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.

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.

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.

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.

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.

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)