The presence of biologically-active estrogenic endocrine disrupting compounds (EDCs) in treated effluents from biological wastewater treatment facilities has prompted wide-spread interest in the behavior of these contaminants during the activated sludge process. The yeast-estrogen screen (YES) was used to quantify the estrogenic activity of samples taken from different areas of three wastewater treatment facilities. An estrogenic mass-balance around these facilities revealed that the majority of influent estrogenic activity was removed in the activated sludge process, but the main route for EDC discharge to the natural environment was via the treated effluent. The estrogenic activity in the effluent from a membrane bioreactor (MBR) was lower compared to a fully aerobic activated sludge process using secondary clarification, suggesting that enhanced removal of particulate and colloidal material may improve EDC removal efficiency. Colloidal material was obtained from settled mixed liquor suspended solids (MLSS) collected from a pilot MBR and a full-scale activated sludge process that included anoxic and aerobic zones. The MLSS was sized fractionated by filtration, and used to quantify the sorption coefficients for pyrene, 17&#946;-estradiol (E2), and 17α-ethinylestradiol (EE2) by fluorescence quenching. The MLSS-derived colloidal organic carbon (COC) sorption coefficient (Kcoc) for pyrene ranged from (< 1 to 80) L/kgcoc, indicating a similar affinity for pyrene compared to natural organic matter. Kcoc coefficients for E2 ranged between (< 1 to 158) L/kgcoc for E2 and (< 1 to 228) L/kgcoc for EE2, and are the highest E2 and EE2 sorption coefficients reported in the literature to date. There was a strong correlation between the Kcoc coefficients and molar extinction coefficient at 280 nm (e280) for pyrene and E2, suggesting that the interaction of the π;-electrons is an important factor in determining overall sorption behavior. There was no such correlation for EE2. Based on the Kcoc coefficients and COC concentrations of the samples, between 1 and 50% of the aqueous E2 and EE2 concentrations were associated with colloidal material. In a novel application of the YES bioassay, the bioavailability of colloid-associated E2 was quantified by comparing the EC50 values of the dose-response curves generated in the presence and absence of size fractionated COC. An increase in EC50 values as a function of COC concentration was attributed to a reduction in bioavailability of E2, suggesting that MLSS-derived COC can reduce, but not eliminate, the biological impact of EDCs. However, there was a high degree of variability in the EC50 values, and estimates of the colloid-associated E2 fraction based on the Kcoc-e280 correlation were unsuccessful in accurately predicting increases in EC50 values. Nevertheless, the YES bioassay may represent a powerful tool in determining the bioavailability of EDCs in complex environmental samples. Results from this research effort suggest that the colloidal phase derived from activated sludge systems represents an important transport vehicle whereby EDCs and other trace organic compounds can enter into the natural environment. Consequently, wastewater treatment plants discharging to sensitive ecosystems or involved with direct water reuse programs should optimize the treatment process to remove colloidal material. / Ph. D.

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.

The project involved the study of 12 Victorian municipal wastewater treatment plant discharges. These included lagoon-based plants and those with activated sludge based processes. Permission was obtained from all the relevant water authorities to collect samples of final effluent at point of discharge to the environment, whether that was to a creek, a river, the ocean, or the land. Samples were collected in November 2003, and then again in April and June 2004, and subjected to a number of biological and chemical analyses, including toxicity tests, measurement of hormonal (estrogenic) activity using yeast-based bioassays, and the measurement of specific hormonal concentrations (17-estradiol) using enzyme-linked immunosorbent assays (ELISA). Almost all of the effluents examined showed estrogenic activity, to a greater or lesser extent (no response to 55 ng/L 17β-estradiol equivalents). On the whole, the levels of estrogenic activity observed were to the lower end of the range observed overseas in the northern hemisphere, and comparable with that recently reported in Australia and New Zealand using similar, human-estrogen receptor based assays (no response to ~ 10 ng/L 17β-estradiol equivalents). The reassuring low/no assay response is bolstered by the chemical assessment of estradiol concentrations by ELISA, which returned concentrations of these compounds for the most part in the range 2-5 ng/L. From an aquatic environmental perspective, it is difficult to say with any certainty what the potential risk to aquatic organisms in waters receiving these effluents will be. Typically, in environmental risk assessment one first looks to agreed national or international guideline or trigger values for the type of waters being assessed. In this case, there are as yet no guideline values. Without guideline values to drive the assessment, then one compares a chemical’s concentration in a sample (in this case a WWTP effluent) with data obtained from toxicological experiments in which the concentration known to elicit a specific effect has been determined. In this case, levels of 17β-estradiol were typically between the lowest reported level to induce the production of Female-indicative proteins in male fish (plasma vitellogen; 1 ng/L), and the lowest concentration of known to induce intersex in fish (8 ng/L). Consequently, such levels in a WWTP discharge are likely to be an environmental risk if there is little or no dilution of the discharge by the receiving water, i.e. discharge represents major component of stream flow. In short, to truly assess the risk (hormonal impact) of these WWTP effluents, in vivo testing needs to be undertaken, ideally with a representative native species but failing that with a ‘standard’ species such as the fathead minnow. When this programme began, the ‘watching brief’, being held in Australia on the topic of endocrine disrupting chemicals and their potential effects on aquatic wildlife was considered too passive by many. It still is, by some. Despite the assurance the results may provide (of minimal impact in most cases if there is significant dilution), there is still a need for further extensive on-ground, reassurance research to provide data for higher-level risk assessment by industry and government agencies.

<p>This work explores the usefulness of fish bile analysis in combination with biomarkers for identifying and evaluating new environmental contaminants in the aquatic environment. It illustrates how bile analysis can be used together with biomarkers to assess the causes of estrogenic effects, to identify chemicals in the aquatic environment that are taken up by fish, and to monitor environmental exposure.</p><p>In a first application, fish exposed to sewage treatment plant effluent were studied. Elevated levels of vitellogenin in the exposed fish demonstrated that estrogenic effects occurred. Several estrogen disrupting substances were identified in the fish bile, and analysis of water samples confirmed that these substances were present in the effluent. The synthetic estrogen 17a-ethinylestradiol, which is known to be present in sewage treatment plant effluent, was shown for the first time to be taken up by fish. Considering the reported potencies of the detected substances, it was concluded that 17a-ethinylestradiol was the major contributor to the estrogenic effects.</p><p>Chemical analysis of bile was used to identify rubber additives that were released from tires immersed in water. The bile of rainbow trout held in the water contained high levels of metabolites of PAHs and aromatic nitrogen compounds. Several biomarkers were also measured in the exposed fish, and EROD induction and oxidative stress were observed. Based on the bile analysis observations together with knowledge of toxicological mechanisms, it was postulated that the EROD induction was due to the PAHs, while aromatic nitrogen compounds caused the oxidative stress.</p><p>Resin acids in fish bile proved to be a good indicator of exposure in a chronic long-term study of rainbow trout exposed to effluent from a total chlorine free (TCF) pulp mill. Elevated levels of GST (gluthatione-S-transferase) and GR (gluthatione reductase) activity, and the presence of DNA adducts after a two month recovery period, indicated that compounds in the pulp mill effluents have persistent effects. In addition to characterising the exposure of the fish to the effluent, the analysis of the resin acids in the bile provided evidence of accidents in the pulp mill that the existing process monitoring system had not detected.</p><p>Resin acids in bile were also found to be a valuable indicator of exposure to pulp mill effluents for eelpout living in the Baltic Sea. A correlation between resin acid levels in bile and skewed sex ratios provided an important link in the chain of evidence that substances in the pulp mill effluents cause male bias of the eelpout embryos.</p><p>A particularly good example of the potential of bile analysis was the identification of a previously unknown environmental contaminant. A large peak was observed in the bile extracts of fish that had been exposed to sewage treatment plant effluent. This peak was identified as triclosan, which demonstrated its presence in sewage treatment plant effluent. Other work went on to show that it is a common contaminant of the aquatic environment. The ability of fish to concentrate contaminant metabolites in bile to levels very much higher than in the environment, and the comparatively low levels of analytic interferences, make bile a particularly attractive matrix to search for new, unknown organic pollutants</p>

Bisphenol A used in the production of polycarbonate plastics and epoxy resins is ubiquitous in the environment. The raw material is released to the environment during the manufacturing process and by leaching from consumer products. Recent studies are suggesting that low-dose amounts of Bisphenol A may have adverse health effects on humans. The possibility of removing Bisphenol A from natural water sources or from solvents used to extract the material from consumer products before they enter the market has been studied. The use of model compounds and related substances (4-isopropylphenol, 4-(t-butyl) phenol, and nitrophenols) have been used to study their removal from aqueous solutions using column chromatography and Octolig®, a commercially available material with polyethylenediamine moieties covalently bonded to high-surface area silica gel. The experimental results suggest that 2-nitrophenol and 4-nitrophenol can be successfully removed while 3-nitrophenol, 4-isopropylphenol, and 4-(t-butyl) phenol did not yield a high percent removal. A look at the pKa of the compounds provides an interesting explanation of the results. It is suggested that the compounds with a pKa of approximately 8.3 or higher would require the solution to be at a high pH for anion formation. The resulting pH of the solution would simultaneously deprotonate the ethylenediamine moieties of Octolig® rendering it incapable of removing the anions by ionic interaction.

This work explores the usefulness of fish bile analysis in combination with biomarkers for identifying and evaluating new environmental contaminants in the aquatic environment. It illustrates how bile analysis can be used together with biomarkers to assess the causes of estrogenic effects, to identify chemicals in the aquatic environment that are taken up by fish, and to monitor environmental exposure. In a first application, fish exposed to sewage treatment plant effluent were studied. Elevated levels of vitellogenin in the exposed fish demonstrated that estrogenic effects occurred. Several estrogen disrupting substances were identified in the fish bile, and analysis of water samples confirmed that these substances were present in the effluent. The synthetic estrogen 17a-ethinylestradiol, which is known to be present in sewage treatment plant effluent, was shown for the first time to be taken up by fish. Considering the reported potencies of the detected substances, it was concluded that 17a-ethinylestradiol was the major contributor to the estrogenic effects. Chemical analysis of bile was used to identify rubber additives that were released from tires immersed in water. The bile of rainbow trout held in the water contained high levels of metabolites of PAHs and aromatic nitrogen compounds. Several biomarkers were also measured in the exposed fish, and EROD induction and oxidative stress were observed. Based on the bile analysis observations together with knowledge of toxicological mechanisms, it was postulated that the EROD induction was due to the PAHs, while aromatic nitrogen compounds caused the oxidative stress. Resin acids in fish bile proved to be a good indicator of exposure in a chronic long-term study of rainbow trout exposed to effluent from a total chlorine free (TCF) pulp mill. Elevated levels of GST (gluthatione-S-transferase) and GR (gluthatione reductase) activity, and the presence of DNA adducts after a two month recovery period, indicated that compounds in the pulp mill effluents have persistent effects. In addition to characterising the exposure of the fish to the effluent, the analysis of the resin acids in the bile provided evidence of accidents in the pulp mill that the existing process monitoring system had not detected. Resin acids in bile were also found to be a valuable indicator of exposure to pulp mill effluents for eelpout living in the Baltic Sea. A correlation between resin acid levels in bile and skewed sex ratios provided an important link in the chain of evidence that substances in the pulp mill effluents cause male bias of the eelpout embryos. A particularly good example of the potential of bile analysis was the identification of a previously unknown environmental contaminant. A large peak was observed in the bile extracts of fish that had been exposed to sewage treatment plant effluent. This peak was identified as triclosan, which demonstrated its presence in sewage treatment plant effluent. Other work went on to show that it is a common contaminant of the aquatic environment. The ability of fish to concentrate contaminant metabolites in bile to levels very much higher than in the environment, and the comparatively low levels of analytic interferences, make bile a particularly attractive matrix to search for new, unknown organic pollutants

A number of widely used industrial chemicals have been shown to possess endocrine-disrupting properties. In this thesis, a series of in vitro tests, and an in vivo reproductive performance test with fathead minnows, were used to clarify the extent of estrogenic activity exhibited by the phthalate esters - a class of compound hitherto referred to as 'estrogenic'. Using a recombinant yeast estrogen screen, I demonstrated that a small number of commercially available phthalates showed extremely weak estrogenic activity. The most potently estrogenic phthalate of those tested was BBP, which was approximately one million-fold less potent than 17B-estradiol. The phthalates which were estrogenic in the yeast screen were also mitogenic on estrogen-responsive human breast cancer cells (MCF-7 and ZR-75 cell lines). The most prolifically used phthalate, DEHP, was not estrogenic in any of these assays. The small number of metabolites of phthalate metabolites tested (including MBuP, MBzP, MEHP and MnOP) were also not estrogenic in the recombinant yeast assay. The ability of BBP (as the most potently estrogenic phthalate in vitro) to induce a vitellogenic response (an indicator of estrogen exposure) in fathead minnows (Pimephales promelas) exposed via the water was assessed. No induction of vitellogenin was observed, indicating that 100 Jlg BBP/L (a concentration higher than would normally be found in the environment) is not estrogenic to this species of fish under the conditions employed for this experiment. In the same study, fecundity of breeding pairs of fathead minnows was assessed; exposure to BBP was not found to affect reproductive performance in these fish. A possible alternative mechanism of action of the way in which the phthalates induce frequently reported reproductive disorders was observed. Some of the phthalates, and, notably, some of their metabolites, were demonstrated to act as anti-androgens in a recombinant yeast androgen assay. 4-Nonylphenol is another industrial chemical which is used in large volumes, and due to the nature of its use (mainly in detergent formulations), is discharged into water systems via sewage effluents. This chemical has been shown to be estrogenic to fish at the concentrations at which it has been detected in the environment. 4-Nonylphenol was tested for its ability to affect plasma and pituitary gonadotropin levels in female recrudescing rainbow trout (Oncorhynchus mykiss). Plasma and pituitary levels of FSH were suppressed in fish exposed to 10 and 100 Jlg 4-NP/L. In addition, FSH gene expression was reduced in these fish, and also in the fish exposed to 1 Jlg 4-NP/L. Pituitary LH content and gene expression of this hormone were suppressed in the fish exposed to 100-, and 10- and 100 Jlg 4-NP/L respectively. Gonadal development in vertebrates is regulated by FSH. Ovarian development ceased in the fish exposed to 100 JAg 4-NP/L, possibly as a result of the suppression of FSH synthesis and/or release in these fish.

Made available in DSpace on 2014-06-11T19:32:56Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-11-23Bitstream added on 2014-06-13T19:43:48Z : No. of bitstreams: 1 cardoso_crp_dr_arafcf.pdf: 7653632 bytes, checksum: 9661b8f40f394a9e28c5c8784c39c064 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / PROPG / O presente trabalho teve como objetivo estudar quatro espécies relacionadas no Projeto BIOTAFAPESP, as quais têm se destacado quanto às atividades farmacológicas: Byrsonima fagifolia Niedenzu, Byrsonima crassa Niedenzu, Indigofera truxillensis Kunth e Indigofera suffruticosa Miller. Foram realizados ensaios de mutação gênica reversa utilizando-se as linhagens TA98, TA100, TA97a e TA102 de Salmonella typhimurium, com ausência e presença do sistema metabólico de ativação. Foram avaliados extratos, frações e algumas substâncias isoladas, a saber: a) B. crassa: amentoflavona, substância isolada do extrato metanólico; b) B. fagifolia: extrato metanólico e clorofórmico, frações acetato e aquosa, ácido gálico, galato de metila, ácido quínico e o ácido 3,4- digaloilquínico; c) I. truxillensis e I.suffruticosa: extrato metanólico e clorofórmico, frações de alcalóides, flavonóides e de glicerolipídeos, além das substâncias índigo, indirubina e o kaempferol- 3,7-diraminosídeo. Foi também avaliada a isatina, molécula precursora dos alcalóides índigo e indirubina. As atividades anti-bacteriana, anti-Leishmania, citotóxica e fitoestrogênica foram avaliadas para substâncias isoladas. Os ensaios de atividade fitoestrogênica foram realizados através do método de e-screen, utilizando-se células tumorais MCF7. Os ensaios de atividade citotóxica (células MCF-7) foram realizados pela técnica de sulforadamina-B. Os ensaios anti- Leishmania (L. amazonensis) também foram relizados com as técnicas de MTT (sal de tetrazólio) e contagem manual em câmara de Neubauer. A atividade anti-bacteriana foi avaliada através das técnicas de difusão em agar e microdiluição, com as bactérias Staphylococcus aureus ATTC 25923 e Escherichia coli ATCC 25922. Estudos anteriores evidenciaram o potencial... / The present work aimed to investigate four related species in the project BIOTA-FAPESP which are notable for their pharmacological activities: Byrsonima fagifolia Niedenzu Byrsonima crassa Niedenzu, Indigofera truxillensis Kunth and Indigofera Indigofera suffruticosa Miller. Tests of reverse gene mutation were performed using TA98, TA100, TA97 and TA102 strains of S. typhimurium in the absence and presence of metabolic activation system. Extracts, fractions and some isolated compounds were evaluated: a) B. fagifolia: methanolic and chloroformic extracts, acetate and aqueous fractions, gallic acid, methyl gallate, quinic acid and 3,4-digaloilquinico derivative. b) I. truxilensis and I.suffruticosa: chloroformic and methanolic extracts, fractions of alkaloids, flavonoids and glicerolipides, and also the indigo, indirubin and kaempferol-3,7-diraminosídeo substances. The isatin, precursor molecule of alkaloids indigo and indirubin was also evaluated. The antimicrobial, antiparasitic, cytotoxic and phytoestrogenic activities were evaluated for natural isolated substances. The phytoestrogenic activity assays were performed using the e-screen method, using MCF7 tumor cells. Trials of cytotoxic activity (MCF-7 cells) were done using sulforadamina-B. The anti-Leishmania tests (L. amazonensis) were also performed with some alkaloids and flavonoids, using MTT techniques (tetrazolium salt) and manual counting in a Neubauer chamber. Antimicrobial activity was evaluated using disc diffusion techniques and microdilution with S. aureus ATTC 25923 and E. coli ATCC 25922 bacterias. The mutagenic activity assays showed positive results only for the methanol extract of I. truxilensis (strain TA98) and for the alkaloids indirubin and indigo isolated from this specie and I. suffruticosa, in addition to isatin. This precursor is also noted... (Complete abstract click electronic access below)

Orientador: Eliana Aparecida Varanda / Banca: Denise Crispim Tavares / Banca: Miriam Sannomiya / Banca: Lourdes Campaner dos Santos / Banca: Taís Maria Baub / Resumo: O presente trabalho teve como objetivo estudar quatro espécies relacionadas no Projeto BIOTAFAPESP, as quais têm se destacado quanto às atividades farmacológicas: Byrsonima fagifolia Niedenzu, Byrsonima crassa Niedenzu, Indigofera truxillensis Kunth e Indigofera suffruticosa Miller. Foram realizados ensaios de mutação gênica reversa utilizando-se as linhagens TA98, TA100, TA97a e TA102 de Salmonella typhimurium, com ausência e presença do sistema metabólico de ativação. Foram avaliados extratos, frações e algumas substâncias isoladas, a saber: a) B. crassa: amentoflavona, substância isolada do extrato metanólico; b) B. fagifolia: extrato metanólico e clorofórmico, frações acetato e aquosa, ácido gálico, galato de metila, ácido quínico e o ácido 3,4- digaloilquínico; c) I. truxillensis e I.suffruticosa: extrato metanólico e clorofórmico, frações de alcalóides, flavonóides e de glicerolipídeos, além das substâncias índigo, indirubina e o kaempferol- 3,7-diraminosídeo. Foi também avaliada a isatina, molécula precursora dos alcalóides índigo e indirubina. As atividades anti-bacteriana, anti-Leishmania, citotóxica e fitoestrogênica foram avaliadas para substâncias isoladas. Os ensaios de atividade fitoestrogênica foram realizados através do método de e-screen, utilizando-se células tumorais MCF7. Os ensaios de atividade citotóxica (células MCF-7) foram realizados pela técnica de sulforadamina-B. Os ensaios anti- Leishmania (L. amazonensis) também foram relizados com as técnicas de MTT (sal de tetrazólio) e contagem manual em câmara de Neubauer. A atividade anti-bacteriana foi avaliada através das técnicas de difusão em agar e microdiluição, com as bactérias Staphylococcus aureus ATTC 25923 e Escherichia coli ATCC 25922. Estudos anteriores evidenciaram o potencial... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The present work aimed to investigate four related species in the project BIOTA-FAPESP which are notable for their pharmacological activities: Byrsonima fagifolia Niedenzu Byrsonima crassa Niedenzu, Indigofera truxillensis Kunth and Indigofera Indigofera suffruticosa Miller. Tests of reverse gene mutation were performed using TA98, TA100, TA97 and TA102 strains of S. typhimurium in the absence and presence of metabolic activation system. Extracts, fractions and some isolated compounds were evaluated: a) B. fagifolia: methanolic and chloroformic extracts, acetate and aqueous fractions, gallic acid, methyl gallate, quinic acid and 3,4-digaloilquinico derivative. b) I. truxilensis and I.suffruticosa: chloroformic and methanolic extracts, fractions of alkaloids, flavonoids and glicerolipides, and also the indigo, indirubin and kaempferol-3,7-diraminosídeo substances. The isatin, precursor molecule of alkaloids indigo and indirubin was also evaluated. The antimicrobial, antiparasitic, cytotoxic and phytoestrogenic activities were evaluated for natural isolated substances. The phytoestrogenic activity assays were performed using the e-screen method, using MCF7 tumor cells. Trials of cytotoxic activity (MCF-7 cells) were done using sulforadamina-B. The anti-Leishmania tests (L. amazonensis) were also performed with some alkaloids and flavonoids, using MTT techniques (tetrazolium salt) and manual counting in a Neubauer chamber. Antimicrobial activity was evaluated using disc diffusion techniques and microdilution with S. aureus ATTC 25923 and E. coli ATCC 25922 bacterias. The mutagenic activity assays showed positive results only for the methanol extract of I. truxilensis (strain TA98) and for the alkaloids indirubin and indigo isolated from this specie and I. suffruticosa, in addition to isatin. This precursor is also noted... (Complete abstract click electronic access below) / Doutor