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

Muitos oxidantes químicos reativos acarretam na ruptura de estruturas moleculares complexas de vários tipos de compostos orgânicos decompondo-as em estruturas mais simples e propiciando condições melhores para uma efetiva ação de micro-organismos na degradação biológica. A presença de hormônios, entre eles o 17β-estradiol, em estações de tratamento de esgoto e em águas subterrâneas e superficiais mostra a necessidade de uma avaliação dos processos de tratamento convencionais. O objetivo deste trabalho foi a análise e remoção de hormônios através de técnicas de cloração e ozonização de amostras reais de águas de saída de filtro de estações de tratamento de água (ETAs), operadas pelo Serviço Autônomo de Água e Esgoto (SAAE) de São Carlos (ETA Centro), que capta águas dos ribeirão Feijão e córrego Espraiado e pela Sociedade de Abastecimento de Água e Saneamento S/A (SANASA) de Campinas (ETAs 3 e 4), que capta águas do rio Atibaia. Foram realizados ensaios contaminando-se as amostras com 17β-estradiol, que é o hormônio natural mais presente no meio ambiente, em concentração de 6.000 ng L-1, submetendo-se tratamento com dosagens em torno de 0,5 e 2,0 mg L-1 desses oxidantes, em tempos de contato de, respectivamente, 10 e 30 min. As amostras submetidas à contaminação e tratamento e as de controle (sem contaminação e tratamento) foram analisadas através da remoção do 17β-estradiol com a verificação da atividade estrogênica das amostras por ensaios de Sistema de Expressão de Estrogênio Induzida por Levedura Bioluminescente (BLYES), que apresentou-se como uma ferramenta simples. Os resultados apresentados neste trabalho demonstram que a oxidação por ozônio se mostrou mais eficiente do que aquela por cloro para a remoção da atividade estrogênica causada, única e exclusivamente, pelo 17β-estradiol para uma dosagem inicial desse hormônio relativamente alta (6.000 ng L-1). Todavia, em todos os ensaios a concentração final da atividade estrogênica permaneceu acima do limite de quantificação desses hormônio, indicando que a remoção não foi completa, mesmo em condições favoráveis, isto é, matriz limpa, com padrões de potabilidade para os parâmetros físico-químicos. / Many reactive chemical oxidants cause the disruption of the complex molecular structures of various types of organic compounds decomposing them into simpler structures and providing better conditions for effective action of micro-organisms in the biological degradation. The presence of hormones, including 17β-estradiol in sewage treatment plants and groundwater and surface water shows the need for an evaluation of conventional treatment processes. The aim of this study was the analysis and removal of hormones through techniques of chlorination and ozonation of real samples of output filter water in water treatment plants (WTP), operated by the Serviço Autônomo de Água e Esgoto (SAAE) of São Carlos (Center WTP), which captures waters from Espraiado stream and Feijão stream and the Sociedade de Abastecimento de Água e Saneamento S/A (SANASA) de Campinas (WTPs 3 and 4), which captures water from the Atibaia river. Assays were performed contaminating the samples with 17β-estradiol, which is the natural hormone more present in the environment, in a concentration of 6.000 ng L-1 submitting themselves to treatment with dosages of around 0.5 to 2.0 mg L-1 of these oxidants in times of contact, respectively, 10 and 30 min. The samples subjected to contamination and treatment and control samples (without contamination and treatment) were analyzed by removing 17β-estradiol with estrogenic activity verification of the samples by Bioluminescent Assays or Estrogen-Inducible Yeast Expression System (BLYES), which was presented as a simple tool. The results presented here demonstrate that the oxidation by ozone was more effective than with chlorine to remove estrogenic activity caused solely by 17β-estradiol for an initial dosage of the hormone relatively high (6000 ngL -1) However, in all experiments the final concentration of estrogenic activity remained above the limit of quantification of these hormones, indicating that removal was not complete, even under favorable conditions, clean matrix with quality for drinking water physico-chemical parameters.

Muitos oxidantes químicos reativos acarretam na ruptura de estruturas moleculares complexas de vários tipos de compostos orgânicos decompondo-as em estruturas mais simples e propiciando condições melhores para uma efetiva ação de micro-organismos na degradação biológica. A presença de hormônios, entre eles o 17β-estradiol, em estações de tratamento de esgoto e em águas subterrâneas e superficiais mostra a necessidade de uma avaliação dos processos de tratamento convencionais. O objetivo deste trabalho foi a análise e remoção de hormônios através de técnicas de cloração e ozonização de amostras reais de águas de saída de filtro de estações de tratamento de água (ETAs), operadas pelo Serviço Autônomo de Água e Esgoto (SAAE) de São Carlos (ETA Centro), que capta águas dos ribeirão Feijão e córrego Espraiado e pela Sociedade de Abastecimento de Água e Saneamento S/A (SANASA) de Campinas (ETAs 3 e 4), que capta águas do rio Atibaia. Foram realizados ensaios contaminando-se as amostras com 17β-estradiol, que é o hormônio natural mais presente no meio ambiente, em concentração de 6.000 ng L-1, submetendo-se tratamento com dosagens em torno de 0,5 e 2,0 mg L-1 desses oxidantes, em tempos de contato de, respectivamente, 10 e 30 min. As amostras submetidas à contaminação e tratamento e as de controle (sem contaminação e tratamento) foram analisadas através da remoção do 17β-estradiol com a verificação da atividade estrogênica das amostras por ensaios de Sistema de Expressão de Estrogênio Induzida por Levedura Bioluminescente (BLYES), que apresentou-se como uma ferramenta simples. Os resultados apresentados neste trabalho demonstram que a oxidação por ozônio se mostrou mais eficiente do que aquela por cloro para a remoção da atividade estrogênica causada, única e exclusivamente, pelo 17β-estradiol para uma dosagem inicial desse hormônio relativamente alta (6.000 ng L-1). Todavia, em todos os ensaios a concentração final da atividade estrogênica permaneceu acima do limite de quantificação desses hormônio, indicando que a remoção não foi completa, mesmo em condições favoráveis, isto é, matriz limpa, com padrões de potabilidade para os parâmetros físico-químicos. / Many reactive chemical oxidants cause the disruption of the complex molecular structures of various types of organic compounds decomposing them into simpler structures and providing better conditions for effective action of micro-organisms in the biological degradation. The presence of hormones, including 17β-estradiol in sewage treatment plants and groundwater and surface water shows the need for an evaluation of conventional treatment processes. The aim of this study was the analysis and removal of hormones through techniques of chlorination and ozonation of real samples of output filter water in water treatment plants (WTP), operated by the Serviço Autônomo de Água e Esgoto (SAAE) of São Carlos (Center WTP), which captures waters from Espraiado stream and Feijão stream and the Sociedade de Abastecimento de Água e Saneamento S/A (SANASA) de Campinas (WTPs 3 and 4), which captures water from the Atibaia river. Assays were performed contaminating the samples with 17β-estradiol, which is the natural hormone more present in the environment, in a concentration of 6.000 ng L-1 submitting themselves to treatment with dosages of around 0.5 to 2.0 mg L-1 of these oxidants in times of contact, respectively, 10 and 30 min. The samples subjected to contamination and treatment and control samples (without contamination and treatment) were analyzed by removing 17β-estradiol with estrogenic activity verification of the samples by Bioluminescent Assays or Estrogen-Inducible Yeast Expression System (BLYES), which was presented as a simple tool. The results presented here demonstrate that the oxidation by ozone was more effective than with chlorine to remove estrogenic activity caused solely by 17β-estradiol for an initial dosage of the hormone relatively high (6000 ngL -1) However, in all experiments the final concentration of estrogenic activity remained above the limit of quantification of these hormones, indicating that removal was not complete, even under favorable conditions, clean matrix with quality for drinking water physico-chemical parameters.

Les perturbateurs endocriniens (PE) posent des risques pour la santé Humaine et pour la faune. L’utilisation de tests biologiques basés sur des mécanismes d’action spécifiques permet de caractériser le potentiel PE des substances chimiques dans le cadre de l'évaluation toxicologique, mais les capacités de biotransformation de ces modèles sont rarement prises en compte. Or, le métabolisme conditionne le devenir des xénobiotiques (détoxication vs. bioactivation) et donc in fine l'activité biologique mesurée. Dans ce travail, le devenir de deux contaminants œstrogéniques émergents (benzophénone-2, bisphénol S) a été comparé dans de nouveaux modèles in vitro et in vivo de poisson zèbre (cellules ZELH-zfERs, larve transgénique cyp19a1b-GFP) et des lignées humaines. Nos résultats démontrent que les modèles de poissons zèbres sont métaboliquement compétents et soulignent leur pertinence dans une approche intégrée in vivo/in vitro pour le criblage de l'activité PE des substances chimiques. / Endocrine disruptors (ED) are potentially harmful for Human beings and for wildlife species. Species-specific mechanism-based screening tests nowadays allow characterizing the ED potency of chemicals in the context of their toxicological assessment. However, the biotransformation capabilities of the biological models used are seldom taken into consideration, despite they ultimately condition the fate of the tested xenobiotics (detoxification vs. bioactivation), and, consequently, the biological response. In this work, we examined the fate of two emerging ED (benzophenone-2, bisphenol S) using novel in vitro and in vivo zebrafish models (ZELH-zfERs cells and cyp19a1b-GFP transgenic larvae) as well as human cell lines. Our results demonstrate that the zebrafish models are metabolically competent, and underline their relevance, accuracy and usefulness in an integrated in vivo/in vitro screening approach designed to assess ED's biological activity

L’objectif de ce travail de thèse est d’évaluer le potentiel de nouveaux bio-essais in vitro et in vivo basés sur le poisson zèbre pour la biosurveillance de la contamination chimique de l’eau par les xeno-estrogènes. Pour cela, les bio-essais ont été appliqués pour évaluer les effets de polluants aquatiques environnementaux seuls, mais aussi en mélanges simples (reconstitués) et complexes (échantillons environnementaux). L’évaluation d’échantillons d’eau à travers les bioessais in vitro humain (MELN) et poisson zèbre (ZELH-zfERβ2) a montré des différences qualitatives et quantitatives de réponse, non expliquées par les molécules estrogéniques détectées. Afin de mieux comprendre ces différences, l’activité de polluants aquatiques a été caractérisée sur les différents modèles, individuellement et au sein de mélanges de 2 à 12 polluants, combinant molécules estrogéniques et non estrogéniques pour simuler des situations environnementales.Les résultats obtenus montrent que les bio-essais basés sur le poisson zèbre répondent différemment, et parfois de manière opposée, au modèle humain MELN aux mélanges reconstitués. Ces différences s’expliquent par des sensibilités différentes à certaines molécules entrainant des interactions avec la réponse aux xeno-oestrogènes spécifiques à chaque modèle biologique. Dans son ensemble, ce travail montre que les particularités biologiques de chaque bioessai peuvent influencer la réponse des xeno-estrogènes quand présents au sein de mélange avec d’autres polluants. Ces résultats sont discutés au regard de l’utilisation de bio-essais dans l’évaluation de la contamination chimique des masses d’eau. / This PhD thesis aims at assessing the potential of innovative in vitro and in vivo zebrafish based bioassays for biomonitoring of surface water contamination by xeno-estrogens. For this purpose, the bioassays were applied to assess the effect of environmentally relevant surface water pollutants, alone and in simple (artificial) and complex (environmental samples) mixtures. The screening of surface water samples in zebrafish- (ZELH-zfERβ2 cells) and human-based (MELNcells) bioassays revealed qualitative and quantitative differences which could not be entirely explained by the xeno-estrogens identified. To better understand the response of bioassays to complex environmental mixtures, the activity of environmentally relevant surface waterpollutants was characterized across the bioassays, alone and in 2 to 12-component mixtures combining estrogenic and non-estrogenic chemicals to simulate an environmental contamination.The results indicate that zebrafish-based bioassays have a different and even in some cases an opposite response to the simple mixtures compared with the human-based bioassay MELN. These differences are explained by different sensitivity to some pollutants leading to bioassay-specific interactions with estrogen receptor activation. Al together, this work shows that the biological particularities of each bioassay can influence the response to estrogenic chemicals when mixed with other environmental pollutants, opening the discussion regarding their implementation in chemical water biomonitoring.

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

<p>Sintetisana su nova jedinjenja, 16- i 17-supstituisani&nbsp;16,17-sekoestratrienski derivati i D-homoestranski&nbsp;derivati, polazeći od 3-benziloksi-17-hidroksi-16,17-sekoestra-1,3,5(10)-trien-16-nitrila. Ispitana je&nbsp;estrogena i antiestrogena aktivnost u eksperimentima <em>in&nbsp;vivo</em>, antiaromatazna aktivnost <em>in vitro</em>, antioksidantna&nbsp;aktivnost DPPH &nbsp;i TBA testom, kao i antiproliferativna&nbsp;aktivnost prema ćelijskim linijama MCF-7 ATCC,&nbsp;MDA-MB-231, HT-29 i MRC-5 novosintetisanih&nbsp;jedinjenja.</p> / <p>Some new compounds, 16- and 17-substituted 16,17-secoestratriene derivatives, as&nbsp; well as D-homoestratriene derivatives, were synthesized, starting from 3-benzyloxy-17-hydroxy-16,17-secoestra-1,3,5 (10)-triene-16-nitrile. The newly synthesized compounds were tested for their <em>in vivo&nbsp;</em>estrogenic and antiestrogenic activity,&nbsp; <em>in vitro&nbsp;</em>antiaromatase activity, antioxidative activity by DPPH and TBA tests, as well as antiproliferative activity against MCF-7 ATCC, MDA-MB-231, HT-29 i MRC-5 cell lines.</p>

(Oberflächen-) Gewässer weltweit sind mit geringen Mengen (ng/L bis wenige µg/L) humaner Pharmazeutika belastet. Diclofenac (DCF; nicht-steroidal, entzündungshemmend) und Metoprolol (MTP; ß-Blocker) gehören entsprechend ihres hohen Verbrauchs zu den am häufigsten gefundenen Substanzen. Deren biologische Aktivität ist nicht auf den Menschen beschränkt. Gut konservierte Enzyme innerhalb der Vertebraten legen Auswirkungen auf Nicht-Zielorganismen wie Fische nahe, die bisher in Langzeituntersuchungen mit umweltrelevanten Konzentrationen unzureichend untersucht wurden. In der vorliegenden Arbeit wurden die physiologischen Effekte von DCF und MTP auf die Nil-Tilapie (Oreochromis niloticus), einem der wichtigsten Aquakulturfische weltweit, untersucht. In vitro konnte anhand primärer Hepatozyten gezeigt werden, dass bereits umweltrelevante Konzentrationen von DCF zu einer erhöhten Genexpression verschiedener Schlüsselenzyme der Detoxifizierung führten. Nach MTP-Exposition waren die Veränderungen weniger eindeutig. Beide Substanzen induzierten die Vitellogenin Genexpression, nur DCF jedoch bereits in umweltrelevanter Konzentration. In vivo wurden in zwei Langzeit-Expositionsversuchen die physiologischen Effekte vom befruchteten Ei bis 80 Tage nach Schlupf in O. niloticus untersucht. Beide Substanzen hatte keinen Einfluss auf Schlupferfolg und Überleben, das Wachstum war nach 80 Tagen nach Schlupf leicht reduziert. Die deutlichsten Auswirkungen waren histopathologische Veränderungen der Kiemen, veränderte Genexpressionen der Gonadotropine und eine erhöhte Expression von Vitellogenin. Die Ergebnisse legen eine stärkere östrogene Aktivität von DCF im Vergleich zu MTP nahe. Zusammenfassend sind die Bedenken gegenüber den Einzelsubstanzen eher gering, negative Auswirkungen auf die Reproduktion und sich verstärkende Effekte bei zeitgleicher Exposition gegenüber DCF und MTP lassen sich jedoch nicht ausschließen und sollten im Weiteren untersucht werden. / Surface waters worldwide are contaminated with low levels (ng/L up to few µg/L) of human pharmaceuticals. Diclofenac (DCF; non-steroidal, anti-inflammatory) and metoprolol (MTP; ß-blocker) are highly consumed and therefore commonly detected. Their biological activity is not restricted to humans. Well conserved enzymes within the vertebrates suggest effects on non-target organisms such as fish, poorly studied in long-term exposure experiments using environmentally relevant concentrations. In the presented work, physiological effects of DCF and MTP on the Nile tilapia (Oreochromis niloticus), an important aquaculture fish species, were studied. Using primary hepatocytes, it was shown in vitro that environmentally relevant concentrations of DCF increased the gene expression of different key enzymes of the detoxification, while MTP exposure had a less clear effect. Both substances induced vitellogenin gene expression, but only after DCF exposure this was significantly elevated already at the environmentally relevant concentration. In vivo, two long-term exposure studies on the physiological effects from the fertilized egg until 80 days post-hatch were evaluated. Both substances did not affect hatching success and survival, while growth was slightly reduced after 80 days post-hatch. Histopathological alterations of the gills, changed gene expression patterns of the gonadotropins and induced vitellogenin gene expression were the most dominant findings. The results indicate a stronger estrogenic mode of action of DCF compared to MTP. Overall, the risk due to a single substance exposure seems to be relatively low but adverse effects on reproduction and additive effects during simultaneous exposure to DCF and MTP cannot be excluded and should be investigated further.

<p>Hemijska karakterizacija ekstrakata&nbsp; <em>T. pratense&nbsp;</em>L.&nbsp; određena je LC-MS-MS, a etarskih ulja GC-MS analizom, pri čemu je&nbsp; identifikovano vi&scaron;e&nbsp;bioaktivnih jedinjenja.&nbsp; Spektrofotometrijskim&nbsp;metodama ispitan je antioksidantni potencijal&nbsp;ekstrakata i etarskih ulja. Hepatoprotektivni&nbsp;efekat određen je&nbsp; na&nbsp; homogenatu i hemolizatu&nbsp;jetre laboratorijskih mi&scaron;eva.&nbsp; Budući da&nbsp; ova&nbsp;biljka ima visok sadržaj izoflavona, određena je&nbsp;<br />estrogena, antiestrogena aktivnost, kao i&nbsp;citotoksičnost ekstrakata. Antimikrobna&nbsp;<br />aktivnosta ekstrakata i etarskih ulja ispitana je&nbsp;na&nbsp; &scaron;est bakterijskih sojeva.&nbsp; Ekstrakti i etarska&nbsp;ulja su bili umereno aktivni u pogledu biolo&scaron;kih&nbsp;aktivnosti, osim u slučaju antimikrobne&nbsp;aktivnosti koju nije pokazao nijedan ispitivani&nbsp;uzorak.</p> / <p>The chemical characterization of&nbsp; <em>T.&nbsp; pratense</em> extracts was determined by LC-MS-MS, and the essential oils of the GC-MS analysis, wherein the&nbsp; various&nbsp; bioactive compounds were identified.&nbsp; Antioxidant potential of extracts and essential oils was tested&nbsp; by spectrophotometric methods.&nbsp; Hepatoprotective&nbsp; effect&nbsp; was determined&nbsp; in&nbsp; haemolysate and liver homogenate of&nbsp; laboratory&nbsp; mice.&nbsp; Estrogen, antiestrogen activity, as well&nbsp; as the cytotoxicity of the extracts were&nbsp; determined&nbsp; due to the high&nbsp;content of isoflavones.&nbsp; Antimicrobial activity of extracts and essential oils was&nbsp; investigatied&nbsp; on six bacterial strains. Essential oils and extracts were moderately active in terms of biological activity, except&nbsp; in the case of&nbsp;&nbsp; antimicrobial activity&nbsp; where none of the extracts were active against tested bacterial strains.</p>

MSc (Microbiology) / Department of Microbiology / Background: The occurrence of microorganisms and endocrine disrupting chemicals (EDCs) in water poses a serious concern due to their effects on humans, animals and environment. In recent years, EDCs have been increasingly reported in rivers that receive large amounts of wastewater effluents. Of all the EDCs, natural and synthetic hormones are among those that are recognized for their potential to mimic or interfere with normal hormonal functions of humans and animals. The present study aimed at assessing the occurrence of these hormones in relation to the molecular diversity of Aeromonas and evaluating the resistance of Aeromonas to antibiotics as well as to assess anti-bacterial activity of two selected traditional medicinal plants. Methods: Wastewater, water and fish samples were collected from various sources (rivers, wastewater treatment plants, taps, and dams) for the detection of hormones and isolation of Aeromonas species. The analysis of hormones from various organs of the fish and from water samples was conducted, after extraction using enzymelinked immunosorbent assays (ELISA). Different types of hormones including Estriol, Estradiol, Ethinylesradiol and Testosterone were detected, and their concentrations determined. Aeromonas spp were isolated rom the samples using microbiological methods and Conventional PCR was used for genotyping as well as for detection of the beta-lactamase genes. Kirby-bauer method was used to determine the susceptibility profiles of Aeromonas to different antibiotics. Microdilution assay was used to determine the Anti-bacterial activity of the plant (Annoniceae and Zornia milneana) extracts against Aeromonas species. Results: A total of 144 samples were collected from 23 different locations in two countries: South Africa and Zambia. These included wastewater and treated wastewater, River water, fish and tap water. 17α-ethinylestradiol (EE2) was detected in most of the samples (92.7%) with concentrations varying from 0.59 ng/ml to 65 ng/ml. The hormones were also detected from drinking water, with testosterone detected at high concentrations of up to 140 ng/ml in tap water. Most sewage treatment plants were not able to remove the EE2 from the wastewater as the concentration of this hormone in the final effluent was almost always higher than that in the influent. These homones were also detected in drinking water at high concentrations of up to 53.49 ng/ml in the tap water for EE2 and 1777 ng/ml for E2. The overall detection of Aeromonas species in the samples was 84.5%. A. caviae was the most prevalent species accounting for 73.6%, followed by A. veronii with 64.6%. The bacteria were completely resistant to cefuroxime accounting for 100% resistance. Aeromonas isolates also showed high resistance to trimethroprim (88.7% for A. hydrophila), cefazolin (highest 97.8% for A. cavie), and ceftazidime (83.9% for A. sobria). TEM was the most prevalent beta-lactamase gene with detection rate of 87%. All isolates lacked the presence of the CTX-M3 gene. Also, wastewater had the highest prevalence of A. veronni and A. caviae accounting for 87.5% and 82.5% respectively. Multiple antibiotic resistance was also observed with the Aeromonas isolates being resistant to up to 11 antibiotics. High prevalence of 77.1% of Aeromonas hydrophila was observed in the presence of ethinylestradiol (EE2). Aeromonas veronii and Aeromonas caviae were the most predominant species in the presence of total estriol, A. veronii had a prevalence of 57.1% and A. caviae had a prevalence of 52.8%. Aeromonas hydrophila and Aeromonas caviae had the lower prevalence in the presence of hormones with the percentages of 26.1% and 27.8% respectively. The methanol extracts of both Zornia milneana and Annona species showed good activity against the Aeromonas spp with the lowest MIC of 0.078 mg/ml. Ethyl acetate extracts were the least effective. Conclusion: This study has shown high occurrence of steroid hormones in all types of environmental samples tested. These included tap water, river water, wastewater and fish both in Zambia and South Africa. Therefore, steroid hormones constitute and important health problem in the Southern African Sub-Region. The incapacity of the wastewater treatment plants to remove EE2 is an important problem that needs to be tackled immediately. The prevalence of Aeromonas species is very high in our environmental water as well as in drinking water, with the highest prevalence observed in fish and wastewater. It was also revealed that there is relationship between steroid hormones and Aeromonas species, with the hormones supporting the growth of Aeromonas species. The presence of beta-lactamase genes which causes Aeromonas to be resistant to antibiotics was also noted. Methanol extracts of Zornia milneana and Annona spp were the most effective against Aeromonas spp and could serve as primary sources for the isolation of lead compounds. / NRF