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Thesis (M. Phil.)--Hong Kong University of Science and Technology, 2002. / Includes bibliographical references. Also available in electronic version. Access restricted to campus users.

Zugl.: Cambridge, Mass., Harvard University, Diss., 1972.

Thesis (Ph.D.)--Hong Kong University of Science and Technology, 2006. / Includes bibliographical references. Also available in electronic version.

Thesis (Ph.D.)--Hong Kong University of Science and Technology, 2006. / Includes bibliographical references. Also available in electronic version.

Tesis presentada a la Universidad de Chile para optar al grado de Doctor en Ciencias Farmacéuticas / La planta Matico, Buddleja globosa Hope, Budlejaceae es una especie nativa en Chile de la cual se usan sus hojas por sus múltiples propiedades terapéuticas. Las investigaciones más recientes han permitido demostrar que el extracto etanólico (EMATst) obtenido a partir de sus hojas y estandarizado en su principal componente (verbascósido) presentó efectos antiinflamatorio, analgésico y cicatrizante. Este compuesto ha sido identificado en distintas especies vegetales y se le ha demostrado mediante ensayos in vitro efecto estrogénico y antiestrogénico dependiente de la concentración. En esta tesis se estudió la propiedad estrogénica y el posible efecto disruptor endocrino (DE) del EMATst y del verbascósido usando distintos modelos experimentales tanto in vivo como in vitro. Esta investigación se inició con la recolección de las hojas de matico en el mes de enero de 2008, en el campus Antumapu de la Universidad de Chile, con el objetivo de obtener el EMATst que fue elaborado por extracción sucesiva del material vegetal con solventes de polaridad creciente (hexano, diclorometano y etanol). La cuantificación del componente mayoritario (verbascósido) del extracto etanólico se realizó por CLAE-DAD y se expresó en ácido cafeíco. Posteriormente a partir del EMATst por sucesivas columnas cromatográficas de silicagel 60 y Sephadex LH20, se aisló el verbascósido. Tanto EMATst como verbascósido fueron utilizados para realizar los trabajos experimentales con el fin de determinar la alteración del ciclo estral de ratas de laboratorio despu es de la administración subcutánea de dos diferentes dosis de EMATst; posible unión competitiva de EMATst y verbacósido al receptor estrogénico (RE) mediante un ensayo in vitro usando citosol de útero de rata como fuente de RE; toxicidad aguda de Daphnia magna mediante la determinación de la concentración letal 50 (CL50) a las 24 h de exposición; toxicidad crónica y efecto DE mediante la determinación de la sobrevivencia, frecuencia de muda y reproducción de dos generaciones a los 14 días de exposición. Nuestros resultados permitieron demostrar que el EMATst en dosis alta (10-5 M) provocó una alteración en el ciclo estral de ratas, lo que podrá atribuirse a un efecto antiestrogénico. Tanto EMATst como verbascósido se unieron levemente de modo competitivo al RE comparado con estradiol. Ambos presentaron una baja toxicidad aguda relativa respecto del t oxico de referencia (dicromato de potasio) y 17 ß-estradiol en D. magna. Los resultados de toxicidad crónica en D. magna, muestran que se produce una disminución de la reproducción en ambas generaciones dependiente de la concentración y más marcada aún en la segunda generación y el efecto DE de ambas muestras se manifestó porque se alteró la reproducción / Matico plant, Buddleja globosa Hope, Budlejaceae is a native species in Chile which used their leaves of its multiples therapeutic properties. More recent investigations have demonstrated that the ethanol extract (EMATst) obtained from leaves and with an standardized main component (verbascoside) presented anti-in ammatory, analgesic and scarring e ects. This compound has been identi ed in di erent plant species and has shown through in vitro assays concentration dependent estrogenic and antiestrogenic e ects. In this thesis we studied the estrogenic property and possible endocrine disruptor (ED) e ect of EMATst and verbascoside using di erent experimental models both in vivo and in vitro. This investigation began with collecting matico leaves in the month of January 2008, in the Antumapu campus of the University of Chile, in order to obtain EMATst which was prepared by the successive extraction of plant material with solvents of increasing polarity (hexane, dichloromethane and ethanol). Quanti cation of the major component (verbascoside) of the ethanol extract was performed by HPLC-DAD and expressed as ca eic acid. Subsequently, verbascoside was isolated from EMATst by successive silica gel 60 and Sephadex LH20 chromatographic columns. Both EMATst and verbascoside were used to perform experimental work to determine the alteration of the estrous cycle of rats after subcutaneous administration of two di erent doses of EMATst; verbacoside and EMATst possible competitive binding to the estrogen receptor (ER) though an in vitro assay using the cytosolic fraction of a rat uterus as ER source; acute toxicity of Daphnia magna by determining the lethal concentration 50 (LC50) after 24 hours of exposure; chronic toxicity and DE e ect by determining the survival, molt frequency and reproduction of two generations at 14 days of exposure. Our results show that the EMATst at a high-dose (10-5 M) caused an alteration in

L'objectif général de ce travail est l étude de la toxicité chronique du chlordane chez un crustacé d eau douce afin d évaluer le risque de ce pesticide sur les organismes aquatiques. Pour ce faire, nous avons choisi le cladocère Daphnia magna comme modèle d étude, à cause de son rôle clé dans la chaîne trophique, sa sensibilité et son cycle de vie qui est mis à profit pour étendre le test de reproduction classique utilisant une seule génération saine sur deux générations successives. Le test chronique (21j) a donné une concentration sans effet observée (NOEC) sur tous les paramètres de 0,18 g/L qui constitue une donnée importante dans l évaluation du risque du chlordane pour le milieu aquatique. L exposition de D.magna aux fortes concentrations de chlordane a induit des malformations et l apparition de mâles chez la descendance. L étude de la bioaccumulation a montré le fort potentiel de bioaccumulation chez D.magna avec un facteur de bioaccumulation de 244000 (poids sec) après 40 jours d exposition à la NOEC. L étude de la toxicité du chlordane sur deux générations successives (F0 et F1) a montré que celle-ci augmente avec la concentration et le temps d exposition. Ainsi, la génération F1 est plus sensible que la génération F0 et la population de la génération F1 issue de la 5ème ponte s est montrée plus sensible que la génération F1 issue de la 2ème ponte. L étude de la capacité de récupération des daphnies de la génération F1, après leur transfert dans un milieu propre a montré l absence d une récupération totale aux plus fortes concentrations testées. La comparaison de la sensibilité de D.magna à un autre crustacé C.dubia suite à une exposition chronique au chlordane a souligné l intérêt de l utilisation de ce dernier dans l étude de la toxicité chronique des polluants persistants à condition de prolonger la durée du test de 7 à 14 jours. L ensemble de ces résultats souligne l intérêt du modèle D.magna dans l étude de la toxicité chronique des polluants persistants et de leurs éventuels effets sur les différents processus de reproduction. / This research aimed at studying the toxicity of chlordane in freshwater crustacean in order to evaluate the risk of this pesticide on aquatic organisms. We choice Daphnia magna as model of study, because her key role in trophic chain, her sensitivity and her short life cycle that are exploited for extended the classic reproduction test using only one healthy generation on two successive generations. In the chronic test (21d), a concentration without observed effect (NOEC) on all parameters of 0.18 g/L was obtained. The exposition of D.magna to the strong concentrations of chlordane led the malformations and the apparition of males at the progeny. The study of the bioacumulation showed the high potential of bioaccumulation at D.magna with a factor of bioaccumulation of 244 000 (dry weight) after 40 days of exposition to the NOEC. The study of the toxicity of the chlordane on two successive generations (F0 and F1) showed that this one increases with the concentration and the time of exposition. So, the F1 generation is more sensitive than the F0 generation and the population of the F1 generation 5th brood is more sensitive than the one of the F1 generation 2nd brood. The study of the recovery of the daphnids after their transfer in clean water showed their capacity to restore reproduction only for the low doses. The comparison of the sensitivity of D.magna in another crustacean C.dubia following a chronic exposition to chlordane underlined the interest of the use of C.dubia in the study of the log term toxicity of the persistent pollutants under the condition to prolong the duration of the test from 7 to 14 days.The present work highlights the interest of our model in the study of the chronic toxicity of persistent pollutants and their possible effects on the different processes of reproduction

Spec. Litt. Inaug.--Leyden. / Includes bibliographical references.

Lead is an omnipresent pollutant, and its contamination in natural waters is an issue of current regulatory concern throughout the world including Canada. The free divalent ion of lead (Pb2+) is considered to be the most bioavailable and toxic form of lead. Pb2+ is known to be a calcium antagonist in vertebrates including fish. It is believed that lead causes toxicity to freshwater fish primarily by disrupting ionic homeostasis both during acute and chronic waterborne exposure. Lead can also potentially act as a respiratory toxicant since it is known to impair hemoglobin synthesis in both vertebrates. To date, the mechanistic underpinnings of lead accumulation and toxicity in aquatic invertebrates are not well understood, particularly during acute exposure. Therefore, the main objectives of the present study were in two folds: (i) to investigate the mechanisms of waterborne lead uptake, and (ii) to understand the physiological basis of lead toxicity during acute exposure. I used freshwater crustacean, <i>Daphnia magna</i>, as a model freshwater invertebrate species for my study. <i>Daphnia</i> are known to be quite sensitive to metals and widely used as a model species for toxicity assessments. The results of my study suggest that lead inhibits waterborne Ca2+ uptake in <i>Daphnia</i> in a concentration dependent manner, and this inhibition occurs predominantly through a direct competitive interaction. The entry of waterborne Pb2+ in <i>Daphnia</i> likely occurs via both lanthanum-sensitive and verapamil-sensitive epithelial calcium channels. Moreover, my results also indicate that acute waterborne lead exposure severely disrupts both Ca2+ and Na+ uptake from water, which are concomitant with the increase in the lead body burden in <i>Daphnia</i>. Interestingly however, acute exposure to lead does not affect the rate of oxygen consumption in <i>Daphnia</i>, indicating no acute respiratory toxicity of lead. Overall, it appears that lead acts as an ionoregulatory toxicant to <i>Daphnia</i> during acute waterborne exposure.

Lead is an omnipresent pollutant, and its contamination in natural waters is an issue of current regulatory concern throughout the world including Canada. The free divalent ion of lead (Pb2+) is considered to be the most bioavailable and toxic form of lead. Pb2+ is known to be a calcium antagonist in vertebrates including fish. It is believed that lead causes toxicity to freshwater fish primarily by disrupting ionic homeostasis both during acute and chronic waterborne exposure. Lead can also potentially act as a respiratory toxicant since it is known to impair hemoglobin synthesis in both vertebrates. To date, the mechanistic underpinnings of lead accumulation and toxicity in aquatic invertebrates are not well understood, particularly during acute exposure. Therefore, the main objectives of the present study were in two folds: (i) to investigate the mechanisms of waterborne lead uptake, and (ii) to understand the physiological basis of lead toxicity during acute exposure. I used freshwater crustacean, <i>Daphnia magna</i>, as a model freshwater invertebrate species for my study. <i>Daphnia</i> are known to be quite sensitive to metals and widely used as a model species for toxicity assessments. The results of my study suggest that lead inhibits waterborne Ca2+ uptake in <i>Daphnia</i> in a concentration dependent manner, and this inhibition occurs predominantly through a direct competitive interaction. The entry of waterborne Pb2+ in <i>Daphnia</i> likely occurs via both lanthanum-sensitive and verapamil-sensitive epithelial calcium channels. Moreover, my results also indicate that acute waterborne lead exposure severely disrupts both Ca2+ and Na+ uptake from water, which are concomitant with the increase in the lead body burden in <i>Daphnia</i>. Interestingly however, acute exposure to lead does not affect the rate of oxygen consumption in <i>Daphnia</i>, indicating no acute respiratory toxicity of lead. Overall, it appears that lead acts as an ionoregulatory toxicant to <i>Daphnia</i> during acute waterborne exposure.