavailability of the chemopreventive components.

There has been an increase in reports of male reproductive disorders that include male infertility and testicular cancer worldwide. It has been suggested that agents such as xenoestrogens could be responsible. Xenoestrogens are chemical compounds that mimic the action of estrogens by binding to the estrogen receptors (ERs). The response ofa testicular cell line to estrogenic pesticides was examined. The effect of estrogenic pesticides on the growth and protein levels of ERα and ERβ of mouse Sertoli cells was investigated. Pyrethroids are widely used insecticides due to their insecticidal potency and low mammalian toxicity. In this study, the estrogenicity ofpyrethroid chemicals were tested using the yeast estrogen screen (YES) assay. The toxic effects of the pyrethroid compounds cypermethrin, 3-(4-hydroxy-phenoxy)benzyl alcohol (metabolite of permethrin), and the commercial product (Ripcord Plus) were evaluated. The Sertoli cells were exposed to pyrethroids at concentrations of 0.36 nM and 36 µM (cypermethrin and Ripcord Plus), and 0.69 nM and 69 µM (metabolite) for 100 h. The expression of the ERs was analysed through the use of Enzyme Linked ImmunoSorbent Assay (ELISA) experiments. The most toxic pyrethroid was the metabolite, followed by Ripcord Plus then cypermethrin. Overall the exposure of the cells to cypermethrin (36 µM), Ripcord Plus (36 µM) and the metabolite (69 µM) caused a significant decrease (p<0.05) in ERα levels. In the cultures exposed to the metabolite (69 µM), there was also a significant increase in ERβ levels. There appears to be a relation between cell toxicity and an increase in ERβ levels, which supports the theory that ERβ promotes apoptosis. Pyrethroids are rapidly excreted from the body, and it is unknown if there is accumulation in the male testes. Male fertility could be affected through molecular mechanisms involving the ERs, should cells in the male testes be exposed to these pyrethroids at physiologically relevant concentrations.

Estrogenic contamination of waterways is of world-wide concern due to the adverse effects observed in aquatic biota. Recently, wastes from agricultural activities have been identified as likely sources of steroid estrogens released into the environment. Wastes from dairying activities are of particular concern in New Zealand. This project included development of analytical methods to measure free and conjugated estrogens, measurement of estrogens from the source to receiving environments and an investigation of effluent treatment technologies. The analytical method developed in this study was based on GC-MS measurement of free estrogens (17α-estradiol (17α-E2), 17β-estradiol (17β-E2) and estrone (E1)) and LC-IT-MS measurement of their sulfate-conjugates (17α-E2-3S, 17β-3S, E1-3S) in raw and treated farm dairy shed effluents (DSE). Effluents from farms in the Canterbury and Waikato Regions, two regions where dairy farming is the dominant land-use, were collected and analysed. All effluents demonstrated high concentrations of steroid estrogens, particularly 17α-E2 (median 760 ng/L). Estrogenic activity was also elevated, at up to 500 ng/L 17β-E2 equivalents using the E-Screen, an in vitro cell proliferation bioassay. Comparison to the chemical data indicated that for most samples, the highest proportion of estrogenic activity was derived from steroid estrogens naturally excreted by dairy cows. Conjugated estrogens were measured in several raw effluent samples, at similar concentrations to those of free estrogens, particularly E1. Dairy effluent treatment systems reduced free estrogen concentrations by 63-99% and reduced estrogenic activity by up to 89%. In spite of high removal efficiencies, estrogens remained elevated in the treated effluents that are discharged into waterways. Steroid estrogens and estrogenic activity were detected in streams and groundwater in areas impacted by dairy farming. Although concentrations were generally low, in two streams the concentrations were above levels regarded as safe for aquatic biota (<1 ng/L). The results demonstrate that dairy effluents are indeed a major source of estrogens to the environment and to waterways.

Hormônios estrogênicos, quando presentes no meio ambiente, podem causar danos ao sistema endócrino de organismos com os quais entram em contato. Estes hormônios são liberados nas fezes e urinas de animais, incluindo os seres humanos, podendo atingir os corpos hídricos e solos. As formas de tratamento aplicadas na maioria das estações de tratamento de esgotos (ETEs), em geral, não são capazes de remover totalmente os hormônios. A remoção dos hormônios tem sido associada ao metabolismo dos microorganismos, ao tempo de detenção hidráulica, à idade do lodo e à carga de alimentação dentre outros fatores. O objetivo deste estudo foi investigar a remoção dos hormônios estrona (E1), 17-?-estradiol (E2), estriol (E3) e 17-?-etinilestradiol (EE2) na ETE da Escola de Engenharia de Lorena/USP operando com o sistema de lodos ativados por bateladas sequenciais (SBR), além de estudar a influência da carga de alimentação e da remoção de nitrogênio na remoção destes hormônios. Foram utilizadas cromatografia líquida acoplada a detector UV para a determinação dos hormônios, e análises químicas, físicas e biológicas para a caracterização da ETE. A fim de garantir a confiabilidade dos resultados, foi necessária a validação do método desenvolvido para o preparo e a quantificação dos hormônios, sendo obtidos valores aceitáveis de precisão, coeficiente de variação (CV) entre 0,17 e 8,48 %; exatidão entre 45 e 116 %; e limites de detecção (LD) e quantificação (LQ) satisfatórios para o tipo de detector empregado (UV), entre 60 e 250 ?g L-1 de LD e entre 250 e 520 ?g L-1 de LQ. Entretanto, durante a validação, foi detectado efeito matriz tendo sido necessária a quantificação dos hormônios pelo método de adição de padrão a cada eluato obtido. As elevadas concentrações dos hormônios no esgoto (5,148 ± 2,747; 7,434 ± 4,356; 5,200 ± 3,331 e 5,638 ± 4,312 ?g L-1 de E1, E2, E3 e EE2, respectivamente) podem ser atribuídas à baixa geração de esgoto per capita e à possível predominância da urina no esgoto, visto a elevada concentração de nitrogênio total Kjeldahl (NTK) nestas amostras. O desempenho da ETE para a remoção dos hormônios estrogênicos não foi satisfatório podendo estar relacionada à ineficiente remoção de NTK (-4 %), ao curto tempo de detenção hidráulica deste sistema (2h05min), além da ocorrência da desconjugação durante o tratamento, dado o curto tempo entre a geração e a ETE, o que pode ser insuficiente para a desconjugação antes da chegada a ETE. / Some damages can be caused to endocrine systems of organisms if environment contains estrogenic hormones. Estrogens are released in feces and urine of animals, including humans, and may reach soils and water bodies. Most of systems in use as wastewater treatments are not capable of removing these hormones. Some factors can impact on it, as the kind of microorganisms metabolism, hydraulic retention time, sludge age and organic load. The objectives of this work were to investigate the removal of estrone (E1), 17?- estradiol (E2), estriol (E3) and 17?-ethynylestradiol (EE2) hormones in sewage treatment plant (STP), at Lorena School of Engineering, with secondary treatment based on activated sludge into a sequencing batch reactor (SBR) and to study the influence of organic load and nitrogen removal on estrogens remotion. Liquid chromatography coupled to UV detector was applied to estrogens determination and chemical, physical and biological analyses were used to STP\'s characterization. Extraction and quantification method was validated by the assays of linearity; precision, with RSD values between 0.17 and 8.48 %; and accuracy, which presented recuperation between 45 and 116% for these four estrogens. Limits of detection values, between 60 and 250 ?g.L-1, and limits of quantification values, between 250 and 520 ?g.L-1, are consistent with the detector used (UV). However, matrix effect was detected along validation, consequently it was necessary to employ postextraction addition method of standards into each eluate. High concentrations of estrogens in sewage (5.148 ± 2.747; 7.434 ± 4.356; 5.200 ± 3.3314 e 5.638 ± 4.312 ?g L-1 of E1, E2, E3 e EE2) could be attributed to the reduced sewage generation and to the possible predominance of urine in the sewage, due to high NTK measured. The poor performance could be associated to inefficient removal of NTK (-4 %); to short hydraulic retention time applied (2h05min); besides the occurrence of deconjungation during biologic treatment, due to short transit between generation and STP which can be insufficient to deconjugate before STP.

Hormônios estrogênicos, quando presentes no meio ambiente, podem causar danos ao sistema endócrino de organismos com os quais entram em contato. Estes hormônios são liberados nas fezes e urinas de animais, incluindo os seres humanos, podendo atingir os corpos hídricos e solos. As formas de tratamento aplicadas na maioria das estações de tratamento de esgotos (ETEs), em geral, não são capazes de remover totalmente os hormônios. A remoção dos hormônios tem sido associada ao metabolismo dos microorganismos, ao tempo de detenção hidráulica, à idade do lodo e à carga de alimentação dentre outros fatores. O objetivo deste estudo foi investigar a remoção dos hormônios estrona (E1), 17-?-estradiol (E2), estriol (E3) e 17-?-etinilestradiol (EE2) na ETE da Escola de Engenharia de Lorena/USP operando com o sistema de lodos ativados por bateladas sequenciais (SBR), além de estudar a influência da carga de alimentação e da remoção de nitrogênio na remoção destes hormônios. Foram utilizadas cromatografia líquida acoplada a detector UV para a determinação dos hormônios, e análises químicas, físicas e biológicas para a caracterização da ETE. A fim de garantir a confiabilidade dos resultados, foi necessária a validação do método desenvolvido para o preparo e a quantificação dos hormônios, sendo obtidos valores aceitáveis de precisão, coeficiente de variação (CV) entre 0,17 e 8,48 %; exatidão entre 45 e 116 %; e limites de detecção (LD) e quantificação (LQ) satisfatórios para o tipo de detector empregado (UV), entre 60 e 250 ?g L-1 de LD e entre 250 e 520 ?g L-1 de LQ. Entretanto, durante a validação, foi detectado efeito matriz tendo sido necessária a quantificação dos hormônios pelo método de adição de padrão a cada eluato obtido. As elevadas concentrações dos hormônios no esgoto (5,148 ± 2,747; 7,434 ± 4,356; 5,200 ± 3,331 e 5,638 ± 4,312 ?g L-1 de E1, E2, E3 e EE2, respectivamente) podem ser atribuídas à baixa geração de esgoto per capita e à possível predominância da urina no esgoto, visto a elevada concentração de nitrogênio total Kjeldahl (NTK) nestas amostras. O desempenho da ETE para a remoção dos hormônios estrogênicos não foi satisfatório podendo estar relacionada à ineficiente remoção de NTK (-4 %), ao curto tempo de detenção hidráulica deste sistema (2h05min), além da ocorrência da desconjugação durante o tratamento, dado o curto tempo entre a geração e a ETE, o que pode ser insuficiente para a desconjugação antes da chegada a ETE. / Some damages can be caused to endocrine systems of organisms if environment contains estrogenic hormones. Estrogens are released in feces and urine of animals, including humans, and may reach soils and water bodies. Most of systems in use as wastewater treatments are not capable of removing these hormones. Some factors can impact on it, as the kind of microorganisms metabolism, hydraulic retention time, sludge age and organic load. The objectives of this work were to investigate the removal of estrone (E1), 17?- estradiol (E2), estriol (E3) and 17?-ethynylestradiol (EE2) hormones in sewage treatment plant (STP), at Lorena School of Engineering, with secondary treatment based on activated sludge into a sequencing batch reactor (SBR) and to study the influence of organic load and nitrogen removal on estrogens remotion. Liquid chromatography coupled to UV detector was applied to estrogens determination and chemical, physical and biological analyses were used to STP\'s characterization. Extraction and quantification method was validated by the assays of linearity; precision, with RSD values between 0.17 and 8.48 %; and accuracy, which presented recuperation between 45 and 116% for these four estrogens. Limits of detection values, between 60 and 250 ?g.L-1, and limits of quantification values, between 250 and 520 ?g.L-1, are consistent with the detector used (UV). However, matrix effect was detected along validation, consequently it was necessary to employ postextraction addition method of standards into each eluate. High concentrations of estrogens in sewage (5.148 ± 2.747; 7.434 ± 4.356; 5.200 ± 3.3314 e 5.638 ± 4.312 ?g L-1 of E1, E2, E3 e EE2) could be attributed to the reduced sewage generation and to the possible predominance of urine in the sewage, due to high NTK measured. The poor performance could be associated to inefficient removal of NTK (-4 %); to short hydraulic retention time applied (2h05min); besides the occurrence of deconjungation during biologic treatment, due to short transit between generation and STP which can be insufficient to deconjugate before STP.

Recent studies have shown that dehydroabietic acid (DHAA), a resin acid present in pulp and paper mills, may have anti-estrogenic effects in fish. A chronic-exposure toxicity experiment using immature rainbow trout (Oncorhynchus mykiss) was conducted in order to assess the endocrine disrupting and liver metabolic effects of the wood extractives DHAA and β- sitosterol (BS) regularly present in pulp and paper mills and the model estrogen 17β-estradiol (E2). It was found that exposure to 5 ppm of E2 significantly increased hepatosomatic index (HSI), vitellogenin (VTG) and plasma sorbitol dehydrogenase (SDH). This effect was reduced by mixing E2 with DHAA, indicating that DHAA does not cause its anti-estrogenic effects indirectly due to liver damage. Exposure to 5 ppm of DHAA caused a significant increase in liver citrate synthase (CS), and liver ethoxyresorufin-O-deethylase (EROD) activity after 7 days, however, the fish recovered by 28 days. This study also determined the effect of 14 different pulp and paper mill effluent extracts on liver enzyme metabolism through alterations in the activity of liver lactate dehydrogenase activity (LDH) and CS. This activity varied greatly between mills but most showed an induction of CS after 28 days exposure through i.p. injection. The results of the study indicate that DHAA may alter energy metabolism as well as cause anti-estrogenic effects in female juvenile rainbow trout. / UOIT

Ecological impacts of natural estrogens and xenoestrogens in treated wastewater include altered sexual development and sex ratios among continuously exposed organisms. The primary sources of estrogenic activity in wastewater are natural estrogens such as estrone, 17β-estradiol and estriol and synthetic compounds like 17α- ethinylestradiol, alkylphenols and alklphenol ethoxylates. Precursors in raw wastewater can yield estrogenic intermediates during wastewater treatment. All these compounds can be destroyed by biochemical processes conventional wastewater treatment processes, suggesting that conventional processes can be optimized for removal of estrogenic activity from wastewater. Sorption to sludges derived from wastewater treatment affects the fates of hydrophobic xenoestrogens such as nonylphenol, in part because the biodegradability of sorbed contaminants is limited. It may also be possible to tailor sludge stabilization processes to remove trace contaminants, including estrogens. For example, there are significant differences in the efficiencies of aerobic and anaerobic digestion for destruction of alkylphenols and probably other estrogenic compounds with aromatic moieties. Because advanced wastewater treatment is not economically feasible for most communities, there is ample incentive to develop accurate relationships between operational parameters and removal of estrogenic compounds during secondary wastewater treatment. Large quantities of polybrominated diphenyl ethers (PBDEs) have been used as flame retardants in clothing and plastic products since the 1970s. A small fraction of the PBDEs in manufactured products subsequently enters municipal wastewater. The resistance of these compounds to chemical and biochemical transformations provides opportunities for accumulation in sediments. Balances developed for PBDE congeners indicate that conventional wastewater treatment processes and soil infiltration of treated wastewater in recharge operations do not discriminate significantly among the major congeners in commercially available PBDE products. Accumulation of PBDEs at near part-per-million levels was measured in the sediments at the Sweetwater Recharge Facility in Tucson, Arizona, during 10-15 years of operation. Half times for loss of major PBDE congeners from sediments were decades or longer. Local agricultural soils amended with biosolids over a 20-year period showed similar accumulation of PBDEs. The widespread use of PBDEs in commercial products, compound persistence and toxicity indicate that additional effort is warranted to better understand fate-determining processes for PBDEs in the environment.

Municipal wastewater treatment plants (MWWTPs) are point sources of environmental contamination that can cause adverse effects on fish species exposed to their effluents (MWWEs). Contaminants, such as endocrine disrupting compounds (EDCs), commonly found in MWWEs have been shown to induce many adverse reproductive effects. In the Grand River watershed of southern Ontario, three MWWTPs from the cities of Guelph, Waterloo and Kitchener have been identified to adversely alter molecular and cellular responses in fish, which have been previously associated with exposure to estrogenic EDCs. Currently these systems are undergoing process upgrades to address aging infrastructure as well as expanding capacities to serve rapidly increasing populations. Studies in this thesis were conducted to determine whether the Guelph and Waterloo MWWEs, as well as surrogate pilot plant effluents that modeled possible treatment process upgrades for MWWTPs were estrogenic in vivo in controlled laboratory exposures. Unfortunately, the Kitchener MWWE (conventional activated sludge and lagoon) was acutely toxic and could not be included in laboratory exposures. Rainbow trout (Oncorhynchus mykiss) were exposed to 20% to 90% effluent from both Guelph and Waterloo, for 2, 8 and 14 days and fish plasma was analyzed for vitellogenin (VTG) a biomarker for estrogenic exposure. These exposures indicated that neither the Guelph nor the Waterloo MWWE were estrogenic in vivo under the exposure conditions in this study. In contrast the total estrogenic equivalence measured using the yeast estrogen screen (YES) of the Guelph, Waterloo and Kitchener MWWEs increased respectively, inversely related to the degree of treatment. Three pilot plant effluents (Burlington MWWTP) including conventional activated sludge (CAS), nitrifying with CAS (CAS-N) and biological nutrient removal with CAS (CAS-BNR) were also tested in rainbow trout in vivo for plasma VTG induction. Only the CAS-BNR effluent caused weak (2-3.5 fold) induction of VTG relative to controls. Although the estrogenicity (YES) of the effluent was variable the VTG induction corresponded with the slightly higher 17??-estradiol equivalents measured in these pilot plant effluents. An effect-directed analysis (EDA) for estrogenic substances was carried out on the Waterloo and Kitchener MWWEs and revealed that the estrogenicity (YES) of the Waterloo MWWE was associated with estrone, while the estrogenicity of the Kitchener MWWE was associated with several chemicals (estrone > 17??-estradiol > bisphenol A ~ testosterone).

There has been an increase in reports of male reproductive disorders that include male infertility and testicular cancer worldwide. It has been suggested that agents such as xenoestrogens could be responsible. Xenoestrogens are chemical compounds that mimic the action of estrogens by binding to the estrogen receptors (ERs). The response ofa testicular cell line to estrogenic pesticides was examined. The effect of estrogenic pesticides on the growth and protein levels of ERα and ERβ of mouse Sertoli cells was investigated. Pyrethroids are widely used insecticides due to their insecticidal potency and low mammalian toxicity. In this study, the estrogenicity ofpyrethroid chemicals were tested using the yeast estrogen screen (YES) assay. The toxic effects of the pyrethroid compounds cypermethrin, 3-(4-hydroxy-phenoxy)benzyl alcohol (metabolite of permethrin), and the commercial product (Ripcord Plus) were evaluated. The Sertoli cells were exposed to pyrethroids at concentrations of 0.36 nM and 36 µM (cypermethrin and Ripcord Plus), and 0.69 nM and 69 µM (metabolite) for 100 h. The expression of the ERs was analysed through the use of Enzyme Linked ImmunoSorbent Assay (ELISA) experiments. The most toxic pyrethroid was the metabolite, followed by Ripcord Plus then cypermethrin. Overall the exposure of the cells to cypermethrin (36 µM), Ripcord Plus (36 µM) and the metabolite (69 µM) caused a significant decrease (p<0.05) in ERα levels. In the cultures exposed to the metabolite (69 µM), there was also a significant increase in ERβ levels. There appears to be a relation between cell toxicity and an increase in ERβ levels, which supports the theory that ERβ promotes apoptosis. Pyrethroids are rapidly excreted from the body, and it is unknown if there is accumulation in the male testes. Male fertility could be affected through molecular mechanisms involving the ERs, should cells in the male testes be exposed to these pyrethroids at physiologically relevant concentrations.

Estrogenic contamination of waterways is of world-wide concern due to the adverse effects observed in aquatic biota. Recently, wastes from agricultural activities have been identified as likely sources of steroid estrogens released into the environment. Wastes from dairying activities are of particular concern in New Zealand. This project included development of analytical methods to measure free and conjugated estrogens, measurement of estrogens from the source to receiving environments and an investigation of effluent treatment technologies. The analytical method developed in this study was based on GC-MS measurement of free estrogens (17α-estradiol (17α-E2), 17β-estradiol (17β-E2) and estrone (E1)) and LC-IT-MS measurement of their sulfate-conjugates (17α-E2-3S, 17β-3S, E1-3S) in raw and treated farm dairy shed effluents (DSE). Effluents from farms in the Canterbury and Waikato Regions, two regions where dairy farming is the dominant land-use, were collected and analysed. All effluents demonstrated high concentrations of steroid estrogens, particularly 17α-E2 (median 760 ng/L). Estrogenic activity was also elevated, at up to 500 ng/L 17β-E2 equivalents using the E-Screen, an in vitro cell proliferation bioassay. Comparison to the chemical data indicated that for most samples, the highest proportion of estrogenic activity was derived from steroid estrogens naturally excreted by dairy cows. Conjugated estrogens were measured in several raw effluent samples, at similar concentrations to those of free estrogens, particularly E1. Dairy effluent treatment systems reduced free estrogen concentrations by 63-99% and reduced estrogenic activity by up to 89%. In spite of high removal efficiencies, estrogens remained elevated in the treated effluents that are discharged into waterways. Steroid estrogens and estrogenic activity were detected in streams and groundwater in areas impacted by dairy farming. Although concentrations were generally low, in two streams the concentrations were above levels regarded as safe for aquatic biota (<1 ng/L). The results demonstrate that dairy effluents are indeed a major source of estrogens to the environment and to waterways.