L’objectif de ce travail a consisté à évaluer différents effets écotoxiques de la deltaméthrine et du malathion sur trois souches du crustacé cladocère Daphnia magna. Deux de ces 3 souches appartiennent au même clone A (souches 1 et 3), ce qui a permis d’observer des différences de sensibilité inter- et intra-clonale. Les tests d’écotoxicité aiguë (48h) ont révélé, aussi bien pour la deltaméthrine que pour le malathion, que la souche 1 est plus sensible que la souche 2, elle-même plus sensible que la souche 3. Les tests chroniques (21 jours) ont montré que le critère commun majoritairement affecté par les deux pesticides est le nombre de nouveaux nés par adulte. Ces tests ont confirmé la sensibilité de la souche 1. La deltaméthrine et le malathion sont embryotoxiques puisque l’exposition des daphnies aux fortes concentrations de deltaméthrine et de malathion a induit des malformations. Pour ces effets, la souche 1 est également plus sensible que la souche 2, elle-même plus sensible que la souche 3. Nous avons aussi observé l’apparition de mâles, mais uniquement dans la descendance de la souche 1 exposée à la deltaméthrine. Le dosage de l’activité spécifique de l’acétylcholinestérase (AChE), pour les deux insecticides, a montré une inversion de l’ordre de sensibilité des souches. L’AChE, biomarqueur d’exposition et d’effet, peut être donc considérée aussi comme biomarqueur de susceptibilité. L’analyse protéomique a permis de révéler plusieurs cibles cellulaires de la deltaméthrine chez la souche 1 par rapport à la souche 2. Elle est surtout venue confirmer la plus grande sensibilité de la souche 1 par rapport à la souche 2. L’ensemble de ces résultats rappelle que le facteur génétique est une source de variation et souligne les perspectives offertes par l’analyse protéomique / The aim of this work was to evaluate different ecotoxic effects of deltamethrin and malathion on three strains of the cladoceran Daphnia magna. Two of the three strains belong to the same clone A (strains 1 and 3), which allowed to observe differences in inter-and intra-clonal sensitivity. The acute ecotoxicity tests (48h) showed for both deltamethrin and malathion, that one strain is more sensitive than strain 2, itself more sensitive than strain 3. Chronic tests (21days) showed that the common criterion mainly affected by both pesticides is the number of neonates per adult. These tests confirmed the sensitivity of the strain 1. Deltamethrin and malathion are embryotoxic as daphnid exposure to high concentrations of deltamethrin and malathion induced malformations. For these effects, strain 1 is also more sensitive than strain 2, itself more sensitive than strain 3. We also observed the appearance of males, but only in the offspring of the strain 1 exposed to deltamethrin. The determination of the specific activity of acetylcholinesterase (AChE), for both insecticides, showed a reversal of the order of susceptibility testing. Thus, as a biomarker of exposure and effect, AChE can also be considered as a biomarker of susceptibility. Proteomic analysis revealed several cellular targets of deltamethrin in strain 1 compared to strain 2. It confirms the greater sensitivity of strain 1 compared to strain 2. All these results point out that the genetic factor is a source of variation and highlight the opportunities offered by proteomic analysis

Introduction: Prostate cancer is the most common type of cancer in Sweden and is often treated using antiandrogenic drug therapy. Two substances belonging to this class of pharmaceuticals are bicalutamide and flutamide. After excretion from the human body, the drug molecules enter the wastewater treatment plant (WWTP). The WWTPs are not effective enough to completely remove pharmaceutical residues, why presence of both bicalutamide and flutamide can be detected in WWTP effluent water. Previous findings: Antiandrogens have been reported to affect reproduction in adult fish, but studies regarding possible effects on the embryonic development of fish are few. Aim: The present study sought to investigate if exposure to bicalutamide or flutamide cause toxicity in the early developmental stages of zebrafish embryos, and whether negative effects occur within concentrations relevant to measured environmental levels. Method: A modified OECD FET-test was used, where additional sublethal endpoints were included and the time period for assessment extended to 144 hours post fertilization (hpf). In addition, a locomotor activity assay was performed at 144 hpf in order to observe any sub-lethal swimming behavioral effects. Results: High doses (10 mg/L) of flutamide led to 100% lethality of the zebrafish embryos but the results suggest no acute toxic effects in the high dose treatment group of bicalutamide, or of either flutamide or bicalutamide within in the low (0.1 mg/L) or intermediate (1 mg/L) treatment groups. Neither did the locomotor activity assay result in statistically significant results, although the pattern of swimming activity in the low dose groups suggests that behavioral developmental effects could be present. Conclusions: High doses of flutamide caused mortality of the embryos, but no lethal or sublethal effects were present at environmentally relevant concentrations. The modest outcome of present study however suggests that further investigation of behavioral developmental effects of antiandrogens could be of future relevance. Analysis of the expression of genes related to neuronal growth, memory and other cognitive behaviors associated with behavioral changes, would then be of interest for further studies.

Les oeufs d’escargot terrestre de l’espèce petit-gris Helix aspersa (syn. Cantareusaspersus) peuvent être utilisés pour évaluer l’écotoxicité de substances chimiques pures ou enmélange. La mesure des effets embryotoxiques classiquement réalisée est le succès d’éclosionaprès 15 à 20 jours d’exposition (Druart et al., 2012). Cependant, les mécanismes impliquésdans la mise en place des effets toxiques à différents niveaux d’organisation biologique chezl’embryon ne sont pas connus. Des oeufs d’escargots ont été exposés à des solutions decontaminants métallique (Cd) ou organiques (pesticides: le Round Up® flash, le Corail® et laBouillie Bordelaise) selon deux modalités différentes (en continu sur la totalité dudéveloppement embryonnaire ou sur une période de 24 heures) afin de 1/ déterminer denouveaux paramètres de mesure au cours du développement embryonnaire pouvant rendrecompte d’un effet toxique, 2/ détecter des effets génotoxiques de divers contaminants(solution métallique de Cd ou de formulations commerciales de pesticides) par la méthodeRandom Amplified Polymorphic DNA (RAPD) et 3/ d’étudier des systèmes de défense métalspécifiques(métallothionéines).Les paramètres morphologiques et physiologiques suivis au cours d’expositionscontinues au Cd ont montré des effets néfastes sur le rythme cardiaque, la durée del’incubation, la taille et le poids à l’éclosion chez les exposés à la plus forte concentrationtestée. Chez ces derniers des signes de fragmentation de l’ADN ont également été détectés enfin d’exposition. Le couplage de la méthode RAPD avec un système d’électrophorèse hauterésolution (SHR) a permis de détecter des effets génotoxiques suite à des expositionscontinues au Cd, au Round Up® et au Corail®. L’étude par PCR quantitative de l’expressiondes gènes des métallothionéines (MTs) a mis en évidence une expression constitutive des MTsainsi qu’un haut niveau d’expression du gène mixte CdCuMT chez les embryons non exposés.Chez les embryons exposés au Cd durant 24 heures, une surexpression du gène spécifiqueCdMT a été mise en évidence alors qu’aucune augmentation significative des taux detranscrits des 2 autres isogènes étudiés (CuMT et CdCuMT) n’a été démontrée.Les résultats de toxicité du Cd basés sur le taux d’éclosion et l’expression des gènes desMTs ont démontré que des facteurs comme le régime d’exposition (24 heures ou en continu)ou le stade de développement (âge des embryons lors de l’exposition) peuvent modulerl’embryotoxicité des substances chimiques.206Les données obtenues durant cette étude intégrative permettent de proposer un largepanel de paramètres de mesure des effets toxiques des substances chimiques chez l’embryond’escargot terrestre H. aspersa au niveau individuel (rythme cardiaque, taille, durée dedéveloppement et succès d’éclosion) et au niveau moléculaire (expression de gènes dessystèmes de défense, détection des signes de génotoxicité et de la fragmentation de l’ADN)pour l’évaluation de la toxicité des substances chimiques. L’approche RAPD-SHR, bien quenécessitant une certaine expertise pour l’analyse des profils d’amplifications obtenus, apparaîtadaptée pour une détection rapide et efficace du potentiel embryogénotoxiques de substancesvariées (métaux, pesticides. / The land snail species Helix aspersa (syn. Cantareus aspersus) eggs can be used to assess theecotoxicity of chemicals. Measurement of embryotoxic effect is classically based on hatching successafter 15-20 days of exposure (Druart et al., 2012). However, the mechanisms involved in toxic effectsin embryos at different levels of biological organization are not known. Eggs of snails were exposedto solutions metallic contaminants (Cd) or organic (pesticides: Round Up® Flash, Corail® andBordeaux Mixture) in two different regimes (continuous over the entire embryonic development orduring a period of 24 hours), in order to 1 / identify of new endpoints of toxic effect measurementsduring embryonic development, 2 / detect of genotoxic effects of metal solution (Cd) or threepesticides commercial formulations by Random Amplified Polymorphic DNA method (RAPD) and 3 /study metal-specific defense systems (metallothionein).Morphological and physiological parameters monitored during Cd continuous exposures showedadverse effects on heart rate, duration of incubation, size and weight of new hatchlings exposed to thehighest concentration tested. In the latter, signs of DNA fragmentation were detected at the end ofexposure. Coupling the RAPD with a high-resolution electrophoresis system (SHR) has enabled todetect genotoxic effects of Cd, Round Up® and Corail® after continuous exposures. Quantitative PCRstudy of metallothioneins (MTs) gene expression has showed constitutive expression of MTs genesand a high level of mRNA for the mixed gene CdCuMT in unexposed embryos. In embryos exposedto Cd for 24 hours, an overexpression of the specific gene CdMT has been demonstrated whereas thetwo other isogenes (CuMT and CdCuMT) didn’t show significant induction of expression rates.The toxicity results based on the hatching rate and MTs genes expression obtained with Cd haveshowed that factors such as the exposure regime (24 hours or continuous) or the stage of development(age of embryos upon exposure) can modulate embryotoxicity of chemicals. This thesis provides awide range of endpoints usable at the individual level (heart rate, height, hatching monitoring) and atthe molecular level (gene expression of defense systems, detection of genotoxicity signs and DNAladdering) for the assessment of the ecotoxicity of chemical substances. The RAPD-SHR, althoughrequiring some expertise to analyze profiles obtained, appears suitable for rapid and efficientdetection of potential embryogenotoxic effects of various substances (metals, pesticides).

L’utilisation excessive de pesticides engendre des pollutions et des dégradations importantes de l’environnement y compris sur les écosystèmes aquatiques. L’activité viticole ne fait pas exception à cette règle, et c’est pour cette raison que ce travail de thèse s’est intéressé aux réponses adaptatives ou délétères aux premiers stades de vie de poissons exposés à trois produits phytosanitaires utilisés en viticulture ainsi qu’à leur mélange et à des échantillons environnementaux. La thèse s’intéresse aux stades précoces de développement de la truite arc-en-ciel, mais également à la lignée cellulaire de foie de truite RTL-W1. Les embryons de truite ont été exposés au cuivre, au glyphosate et au chlorpyrifos seuls ou en mélange. Une autre partie de ce travail a consisté à étudier les effets toxiques des échantillons environnementaux d’eau et de sédiment provenant d’un cours d’eau, La Livenne, à proximité de parcelles viticoles. Toutes les expériences ont été faites en conditions contrôlées de laboratoire. Les réponses induites par ces expositions ont été mesurées à différents niveaux d’organisation biologique : au niveau moléculaire, phénotypique et comportemental pour l’étude in vivo (larves de la truite arc-en-ciel) ; et l’analyse des effets cytotoxiques de l’induction des espèces réactives de l’oxygène (ROS) et des dommages à l’ADN pour l’étude in vitro (lignée RTL-W1). Les résultats de ce travail ont montré que l’exposition aux pesticides individuels, ou en mélange, chez les larves de truites arc-en-ciel n’a pas produit d’effets létaux aux concentrations testés. En revanche, ces substances ont provoqués différents effets sub-létaux, selon le composé et les concentrations étudiés, dont des effets tératogènes, des perturbations du comportement natatoire, des effets génotoxiques et l’expression différentielle de gènes cibles. Le cuivre s’est avéré le plus toxique provoquant un échec d’éclosion important. Des effets cytotoxiques et une production d’espèces réactives de l’oxygène (ERO) ont été observés sur cellules de truite exposées à des extraits d’eau de rivière. Par ailleurs, des effets comportementaux ont été observés sur les larves de truites exposées pendant 48 h à des sédiments et de l’eau de la Livenne. En conclusion, ces travaux de thèse ont mis en évidence des effets sub-létaux sur les cellules et les stades précoces de développement de truite arc-en-ciel exposés à des concentrations environnementales de pesticides utilisés en viticulture. / The excessive use of pesticides generates significant pollution and degradation of the environment, including aquatic ecosystems. The viticultural activity is not an exception for this rule, and this is why the aim of this work is to study the adaptive or deleterious responses in the early life stages of fish exposed to three phytosanitary products used in viticulture as well as to their mixture and environmental samples. The thesis focuses on the early stages of development of rainbow trout, but also on the trout liver cell line RTL-W1. Trout embryos were exposed to copper, glyphosate and chlorpyrifos alone or as a mixture. Another part of this work consisted in studying the toxic effects of environmental samples of water and sediment coming from a river, La Livenne, close to vineyard plots. All experiments were done under controlled laboratory conditions. The responses induced by these exposures were measured at different levels of biological organization: at the molecular, phenotypic and behavioral level for the in vivo study (rainbow trout larvae); and the analysis of cytotoxic effects and induction of reactive oxygen species (ROS) and DNA damage for the in vitro study (RTL-W1 line). The results of this work showed that exposure to individual or mixed pesticides in rainbow trout larvae did not produce lethal effects at the tested concentrations. In contrast, these substances caused different sub-lethal effects, depending on the compound and concentrations studied, including teratogenic effects, swim behavior disturbances, genotoxic effects, and differential expression of target genes. Copper was found to be the most toxic causing a major hatching failure. Cytotoxic effects and production of reactive oxygen species (ROS) were observed on trout cells exposed to river water extracts. In addition, behavioral effects were observed on trout larvae exposed for 48 h to sediments and Livenne water. In conclusion, these thesis studies revealed sub-lethal effects on cells and early stages of development of rainbow trout exposed to environmental concentrations of pesticides.

Orientador: Ione Pellegatti Lemonica / Banca: João Lauro Viana de Camargo / Banca: Márcia Guimarães da Silva / Banca: Silvana Lima Górniak / Banca: Regiane Kawakami / Tese não possui um resumo geral, possue um resumo para cada capítulo / Resumo : O objetivo do presente estudo foi verificar, experimentalmente, o possível mecanismo pelo qual o extrato aquoso de Plectranthus barbatus (boldo-brasileiro), planta utilizada popularmente como abortiva, atua sobre o organismo materno ou sobre o desenvolvimento do concepto durante o período de pré-implantação, correlacionando sua ingestão com possíveis alterações no transporte e desenvolvimento embrionário ou com alterações hormonais maternas...(Resumo completo, clicar acesso eletrônico abaixo) / Abstract : The present study was conducted to determine the possible mechanism by which the aqueous extract of Plectranthus barbatus (brazilian-boldo), a plant used popularly as abortive agent, can lead to early loss of pregnancy, correlating this possible effect with morphological alterations in the embryo, oviductal motility dysfunctions or maternal hormonal level modifications...(Complete abstract, access undermentioned electronic address) / Doutor

Die derzeit angewendete Formalinbegasung von Bruteiern zur Keimreduktion stellt ein wichtiges Mittel zum Schutz des Verbrauchers vor dem Eintrag der Salmonellose aus der Geflügelindustrie in die Lebensmittelkette dar. Jedoch verlangt sein kanzerogenes Potenzial die Suche nach einer ebenso effektiven und einfach zu praktizierenden, aber weniger gesundheitlich bedenklichen alternativen Methode. In dieser Arbeit wurde die Eignung einer in ovo-Ozonbegasung zur Bruteidesinfektion hinsichtlich ihrer Auswirkung auf die Embryonalentwicklung untersucht. Dafür wurden befruchtete Eier vor ihrem Einsatz in den Brüter mit unterschiedlichen Ozonkonzentrationen zwischen 0,5% bis >5,0% (wt/ wt O3 in O2) bei einer relativen Luftfeuchte von 70% in einer Laborkammer begast. Die verwendeten Ozonkonzentrationen wurden dabei in drei Konzentrationsgruppen eingeteilt: hoch (2,8% bis 5,0%), mittel (1,1% bis 2,5%) und niedrig (0,5% bis 1,0%). Nach Erreichen der Zielkonzentration blieben die Eier für eine definierte Einwirkzeit (EWZ) zwischen 0 bis 24 h in der Kammer. Am Bruttag (BT) 18, 19 oder 20 wurden die Überlebensrate (ÜLR), Gewicht und Länge erhoben sowie histologische Untersuchungen der Organe Herz, Leber, Milz und Niere vorgenommen. Bei vier Versuchen erfolgte zusätzlich die Untersuchung am BT 6 und BT 12. Insgesamt wurden 13 Versuchsreihen mit Begasungen in der Laborkammer in den drei genannten Konzentrationsgruppen durchgeführt. Des Weiteren sollte die Übertragbarkeit der ermittelten Ergebnisse auf eine großtechnische Lösung, durch die Anwendung in einer projektintern entwickelten Prototyp-Kammer überprüft werden. Die Konzipierung dieses Prototyps folgte den technischen Gegebenheiten unter Einbeziehung der ermittelten Ergebnisse zur Embryotoxizität, zur Effektivität der Keimreduktion sowie der Veränderung der Eiinhaltsstoffe. Im Prototyp wurde daher die Begasungskonzentration von 0,7% Ozon bei einer EWZ von 2 h angewendet und in 2 Versuchsreihen hinsichtlich ihrer Auswirkungen auf die Lebensfähigkeit und die morphologische Entwicklung getestet. Um zusätzlich eine Aussage treffen zu können, welche Wirkung eine hohe Ozonkonzentration bei der Applikation während der Bebrütung zur Folge hat, wurde in zwei weiteren Versuchsreihen jeweils am BT 3, 4 und 5 eine Ozondosis von 5,0% in Kombination mit 1 h EWZ appliziert. Nach der Entnahme der Embryonen am BT 6 bzw. BT 8 erfolgte die morphologische Untersuchung. Aus der vorliegenden Arbeit wird zusammenfassend geschlussfolgert:  Ozon besitzt einen dosisabhängigen Effekt auf die ÜLR: je höher die Dosis, desto geringer die Überlebensrate. Bei niedrigen Ozonkonzentrationen zwischen 0,5% und 1,0% kommt es zu ÜLR von 90% und 100%.  Die Einwirkzeit stellt einen wichtigen Einflussfaktor auf die Überlebensrate dar. Eine Kombination einer hohen Ozondosis mit langer EWZ hat eine höhere Embryomortalität zur Folge, als eine hohe Ozondosis ohne EWZ. Eine erhöhte Mortalitätsrate zeigt sich auch bei der Kombination einer mittleren Ozondosis mit einer langen EWZ. Keinen Einfluss zeigt sie bei der Kombination mit einer niedrigen Ozonkonzentration.  Ozonierte Embryonen zeigen dosisabhängig eine geringere Längen- oder Gewichtsentwicklung im Vergleich zur Kontrollgruppe. Signifikante Gewichts- und Längenunterschiede lagen nur vereinzelt bei der Begasung mit hoher Ozonkonzentration vor.  Pathohistologische Befunde an Organen, die mit einer Ozonbegasung in Zusammenhang gebracht werden können, konnten nicht erhoben werden.  Die Versuche zur Begasung während der Organogenese wiesen nicht auf ein teratogenes Potenzial von Ozon hin. Korrespondierend mit den Ergebnissen der Begasung am BT 0 zeigte sich ebenfalls eine deutliche Embryotoxizität.  Die Wirkung von Ozon folgt einem Alles-oder-Nichts-Prinzip: entweder die Schädigung ist so stark, dass es zu keiner Entwicklung mehr kommt, oder aber der überlebende Embryo bzw. Fetus zeigt ein phänotypisch normales Aussehen.  Mit den vorgestellten Untersuchungen konnte bewiesen werden, dass bei einer in ovo-Begasung von befruchteten Hühnereiern am Bruttag 0 mit einer niedrigen Ozonkonzentration von 0,5% bis 1,0% in Kombination mit einer mittleren und geringen EWZ keine teratogenen oder embryotoxischen Veränderungen zu erwarten sind.  Aus diesen Befunden ergibt sich ein unbedenklicher Einsatze von Ozon als alternative Methode zur Bruteidesinfektion. Die Voraussetzung dafür ist, dass die großtechnische Lösung die Anwendung von 1,0% Ozonkonzentration in Kombination mit 2 h EWZ ermöglicht, um eine sehr gute ÜLR, Entwicklung und eine 100%ige Inaktivierung des in Legehennenbeständen vorherrschenden Serovars Salmonella Enteritidis zu gewährleisten.

No description available.

Passive sampling, as opposed to the conventional spot or bottle water sampling technique, has shown to be reliable and efficient in monitoring the toxicologically relevant, freely dissolved (e.g. bioavaialable) concentrations of a wide range of organic contaminants in water. At the same time, partitioning controlled delivery (passive dosing; PD) techniques promise to overcome many of the challenges associated with toxicity testing of hydrophobic substances that may bias the interpretation of toxicity data. The present study investigated the feasibility of coupling silicone rubber passive sampling devices (SR-PSDs) with bioassay techniques for both chemical and ecotoxicological assessment of complex mixtures of organic contaminants in the aquatic environment. SR-PSDs were deployed in water at various locations within the Ythan catchment (north east, Scotland, UK), Forth estuary and the Firth of Forth (east coast of central Scotland, UK) for 7 to 9 weeks. Following retrieval, extracts from the SR-PSDs were analysed for dissolved concentrations of a variety of organic contaminants including PAHs and PCBs using GC-MS and GC-ECD respectively and were screened for a wide range of pesticides using GC-MS/MS and LC-MS/MS. The extracts were further evaluated for acute cytotoxicity (i.e. neutral red uptake assay) and EROD induction potential using rainbow trout liver cell line (Oncorhynchus mykiss; RTL-W1) and for phytotoxicity and developmental toxicity potential using algal growth inhibition test (with a marine phytoplankton, Diacronema lutheri) and fish embryo toxicity test (with embryos from zebrafish Danio rerio) respectively. Overall, the individual and total dissolved concentrations of PAHs (ΣPAH40; parent and branched) and PCBs (ΣPCB32; ortho and mono-ortho) measured in water from the Ythan, Forth estuary and Firth of Forth were relatively low compared with other studies using PSDs. A number and level of pesticides, including insecticides, herbicides and fungicides of varying hydrophobicity (log KOWs ~2.25 to ~5.31) were detected in the silicone rubber (SR) extracts from the Ythan catchment, the Forth estuary and the Firth of Forth, suggesting input mainly from agricultural run-off and possibly from direct discharges. No statistically significant (p<0.05) acute cytotoxicity was observed following 48 h exposure of RTL-W1 cells to SR extracts from the Ythan catchment. But, on a sublethal level, for every site, statistically significant EROD activity was observed to some degree following 72 h exposure. In addition, developmental and algal toxicities on embryos of D. rerio and D. lutheri respectively, were measured in all the deployed samples compared with the procedural controls (undeployed samples). Interestingly, extracts of SR-PSDs from the Forth estuary and the Firth of Forth exhibited growth inhibitions on D. lutheri that were similar to those of extracts from the Ythan, even though, fewer numbers of pesticides were detected in the Forth estuary and Firth of Forth than the Ythan. This suggests that pesticides were not solely responsible for the observed effects in the Ythan catchment. To further improve data from toxicity testing of hydrophobic substances, the study identified the use of SR O-rings as a suitable passive dosing format in in vitro toxicity tests and was partially validated through their use in dosing RTL-W1 cells with two individual PAHs and subsequently determining cytotoxicity and EROD-activity.

Made available in DSpace on 2014-06-11T19:33:25Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-08-24Bitstream added on 2014-06-13T18:45:06Z : No. of bitstreams: 1 alvarenga_cmd_dr_botfm_prot.pdf: 1786103 bytes, checksum: 14d9f0d294fb6c639b79eab0f8e823c6 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O objetivo do presente estudo foi verificar, experimentalmente, o possível mecanismo pelo qual o extrato aquoso de Plectranthus barbatus (boldo-brasileiro), planta utilizada popularmente como abortiva, atua sobre o organismo materno ou sobre o desenvolvimento do concepto durante o período de pré-implantação, correlacionando sua ingestão com possíveis alterações no transporte e desenvolvimento embrionário ou com alterações hormonais maternas... / The present study was conducted to determine the possible mechanism by which the aqueous extract of Plectranthus barbatus (brazilian-boldo), a plant used popularly as abortive agent, can lead to early loss of pregnancy, correlating this possible effect with morphological alterations in the embryo, oviductal motility dysfunctions or maternal hormonal level modifications...(Complete abstract, access undermentioned electronic address)

PhD (Department of Biotechnology, Faculty of Applied and Computer Sciences), Vaal Universtiy of Technology / The genus Tulbaghia has been used in traditional medicine to treat various ailments such as fever, earache, tuberculosis and esophageal cancer. However, there is limited scientific evidence to support its use. Therefore the objectives of this study were to perform phytochemical analysis, investigate the antioxidant, antimicrobial, anticancer, immunomodulatory activities and toxicity of crude acetone and water extracts from selected Tulbaghia species. Standard methods were used for preliminary phytochemical analysis. The total phenolic content of the plant extracts was determined using the folin ciocalteu method whereas the total flavonoids were determined by using the aluminium chloride colorimetric method. DPPH and ABTS assays were used to evaluate the antioxidant activity. The antimicrobial activity was assessed by agar well diffusion, microtiter dilution and time kill assays. For anticancer studies, the antiproliferative activity of the extracts was evaluated using the MTT assay on Hkesc-1 and KB cells. Morphological changes of the cancer cells treated with extracts were examined using light microscopy. Induction of apoptosis was assessed using fluorescence microscopy and acridine orange/ethidium bromide staining. Flow cytometry analysis was conducted to examine the multicaspase activity and cell cycle arrest. For immunomodulatory activity, the Greiss reagent and Luminex cytokine assays were used to determine the effect of the extracts on NO production and the concentration of the cytokines in the treated cells, respectively. Toxicity of selected Tulbaghia species was examined by investigating the effect of the extracts on the metabolic activity and cell membrane integrity on the treated RAW264.7 cells using the MTT and LDH assays, respectively. The zebrafish assay was used to evaluate the embryotoxicity and teratogenic effects of crude acetone and water extracts of T. violacea at 24 h intervals for 96 h post fertilisation (hpf). The percentage mortality, hatchability and heart rate were examined. Phytochemical screening of eight Tulbaghia species demonstrated the presence of flavonoids, glycosides, tannins, terpenoids, saponins and steroids. The amount of total phenol and flavonoid content varied in different plant extracts ranging from 4.50 to 11.10 milligrams gallic acid equivalent per gram (mg GAE/g) of fresh material and 3.04 to 9.65 milligrams quercetin equivalent per gram (mg QE/g) of fresh material respectively. The IC50 values based on DPPH and ABTS for T. alliacea (0.06 and 0.06 mg/mL) and T. violacea (0.08 and 0.03 mg/mL) were generally lower showing potential antioxidant activities. For antimicrobial activity, the acetone extracts of T. acutiloba, T. alliacea, T. leucantha, T. ludwigiana, T. natalensis and T. simmleri showed moderate antimicrobial activity against all test organisms while the water extracts showed moderate to no activity. One species, T. cernua, showed poor activity against all the tested microbes. The acetone and water extracts of T. violacea showed the greatest antibacterial and antifungal activity against all the tested microorganisms with minimum inhibitory concentration ranging from 0.1 mg/mL to 3.13 mg/mL. The acetone extracts of T. violacea also exhibited both bacteriostatic/fungistatic and bactericidal/fungicidal activity depending on the incubation time and concentration of the extract. The bactericidal/fungicidal activity was observed at x2 MIC. The results for anticancer activity showed that treatment of Hkesc-1 cells with acetone and water crude extracts had anti-proliferative activity with IC50 values of 0.4 mg/mL and 1.625 mg/mL, respectively while KB had 0.2 mg/mL and 1 mg/mL, respectively. Morphological changes such as blebbing, cell shrinkage and rounding were observed in the treated cells suggesting that apoptosis was taking place. AOEB staining showed that the level of apoptosis was dependent on the concentration of the extracts. The activation of multicaspase activity in both Hkesc-1 and KB treated cells was also concentration dependent leading to cell death by apoptosis and the induction of cell cycle arrest at the G2/M phase. Immunomodulatory activity results indicated that cell viability was above 80% when concentrations of 50 µg/mL or less of both acetone and water crude was used. Treatment with the acetone extract had no significant effect (p>0.05) on the LPS induced NO production in RAW264.7 cells except at 50 µg/mL where significant inhibition was observed. The water extract had no significant effect (p>0.05) on NO production at all the concentrations. Treatment of LPS–induced RAW264.7 cells with acetone extract stimulated the production of IL-1α, IL-6 and TNF-α, but had no significant effect (p > 0.05) on IL-1β. On the other hand, treatment with the water extracts stimulated the production of IL-1α, IL-6 but had no significant effect (p>0.05) on TNF-α and IL-1β. Treatment of LPS-induced RAW264.7 cells with the acetone extract had very little stimulatory effect on IL-4, IL-5 and IL-13 and no significant effect on IL-10 whereas for the water extract a significant stimulatory effect was only observed for IL-4 after 48 h of treatment. High concentrations (>10000 pg/mL) of MCP-1, MIP1-α, MIP1-β, MIP-2, GCSF, GM-CSF, RANTES and IP-10 were also observed in acetone and water extract treated RAW264.7 cells. For toxicity studies, acetone and aqueous crude leaf extracts from T. alliacea, T. simmleri, and T. violacea had a significant inhibitory (p<0.05) effect on the RAW264.7 cells after 48h treatment. Acetone extracts from T. alliacea, T. simmleri and T. violacea resulted in IC50 values of 0.48 mg/mL, 0.72 mg/mL and 0.1 mg/mL, respectively. Treatment with water extracts showed minimal toxic effect indicated by higher IC50 values of 0.95 mg/mL, 2.49 mg/mL and 0.3 mg/mL for T. alliacea, T. simmleri and T. violacea, respectively. The LDH release by macrophages after 24 h treatment with acetone extracts was observed to be concentration dependent while treatment with water extracts did not induce LDH release. The zebra fish assay showed a lethal dose (LD50) for the T. violacea acetone crude extract of 20 μg/mL whereas that for water extract was 85 μg/mL. The observed teratogenic effects included scoliosis, edema of the pericardial cavity, retarded yolk resorption, hook-like/bent tail and shorter body length. In conclusion, the results from this study indicate that the extracts from the eight Tulbaghia species examined contain phytochemicals that may have the antioxidant, antimicrobial, anticancer and immunomodulatory properties. Extracts from T. violacea were observed to be the most potent. This study thus supports the use of T. violacea in treating bacterial and fungal infections in traditional medicine. The results of this study also confirm the anticancer potential of T. violacea. The immunomodulatory activity of the acetone and water extracts from T. violacea indicated a dominantly pro-inflammatory activity. Traditional medicine prepared form T. violacea may be of benefit to individuals with weak immune systems. The toxicity of selected Tulbaghia species was observed to be concentration, extract and time dependent. Therefore, traditional medicine prepared from Tulbaghia extracts should be taken with caution preferably in small doses over a short period of time. Future studies will focus on the identification of the bioactive compound(s) responsible for the antimicrobial, anticancer and immunomodulatory activities.