Dissertations / Theses on the topic 'Cadmium chloride'

Assessement of effects of cadium chloride exposure on the anaphylaxis reaction to food was done on six week old Swiss and BALB/c female mice. The animals were exposed to cadium as cadium chloride for either three days or six weeks. Intra-peritonal dose of cadium chloride was injected once a day, five days per week for three successive weeks. The animals were then sensitised to cow's milk by force-feeding with cow's milk for three consecutive days. Oral exposure of mice to a high dose of cadium resulted in cytotoxicity of liver and kidney cells. Retardation in growth rate and haematology change were detected. Proliferative response to the T-cell epitope from the circumsporozoite protein of plasmodium falsiparum was decreased in cultures of lymph node cells from cadium chronically treated mice and sensitised with the same peptide. In contrast, an increase of cell proliferation was observed when cow's milk was used instead. Significant increase in Immunoglobulin E level and Anaphylactic reaction dependent on the quantity of cadium exposed were recorded. No protective effect of ascorbic acid or zinc acetate on cadium alteration of immune response was observed
Master of Science (Hons) (Food Science)

The amount of Municipal Solid Waste (MSW) produced in Canada was 12.6 million tonnes in 1980. Cadmium, which is one heavy metal characteristically present in leachate from MSW, is an extremely toxic metal to all living organisms. Clay liners are used to contain contaminants such as heavy metals (aluminum, mercury, manganese, lead, and zinc) present in leachate from landfill sites containing MSW. Consequently, an investigation into the mechanisms of cadmium adsorption from leachate onto clay was pursued.
Characteristically, such leachates contain trace quantities of heavy metals and abundant quantities of chloride ions. For this study, leachate samples were collected from the Meloche landfill site in Kirkland, Quebec. This leachate is characteristic of other leachates, and in one monitoring well it contained cadmium (Cd) at 0.069 mg/L (ppm) and chloride ions (Cl$ sp-$) at 677 mg/L (ppm). Under these conditions, the formation of complex ions between cadmium and chloride has the potential to interfere with the adsorption of cadmium by clay minerals.

Increased releases of cadmium to the aquatic environment have raised concern over the potential for adverse impacts on freshwater organisms in affected aquatic bodies. This thesis explored the responses of a common freshwater amphipod, Gammarus pulex (G. pulex) to sublethal concentrations of cadmium under different environmental conditions and at various stages of the lifestyle. Endpoints studied encompassed molecular, cellular, physiological and behavioural changes, to enable a comprehensive analysis of the effects of the organism. Exposure to sublethal concentrations of cadmium (Cd) (0.001, 0.005 and 0.01 mg Cd L–1) for 7 and 14 days, influenced the percent survival, induced lipid peroxidation and damaged DNA in haemolymph cells of G. pulex. These concentrations also reduced feeding and ventilation rates as well as the motility, in particular of the females, with increasing Cd concentration and time of exposure. Furthermore, Cd accumulation from water in the body tissues of the amphipods was lower than in their food, with increasing Cd concentrations. These results illustrate how concentrations of Cd below the Environmental Quality Standard for European waters can cause sublethal molecular and cellular damage after relatively short periods of exposure. Histopathological effects of Cd on the gills and hepatopancreas (mid-gut gland) were examined using light, scanning and transmission electron microscopy. Multiple effects were observed on the tissues and cell organelles including external alternations in the muscular fibres of the hepatopancreas, lysis of microvilli and morphological changes in mitochondria. Exposure to 0.01 mg Cd L–1 altered the epithelial layer of the gill, causing vacuolation and lysis across the whole gill structure. Mitochondria showed damage to the inner membrane, shortened cristae and swelling, with an increase in apoptosis at concentrations of 0.005 and 0.01 mg Cd L–1. Collectively, these results document the adverse effects of Cd on target organs at concentrations within the range found in freshwater bodies. An investigation of the effects of water hardness on bioaccumulation and toxicity showed that hardness of water inhibited Cd toxicity and protected the juveniles during long term exposures, without affecting growth rate and food consumption. Soft water reduced the rate of survival, growth rate and food consumption during chronic exposure to low Cd concentrations, and facilitated Cd accumulation in the body parts compared with juveniles exposed in hard water. The results show that water quality plays a vital role in reducing or increasing detrimental effects of low Cd concentrations on the early life stage of amphipods which are a source of food for many species in aquatic environments. Exposure to Cd led to an increase in metallothionein concentrations in the amphipods in both hard and soft water. It also caused disruption to ion/osmoregulation, which may represent one mechanism of compensation for the ions lost in the amphipods exposed to Cd in soft water. In conclusion, these results add to the body of evidence describing the sublethal toxicity of Cd, a priority pollutant, to a common freshwater sentinel species. These results are of relevance for future environmental management and remediation approaches, because they provide scientific data to help in assessing, interpreting and understanding the effects of the heavy metal Cd in freshwater environments.

Heavy metal contamination is a serious concern throughout the world. Increased concentrations in drinking water have many negative impacts on human health. Limestone is an inexpensive and simple media for removing high concentrations of heavy metals from drinking water supplies. Ferric based media is commonly used to remove zinc, cadmium, lead, arsenic and other heavy metals. The drinking water standards set by the US EPA for cadmium, zinc and arsenic are 0.005 mg/L, 5 mg/L and 0.010 mg/L respectively. Bangladesh, parts of India, China and the United States have high concentrations of arsenic in drinking water. Although many technologies exist for heavy metal removal, most of these are complicated and are associated with high costs making them ineffective and unfavorable to be used in impoverished areas. We propose a novel method that combines the benefits of limestone with the capacity of ferric media in an iron-coated limestone based material. Samples of water with various concentrations of zinc and cadmium were prepared and batch tests were performed using both uncoated and iron coated limestone and are compared in removal efficiency. Kinetics studies showed that zinc is removed to a maximum level after 24 hours, while cadmium takes only 15 minutes. The effect of pH on removal of heavy metals was also studied. Metals are analyzed using Inductively Coupled Plasma Emission Spectroscopy (ICP-ES). Limestone is readily available and is also easy to coat with iron, making this material a cost effective and affordable method to be used by developing countries.

Electric cell-substrate impedance sensing (ECIS) pioneered by Giaever and Keese is suitable for continuous, automatic and real-time cell attachment analysis. ECIS is a novel electrical method to study, in real time, many of the activities of animal cells when grown in tissue culture. These include morphological changes, cell locomotion, and other behaviors directed by the cell's cytoskeleton. One of the most direct ECIS measurements is that of the attachment and spreading behaviors of cells. These measurements allow one to study and quantify the interaction of cultured cells with extracellular matrix (ECM) proteins and other macromolecules continuously and in real time. Traditionally, cell attachment and spreading measurements are labor intensive, requiring many manipulations of the cultures for microscopic evaluation of cell behavior. With ECIS, these same measurements can be made in an automated approach without opening the door of the incubator. The ECIS core technology is based on a technique of measuring the change in impedance of a small electrode to AC current flow. The heart of the measurement is a specialized slide that has 8 individual wells for cell culturing. The base of the device has an array of gold film electrodes that connect to the ECIS electronics to each of the 8 wells. In our work we used ECIS to study the attachment and spread of HUVEC and 3T3 cells. The curve of HUVEC showed higher resistances than that of 3T3 cells. This was due to the fact we used gelatin to aid in attachment of HUVECs which accounted for the high resistances. 3T3 cells attached easily without help of gelatin. We also studied the cytotoxicity of HUVEC and 3T3 cells. The drugs that we used were CB, H7 and CdCl2. We found that the best drug was CB since it affected the cells even at low concentrations. H7 effects were mild while CdCl2 only worked at high concentrations. HUVEC cells make loose contact on electrodes and are easily detached by drugs. 3T3 makes firm at tachment to the electrodes and are not easily detached from the electrodes. Electrical impedance measurements on multiple electrodes are highly attractive in this application because of the potential for direct computer control.

Orientador: Mary Anne Heidi Dolder
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: O cádmio é um desregulador endócrino ressaltado por causar significativas disfunções fisiológicas e bioquímicas em animais e humanos. Está presente em pesticidas e no cigarro, sendo comum a contaminação de humanos. O testículo é um dos órgãos mais afetados pela toxicidade do cádmio, sendo muito suscetível ao seu acúmulo, que causa degeneração tubular, atrofia das células de Leydig, redução da qualidade espermática, entre outras alterações. Na tentativa de reverter ou amenizar os danos causados por este metal vários antioxidantes tem sido estudados, como os polifenóis, presentes na uva e seus derivados. Efeitos positivos destas substâncias têm sido relatados, como redução da pressão sanguínea, capacidade de modular enzimas e propriedade de quelar metais pesados. Já que essas substâncias quando ingeridas diariamente em determinadas quantidades podem modificar positivamente o metabolismo, neste estudo o concentrado de suco de uva foi administrado como um hábito alimentar, antes e após a intoxicação com o metal. Para isto, 24 ratos Wistar foram divididos em 4 grupos: GC (sem tratamento), GCd (CdCl2 - 1,2mg/Kg), GCdJ (CdCl2+G8000® ¿ 2g/Kg) e GJ (G8000® ¿ 2g/Kg). O suco de uva foi administrado diariamente por gavagem desde os 50 dias até 136 dias de idade. O CdCl2 foi injetado intraperitonealmente em uma dose única quando os animais tinham 80 dias. Depois do tratamento, os animais foram eutanaziados sob anestesia (xilazina e cetamina, 10 e 80mg/kg, respectivamente). Para avaliar os efeitos do tratamento foram realizadas análises biométricas, morfológicas (morfometria, estereologia e microscopia eletrônica de transmissão), dosagem de marcadores antioxidantes (CAT, SOD, GSH, MDA), acúmulo de cádmio tecidual e análises da qualidade e contagem espermática. A resposta testicular ao cádmio foi um pouco diferente do regularmente encontrado na literatura, considerando que esta dosagem foi incapaz de alterar níveis das enzimas relacionadas com estresse oxidativo, mas isto pode ser devido ao tempo decorrido entre a contaminação e a realização das análises. Apesar disso, um efeito devastador na morfologia testicular e perfil espermático foram observados. O acúmulo de metal foi evidente no grupo GCd, reduzindo a contagem e qualidade espermática, destruindo a arquitetura testicular e ultraestrutura. O efeito positivo do consumo de suco de uva foi confirmado em nosso estudo, sendo capaz de proteger a morfologia testicular e desenvolvimento espermático, levando-se em consideração a produção e morfologia, alterados pelo metal. O suco de uva isoladamente foi capaz de reduzir o ganho de peso, diâmetro tubular e altura do epitélio seminífero, mas considerando nossas evidências, este efeito não foi devido à toxicidade. Em conclusão, o suco de uva é um agente positivo na proteção do sistema reprodutor masculino contra intoxicação por cádmio
Abstract: Cadmium is an endocrine disruptor, highlighted in that it causes significant physiological and biochemical dysfunction in animals and humans. This metal is present in pesticides and cigarettes, so that human contamination is common. In cadmium intoxication, the testis is one of the most strongly affected organs, being very susceptible to accumulation of this metal, which causes tubular degeneration, Leydig cell atrophy and decreases in semen quality, among other alterations. In order to reverse or diminish the modifications generated by this metal, various antioxidants have been studied, such as polyphenols, present in grapes, and its derivatives. Positive effects of these substances have been reported, such as reduction of blood pressure, capacity to modulate some enzymes, and metal chelating properties. Based on the fact that daily ingestion of polyphenols in certain quantities can favorably modify the metabolism, preventing changes that can lead to severe damage, grape juice was administered as an eating pattern, before and after injection of the metal, from the beginning of sexual maturity to the end of a full spermatogenic cycle. For this study, 24 Wistar rats were divided into 4 groups: GC (without treatment), GCd (CdCl2 - 1,2mg/Kg), GCdJ (CdCl2+G8000® ¿ 2g/kg) and GJ (G8000® ¿ 2g/kg). The grape juice was administrated daily by gavage from 50 days of age until the rats were 136 days old. The CdCl2 was intraperitoneally injected when the animals were 80 days old. After the treatments, the animals were euthanized using a mixture of ketamine and xylazine (10 and 80mg/kg, respectively). In order to evaluate this effect, analyses were performed, including biometric analysis, morphological analyses, such as morphometry, stereology and transmission electron microscopy evaluation, dosage of antioxidant markers including CAT, GSH, SOD, MDA, dosage of cadmium accumulation in the testis and sperm quality analysis. Testis response to cadmium was different from that described in the literature, considering that this dosage did not alter enzymes related to oxidative stress, although a devastating morphological effect was observed. Metal accumulation was evident in GCd, reducing sperm count and quality, disrupting the architectural structure and ultrastructure of the testis. The positive effect of the grape juice administration was confirmed in our research considering it was able to protect testis morphology and sperm development, considering sperm production and morphology. Grape juice by itself reduced body weight gain, tubular diameter and seminiferous epithelium height, but considering our evidences its effects were non toxic. In conclusion, grape juice administration is a confirmed positive agent in relation to reproductive cadmium toxicity
Mestrado
Biologia Celular
Mestra em Biologia Celular e Estrutural

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
We investigated the action of butane-2,3-dione thiosemicarbazone oxime against the testicular damage caused by cadmium chloride (CdCl2) in mice swiss adult male. The animals received a single injection of CdCl2 at the dose of 5 mg/kg, intraperitoneally, and after thirty minutes, the oxime was administered subcutaneously at the dose of 10 mg/kg. Twenty four hours after administration of the oxime, the animals were euthanized the blood were collected and after killed the testes were removed for analysis. The parameters determined were δ-aminolevulinate dehydratase (δ-ALA-D), myeloperoxidase (MPO), glutathione-S-transferase (GST) and glutathione peroxidase (GPx) activities, in testicular tissue. The levels of thiobarbituric acid-reactive substances (TBARS), nonprotein thiols (NPSH), ascorbic acid and the quantity of cadmium in testes were also evaluated. In addition, levels of testosterone in serum and cytokines (proinflammatory, IL-1, IL-6, TNF-α, IFN-γ and anti-inflammatory IL-10) were determined. Histological analysis of testicular tissue was also performed. Our results demonstrated that the oxime was effective in restoring partially the inhibition in δ-ALA-D activity induced by CdCl2. The activation of MPO and increase in IL-1, IL-6, TNF-α and IFN-γ levels induced by CdCl2 were also reduced by oxime. IL-10, which was reduced by cadmium, was partially restored by oxime administration. In addition, the oxime was effective in restoring the increase in TBARS levels and partially the reduction on NPSH levels induced by CdCl2. However, the oxime did not have action on the decreased levels of ascorbic acid induced by CdCl2 or on the decrease in enzymatic activity of GST and the increased enzymatic activity of GPx caused by CdCl2. Our results demonstrated that oxime was effective in restoring the histological alterations induced by CdCl2, preventing the loss of elongated spermatids. In addition, oxime was able to increase the testosterone levels reduced by cadmium exposure. In conclusion, the oxime tested was effective in reducing the testicular damage induced by CdCl2 in mice. The beneficial effects of this oxime are related to its antioxidant and anti-inflammatory action.
Investigamos a ação da oxima butano-2,3-dionatiossemicarbazona contra o dano testicular causado por cloreto de cádmio (CdCl2) em camundongos swiss adultos machos. Os animais receberam uma única injeção de CdCl2 na dose de 5mg/kg, intraperitonialmente e, após trinta minutos, foi administrada a oxima subcutâneamente na dose de 10 mg/kg. Vinte quarto horas após a administração da oxima, os animais foram anestesiados o sangue foi coletado, após a eutanásia tiveram os testículos removidos para as análises. Os parâmetros determinados foram as atividades das enzimas δ-aminolevulinato desidratase (δ-ALA-D), mieloperoxidase (MPO), glutationa-S-transferase (GST) e glutationa peroxidase (GPx), no tecido testicular. Os níveis de substâncias reativas ao ácido tiobarbitúrico (TBARS), tióis não protéicos (NPSH), ácido ascórbico e quantidade presente de cádmio nos testículos também foram avaliados. Além dos níveis de testosterona no soro, onde também se quantificou as citocinas (pró-inflamatórias IL-1, IL-6, TNF-α, IFN-γ e anti-inflamatória, IL-10). Também foi realizada a análise histológica de tecido testicular. Nossos resultados demonstraram que a oxima foi efetiva em restaurar parcialmente a inibição da atividade da enzima δ-ALA-D induzida por CdCl2. A ativação da MPO e o aumento dos níveis de IL-1, IL-6, TNF-α e IFN-γ induzidos por CdCl2 também foram reduzidos pela oxima. A IL-10, que foi reduzida pelo cádmio, teve seus níveis restaurados parcialmente pela oxima. Além disso, a oxima foi efetiva em restaurar o aumento nos níveis de TBARS e parcialmente a redução nos níveis de NPSH induzidos pelo CdCl2. Porém, não teve ação sobre a queda dos níveis de ácido ascórbico provocado pelo CdCl2, nem sobre a diminuição da atividade enzimática da GST e sobre o aumento da atividade enzimática da GPx causados pelo CdCl2. Nossos resultados demonstraram que a oxima foi efetiva em conter as alterações histológicas causadas pelo CdCl2, prevenindo a perda de espermátides alongadas. A oxima também foi capaz de aumentar os níveis de testosterona reduzidos pela exposição ao cádmio. Em conclusão, a oxima testada foi efetiva em reduzir os danos testiculares induzidos pelo CdCl2. Os efeitos benéficos desta oxima estão relacionados às suas propriedades antioxidantes e sua ação anti-inflamatória.

Biotests sind für die toxikologische Bewertung von Chemikalien, Pflanzenschutzmitteln und schadstoffbelasteten Gewässern oder Böden von besonderer Bedeutung, da sie Auskünfte über die biologische Wirksamkeit eines Stoffes auf Organismen geben. Bislang gibt es für die ökotoxikologische Bewertung, insbesondere von Chemikalien und Pflanzenschutzmitteln, für die autotrophe Organismenebene neben verschiedenen Biotests mit höheren Pflanzen den DIN 28 692 Biotest "Wachstumshemmtest mit den Süßwasseralgen Scenedesmus subspicatus und Selenastrum capricornutum", der auch als OECD 201 Biotest "Algal, Growth Inhibition Test" vorliegt. Dieser aquatische Biotest wird nur mit einer Süßwasseralgenart durchgeführt und trotzdem zunehmend für die Bewertung von belasteten Böden und Sedimenten eingesetzt. Untersuchungen über aquatische Biotests, die Bodenalgen als Testorganismen nutzen, oder Boden-Biotests mit Bodenalgen gibt es nur vereinzelt. Ein Biotestsystem, das sowohl aus einem aquatischen als auch aus einem terrestrischen Biotest besteht und mehrere Bodenalgenarten als Testorganismen nutzt, existiert bisher nicht. Dieses wurde in vorliegender Arbeit entwickelt und an dem Schwermetall Cadmium als Cadmiumchlorid und dem Herbizid Arelon, Wirkstoff Isoproturon erprobt. Um Bodenalgen, die keine Resistenzen oder Toleranzen gegenüber Schadstoffen aufweisen, als Testorganismen nutzen zu können, wurden aus unbelasteten Böden Algen isoliert, Klonkulturen erstellt und die Arten bestimmt. Dies führte zu einer Sammlung mit 35 Algenarten. Aus den in die Bodenalgensammlung aufgenommenen Arten wurden Xanthonema tribonematoides, Stichococcus bacillaris, Klebsormidium flaccidum, Xanthonema montanum und Chlamydomonas noctigama für das Testsystem ausgewählt. Zusätzlich zu diesen wurde die Süßwasseralge Scenedesmus subspicatus als Referenzalge ausgewählt. Mit diesen Algen wurde der Gel-Biotest, bestehend aus einem flüssigen gelartigen Medium, das die Kontaminationspfade im Wasser nachbildet, und ein Boden-Biotest mit einem naturnahen sorptionsschwachen Boden entwickelt, der die Kontaminationspfade über Gas-, Wasser- und Festphase im Boden nachbildet. Bei der Erprobung dieses Biotestsystems mit Cadmiumchlorid und Isoproturon zeigte sich, daß Bodenalgen gegenüber Cadmiumchlorid im Gel-Biotest eine geringe bis mittlere Sensibilität aufwiesen. Im Boden-Biotest lag eine sehr geringe Sensibilität vor, wie dies auch bei anderen Bodenorganismengruppen in Biotests festgestellt wurde. Dies kann mit der Sorption der Cadmiumionen im Boden erklärt werden und dem damit geringen für die Organismen bioverfügbaren Cadmiumionenanteil. Für Isoproturon lag sowohl im Gel- als auch im Boden-Biotest eine hohe Sensibilität der Bodenalgen vor. Erstaunlich war, daß die Sensibilität in beiden Biotests nahezu identisch war, obwohl Isoproturon in sorptionsschwachen Böden zu ca. 30 % adsorbiert wird. Im Vergleich zur Sensibilität von Scenedesmus subspicatus waren die Bodenalgen bei Cadmiumchlorid bis auf zwei Ausnahmen um den Faktor 5 bis 10 unsensibler. Die Bodenalge Klebsormidium flaccidum besaß eine vergleichbare Sensibilität und Xanthonema montanum war um den Faktor 20 unsensibler. Für Isoproturon konnten keine Unterschiede in der Sensibilität zwischen Scenedesmus subspicatus und den geprüften Bodenalgen ermittelt werden, außer bei Stichococcus bacillaris, die um den Faktor 5 unempfindlicher war. Das entwickelte miniaturisierte Biotestsystem eignet sich dazu, differenzierte Aussagen über das ökotoxische Potential von Stoffen auf Bodenalgen und der Süßwasseralge Scenedesmus subspicatus zu erhalten. Durch den Einsatz von zwei unterschiedlichen Testsubstraten (Flüssigmedium und naturnaher Boden) werden der Einfluß dieser Substrate sowie die daraus resultierenden Kontaminationspfade der Teststoffe und ihre ökotoxikologische Wirkung auf Algen feststellbar und vergleichbar. Ein Normenentwurf des Biotestsystems wurde inzwischen in das "Technical Committee 190 - Soil Quality" der International Standards Organization (ISO) eingereicht.
Biotests are an important device to assess the toxicity of chemicals, pesticides, polluted water, and soils because they can provide direct information about the influence of a compound on the organism level. Besides various biotests using higher plants there is only the DIN 28 692 biotest "Growth-inhibition test using fresh water algae Scenedesmus subspicatus and Selenastrum capricornutum" (DIN 28 692) also known as the OECD 201 biotest "Algal, Growth Inhibition Test" which is currently available for an ecotoxicological assessment of chemicals such as pesticides on the autotrophic organism level. This aquatic biotest is based on a single specie of fresh water algae and is increasingly applied to evaluate polluted soils and sediments. There is almost no information on aquatic biotests which are using soil algae as test organisms instead. A more comprehensive biotest system which actually combines aquatic and terrestric biotests using several soil algae species as test organisms has not been reported, yet. Thus, a biotest system was developed and subsequently evaluated by using cadmium (cadmium chloride) as a heavy metal, and the herbicide arelon containing isoproturon as the active ingredient. Soil algae were isolated from unpolluted soil in order to obtain test organisms which are not resistant or tolerant to pollutants. The algae isolates were then cultivated, and subsequently identified. A total of 35 algae species was collected. Algae species used in the biotest system were Xanthonema tribonematoides, Stichococcus bacillaris, Klebsormidium flaccidum, Xanthonema montanum, Chlamydomonas noctigama. In addition, the fresh water specie Scenedesmus subspicatus served as a reference algae. Based on these different algae species a gel biotest using liquid gel medium was developed to investigate the contamination path via water, and also a soil biotest with a pre-treated soil of low sorption capacity was deviced to simulate the contamination path through gas, water, and solid phase. The evaluation of the biotest system using cadmium chloride and isoproturon did reveal that soil algae have had only low to medium sensitivity to cadmium chloride in the gel biotest. Algae sensitivity in the soil biotest was very low which was in accordance with data from other biotests using different soil organisms. The weak response of the algae was most likely caused by the sorption of the cadmium ions to the soil matrix what may have decreased the bioavailability of cadmium. In comparison, soil algae were very sensitive to isoproturon in both, the gel biotest and the soil biotest. Both biotests indicated almost identical sensitivities of the tested soil algae which was surprising since 30 % of the isoproturon was sorbed even in soils with a low sorption capacity. Soil algae when compared to the water algae Scenedesmus subspicatus were generally 5 to 10-fold less sensitive to cadmium chloride. Only Klebsormidium flaccidum has proved to have a similar sensitivity as Scenedesmus subspicatus had, whereas Xanthonema montanum was about 20-fold less sensitive. With isoproturon, however, no differences in sensitivity could be seen between Scenedesmus subspicatus and the tested soil algae, except Stichococcus bacillaris which was about 5-fold less sensitive. The biotest system as developed in this study has shown to be suitable for obtaining valuable information about ecotoxicological effects of chemicals on soil and water algae. Since the biotest system consists of two different test media (liquid gel and soil) it is possible to determine ecotoxicological effects on algae in both, water and soil. A first draft of the developed biotest system has been submitted to the "Technical Committee 190 - Soil Quality" of the International Standards Organization (ISO) for review.

Orientador: Maria Cristina Cintra Gomes Marcondes
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: Metais pesados como o cádmio são considerados agentes tóxicos devido sua extensiva utilização nas indústrias e agropecuária e, como conseqüência, são amplamente dispersados no meio ambiente. No entanto, o cádmio tem sido foco, também, de inúmeras pesquisas relacionadas a exposição humana e suas conseqüências patológicas como o câncer. Estudos, claramente, caracterizam as relações de tumor de pulmão com a inalação do cádmio e mostram a possível participação deste metal tanto na iniciação quanto na progressão tumoral. Por outro lado, são raros os relatos da literatura envolvendo o mecanismo de ação do cádmio em tecido muscular, uma vez que já foi observado acúmulo desse metal em musculatura esquelética de animais. A administração do cloreto de cádmio, metal pesado designado como carcinogênico, em linhagem de células musculares esqueléticas C2C12 promoveu lesões consistentes com estresse oxidativo, observado pela diminuição da viabilidade celular, aumento da peroxidação de lipídios (conteúdo de malondialdeído) e conseqüente diminuição da enzima antioxidante glutationa transferase (GST). O estresse oxidativo, possivelmente, alterou a adesão celular e, conseqüentemente, houve retração dos miotúbulos, observada através de microscopia de luz e microscopia eletrônica de varredura (Capítulo I- Trabalho publicado no periódico Free Radical Biology & Medicine, 2005). A atenuação das lesões promovidas pelo cloreto de cádmio em linhagem de células C2C12 foi verificada com o pré-tratamento com o sulfato de zinco antecedendo o tratamento com cloreto de cádmio. Os efeitos protetores foram observados através da preservação da viabilidade celular, da GST, e diminuição do conteúdo de malondialdeído. A ação protetora foi verificada, também, na maior preservação da adesão celular, principalmente, contra as maiores concentrações de cádmio (Capítulo II- Trabalho a ser submetido ao periódico Free Radical Biology & Medicine). Por outro lado, a exposição crônica de células tumorais, linhagem de adenocarcinoma de cólon MAC13, ao cloreto de cádmio promoveu alterações morfológicas associadas ao aumento da atividade mitocondrial, interferência quanto à atividade lisossomal e diminuição da viabilidade celular, principalmente, na maior concentração de cádmio, após 24hs de exposição (Capítulo III- Trabalho a ser submetido ao periódico International Journal of Cancer)
Abstract: The heavy metals as cadmium are a toxic agent since it is extensively utilized in industry and can be amply distributed in environment. The cadmium is research focused as its pathological consequences in human exposure as it has been classified as carcinogenic agent. This fact is evident since the cadmium inhalation can be related to lung tumour and many studies show the possible participation of the cadmium on tumoral cells initiation and progression. However, few studies observed that cadmium can be accumulated in animal skeletal muscle cells and its action mechanisms are not completed known. The cadmium chloride exposure promoted oxidative stress and morphologic changes in C2C12 myotubes cell, in vitro, associated to decrease on cellular viability, high lipid peroxidation (increase on malondialdehyde content, MDA) and decrease on glutathione-S-transferase (GST) activity. The cadmium chloride produced chances on the cellular adhesion, integrity and retraction in C2C12 myotubes cells. These effects could be attenuated by zinc sulphate pre-treatment, which maintained the cellular viability, GST activity, reducing the MDA content. The zinc sulphate pre-treatment preserved the cellular adhesion, especially in high cadmium chloride concentration. Additionally, the tumoral cells (colon adenocarcinoma MAC 13) chronically exposed to cadmium chloride showed increase on the mitochondrial activity, and reduction on lysosomal and cellular viability, especially at high cadmium chloride concentration after 24h of treatment, probably indicating the tumoral cell changes
Doutorado
Biologia Celular
Doutor em Biologia Celular e Estrutural

This work concerns the microstructure of CSS-grown CdTe layers used for CdTe/CdS solar cells. Particular attention is given to how the development of microstructure on annealing with CdCl(_2) may correlate with increases in efficiency. By annealing pressed pellets of bulk CdTe powder, it is shown that microstructural change does occur on heating the material, enhanced by the inclusion of CdCl(_2) flux. However, the temperature required to cause significant effects is demonstrated to be higher than that at which heavy oxidation takes place. The dynamics of this oxidation are also examined. To investigate microstructural evolution in thin-films of CdTe, bi-layers of CdTe and CdS are examined by bevelling, thus revealing the microstructure to within ~1 µm of the interface. This allows optical microscopy and subsequent image analysis of grain structure. The work shows that the grain- size, which is well described by the Rayleigh distribution, varies linearly throughout the layer, but is invariant under CdCl(_2) treatment. Electrical measurements on these bi-layers, however, showed increased efficiency, as is widely reported. This demonstrates that the efficiency of these devices is not dictated by the bulk microstructure. Further, the region within 1 µm of the interface, of similar bi-layers to above, is examined by plan-view TEM. This reveals five-fold grain-growth on CdCl(_2) treatment. Moreover, these grains show a considerably smaller grain size than expected from extrapolating the linear trend in the bulk. These observations are explained in terms of the pinning of the CdTe grain size to the underlying CdS, and the small grain size this causes. A simple model was proposed for a link between the grain-growth to the efficiency improvement. The study also examines the behaviour of defects within grains upon CdCl(_2) treatment provided the first direct evidence of recovery on CdCl(_2) treatment in this system. Finally, a computer model is presented to describe the evolution of microstructure during growth. This is shown to be capable of reproducing the observed variation in grain size, but its strict physical accuracy is questioned.

La quantité, le comportement et l'impact de nombreux métaux toxiques rejetés par les divers systèmes de combustion (centrales thermiques, usines d'incinération de déchets,. . . ) constituent un problème grandissant. Le but de cette étude est d'accroître les connaissances relatives à l'adsorption de vapeurs de chlorure de cadmium sur divers substrats tels que la silice, l'alumine, un aluminosilicate, ou la chaux. Ceux-ci représentent des constituants majoritaires des cendres ainsi que des adsorbants économiquement envisageables de métaux lourds, injectables dans ou après la chambre de combustion. Un montage a été conçu afin de réaliser cette étude, dans lequel des particules d'adsorbant sont injectées le long d'un réacteur vertical porté à diverses températures (750°C, 850°C, 950°C), à contre-courant d'un flux d'air contenant des vapeurs de chlorure de cadmium. Les expériences sont menées en faisant varier la concentration en vapeurs de chlorure de cadmium présent dans le flux gazeux, pour chaque substrat étudié à une température donnée. Une simulation au moyen du code de calcul fluent a permis d'étalonner les concentrations en vapeurs de chlorure de cadmium dans le flux gazeux en fonction de la température imposée à la nacelle de CdCl2. Les expériences mettent en évidence l'influence de la nature de l'adsorbant, de la surface spécifique et de la température vis-à-vis de l'adsorption de vapeurs de CdCl2. Elles montrent également la prépondérance du phénomène de chimisorption dans nos expériences sur la silice, l'alumine et l'aluminosilicate. Un modèle simple simulant le processus de chimisorption dans nos expériences permet d'obtenir des constantes de vitesse et des ordres relatifs à cette réaction et de savoir si celle-ci est limitée par la cinétique chimique ou par la diffusion à travers la couche limite.

Nous avons étudié la dynamique de relaxation de spin par des expériences pompe-sonde non-dégénérées. Ceci est possible si les transitions des états dégénérés vers d'autres états sont mutuellement permises ou interdites pour les différentes polarisations de la lumière excitatrice. Nous avons appliqué cette technique à deux types de semi-conducteurs : du CuCl massif et des puits quantiques de CdTe. Dans CuCl, la courbe de dispersion des polaritons est étudié dans une expérience pompe-sonde en regardant différentes longueurs d'onde d'excitation et de détection. Nous avons évalué comment les propriétés optiques près de la résonance excitonique se comportent temporellement depuis l'absorption induite au gain, selon la longueur d'onde et l'intensité de l'excitation. Nous avons montré que le temps de retournement de spin et la dynamique de relaxation des excitons polaritons peuvent être déterminés dans la régions spectrale dite " région de retournement du spin " située juste en-dessous de la région bottleneck où les polaritons sont assez lents et se propagent presque librement. Grâce à cette technique, nous présentons les premières mesures du temps de relaxation de spin dans CuCl. En-dessous de cette région de retournement du spin, la mémoire de spin des polaritons est rapidement détruite à cause des effets de surface. A l’intérieur de la région du « bottleneck », le temps de renversement du spin est réduit de manière significative par des processus de collision exciton-exciton qui deviennent importants à cause de la grande densité d’états et du caractère excitonique du polariton. De plus, nous avons proposé une interprétation théorique du temps de relaxation de spin excitonique dans CuCl. Nous avons considéré deux possibilités pour la relaxation du spin de l’exciton dans CuCl : la première est la relaxation liée aux processus de diffusion, dans lesquels la direction du vecteur Q est modifiée et la deuxième est la relaxation due à l'interaction d'échange entre les électrons et les trous en conjonction avec le mouvement du centre de masse des excitons. Dans les puits quantique CdTe/CdMnTe, le principe de l'expérience se fonde sur deux transitions optiques qui partagent un niveau d’énergie commun. Nous démontrons expérimentalement que dans les puits quantiques piézoélectrique non dopé, les trous lourds relaxent leur pseudo-spin (h =40 ps) plus lentement que les électrons (e=8ps). Nous avons expliqué nos résultats en termes de remplissage de l'espace des phases des bandes d'électron et de trou dont les excitons se composent
We have studied the spin relaxation dynamics using nondegenerate pomp-probe measurements. This is possible if transitions from degenerate states to other states are mutually allowed and forbidden for circularly polarized light with different helicities. We have applied this technique to two types of semiconductors: bulk CuCl and CdTe/CdMnTe quantum wells. In CuCl, the polariton dispersion curve is scanned by looking at different excitation and detection wavelengths. We have performed these studies at low temperatures on monocrystalline samples on a subpicosecond time scale. We have evaluated how the optical properties close to the exciton resonance develop in time from induced absorption to gain, depending on the wavelength and the intensity of excitation. We have shown that the spin-flip time and the relaxation dynamics of exciton polaritons can be determined in the “spin-flip region” situated just below the polariton bottleneck where the studied polaritons are quite slowly propagating. Using this technique, we present the first measurements of spin relaxation times in CuCl. Below the spin-flip region, however, the spin memory of the polaritons is rapidly destroyed due to surface effects. Within the bottleneck region, the spin-flip time shortens significantly through exciton-exciton scattering processes which become important because of the high density of states and the exciton like character of the polaritons. In additions, we have presented a theoretical interpretation of spin relaxation in CuCl based on the invariant expansion method of an effective Hamiltonian. We consider two possibilities for exciton-spin relaxation: first due to scattering processes, in which the direction of Q is changed and second due to the exchange interaction between electrons and holes in conjunction with the center-of-mass motion of the excitons. In CdTe/CdMnTe quantum wells, the principle of the experiment relies on two optical transitions that share a common energy level. We demonstrate experimentally that in our undoped piezoelectric quantum well, the heavy-holes relax their spin (h =40 ps) more slowly than the electrons (e =8ps). We have explained our results in terms of phase space filling of the electron and hole bands, from which the excitons are made up

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Dissertação (Mestrado)
IPEN/D
Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

Determination par diffraction rx des arrangements atomiques de neuf phases cristallines hydratees. Les polyedres de coordination des cations s'organisent en trois types structuraux : chaines simples (cd); empilement en couches (cd); chaines mixtes (cd,ca). Quatre phases possedent des structures caracterisees par une pseudo-symetrie marquee. Les macles et les transformations orientees, observees sur certains cristaux, sont interpretees par l'existence de pseudo-symetrie locale et de parentes structurales entre blocs atomiques des hydrates concernes

Des chromophores non lineaires, derives de la pyridine, sont ancres dans des matrices minerales par un reseau de liaisons hydrogene courtes. Les sels remarquables que nous avons isole sont les dihydrogenoarseniate, chlorure et bromure de 2-amino-5-nitropyridinium dont les signaux de second harmonique sont equivalents ou superieurs a ceux du 3-methyl-4-nitropyridine-n-oxyde (pom). Les composes d'addition, chlorure de cadmium de la bis (4-nitropyridine-n-oxyde) et le bis (bromure mercurique) de la 4-nitropyridine-n-oxyde possedent des structures polaires dont la cohesion de l'entite non lineaire est assuree a la fois par la liaison metal-oxygene et la liaison hydrogene. Leur reponse en generation de second harmonique est equivalente ou superieure a celle du ktiopo#4 (ktp). Des composes possedant un reseau cationique pseudo-centrosymetrique de dimethylaminopyridinium, de guanidinium (cation octupolaire de symetrie d#3#h) et de dimethylammonium-stibazolium-n-hydroxyde sont decrits dans la troisieme partie de ce travail. Cette ingenierie est une voie d'acces a des cristaux non lineaires mecaniquement et chimiquement stables. Il s'avere que le nombre important de sites donneurs et accepteurs d'hydrogene de la matrice dans laquelle est integree la molecule organique est preponderant dans l'obtention de structures non centrosymetriques. Ces matrices se presentent alors generalement sous la forme de chaines ou de couches a caractere polymere

En Afrique, la contamination des zones humides par les métaux et les pesticides va se poursuivre durablement. Pour autant, le statut toxicologique de ces milieux reste trop peu documente. Le but du présent travail est d’estimer la valeur du varan du Nil (Varanus niloticus) en tant qu’espèce sentinelle pour la contamination des zones humides continentales d’Afrique sub-Saharienne. Plomb, cadmium, et pesticides organochlorés et organophosphorés ont été quantifies, par spectrométrie d’absorption atomique et chromatographie gazeuse (respectivement), dans plusieurs tissus provenant de 71 spécimens issus de quatre sites (au Mali et au Niger) juges a priori inégalement contamines. Bien que des différences claires apparaissent, la contamination environnementale s’avère modérée sur les quatre sites, et ne semble pas constituer un risque notable pour les varans ni pour les humains qui s’en nourrissent occasionnellement. Toutefois la variabilité interindividuelle est importante. Les organotropismes des polluants détectés sont cohérents avec ceux préalablement décrits. Si l’on n’a pu mettre en évidence de différence liée au sexe en ce qui concerne les pesticides, les femelles présentaient des charges en métaux supérieures. La relation entre d’autres facteurs (taille, proportion de graisse) et les concentrations tissulaires a également été considérée. Les varans sont susceptibles de révéler des différences subtiles de contamination environnementale entre sites, et la résolution spatiale de l’outil semble très fine. La possibilité pratique d’utiliser cet indicateur se trouve donc validée. Un travail expérimental sur des varans captifs a par ailleurs été mené pour approfondir l’étude
In Africa, metal and pesticide contamination of wetlands is supposed to continue on a permanent basis. However, the ecotoxicological status of these ecosystems remains poorly documented. The aim of the present work is to assess the value of the Nile monitor (Varanus niloticus) as a sentinel species for the environmental contamination of continental wetlands in sub-Saharan Africa. Lead and cadmium on the one hand, and organochlorine and organophosphate pesticides on the other, have been quantified in several tissues by atomic absorption spectrophotometry and gas chromatography, respectively. Samples come from 71 specimens originating from four sites considered unequally contaminated (in Mali and Niger). Although clear differences appear between sites, the environmental contamination turns out to be moderate at the four sites, and does not seem to represent a significant risk neither for the monitors themselves, nor for occasional human consumers. However, the interindividual variability is important. The organotropisms relative to the detected pollutants are consistent with those described in previous studies. Concerning the pesticides, no gender effect has been found, whereas females were more contaminated by metals. The relation between other factors (size, proportion of fat) and tissue concentrations has been considered too. Nile monitors can reveal subtle differences in local pollution and the spatial resolution of this tool seems to be very sharp. Its practical relevance is thus validated. Additionally, an experimental work has been carried out on captive monitors to go into the subject in greater depth

Le traitement des REFIOM par vitrification conduit à l'obtention d'un vitrifiat et d'une petite fraction résiduelle issue du traitement des fumées où se trouvent concentrés les composés toxiques : les cendres secondaires. Celles-ci sont riches en métaux lourds sous forme de chlorures et en chlorures d'alcalins, NaCl et KC1. La forte solubilité et la très grande volatilité des chlorures s'avèrent particulièrement contraignantes pour les opérations d'inertage des métaux lourds. Il apparaît donc nécessaire de procéder à une étape de conversion de ces chlorures de métaux lourds afin de les récupérer sous une forme stable chimiquement et thermiquement. Dans ce cadre, nous avons étudié un procédé original de conversion à haute température des chlorures de métaux lourds utilisant les phosphates. Notre démarche a consisté en l'étude des bilans de matière et de la structure des produits de conversion afin de comprendre les différentes réactions chimiques du mécanisme de conversion. La spectroscopie par Résonance Magnétique Nucléaire (RMN) a été utilisée pour caractériser l'influence des paramètres expérimentaux sur la nature et la structure des matrices phosphates générées par le procédé de conversion à haute température. Dans un premier temps, nous détaillons notamment, grâce à l'étude d'un système modèle ne contenant que PbC12 et CdC12, les différentes réactions chimiques qui se déroulent durant l'étape de conversion. Ensuite, l'influence des chlorures d'alcalins NaCl et KC1 sur la conversion des chlorures de métaux lourds (PbC12 et CdC12) est etudiée de sorte à étendre le procédé de conversion aux cendres secondaires riches en chlorures d'alcalins. Plusieurs techniques RMN ont été utilisées pour caractériser la structure des verres de phosphate générés par la conversion. Pour terminer, nous avons jugé de la potentialité d'utilisation de telles matrices vitreuses en tant que matériaux d'encapsulation de déchets. Le dernier point qui est abordé concerne l'optimisation de la durabilité chimique de ces matériaux par ajout d'oxyde de fer, Fe203
During fly ash vitrification processes, heavy metals volatilize and are recovered into chlorinated salts, called secondary ashes. Due to the volatility of heavy metal chloride compounds, the thermal treatment of these secondary ashes must involve first the conversion of the toxic chlorinated salts into a thermaly stable form. Therefore, we propose a method for the stabilisation of heavy metal wastes generated in fly ashes vitrification processes, based on the batch conversion of çhlorinated species (heavy metal chlorides and alkali chlorides) with ammonium dihydrogenphosphate, that produces phosphate glasses. Our approach of the problem is dual. First, we have stated, thanks to elemental analyses, the different chemical reactions of the conversion mechanism as a fonction of the batch composition. Secondly, the structural study of the phosphate glasses obtained by the thermal conversion process have been achieved using NMR spectroscopy. To set about this study, we have prepared synthetic mixtures to be converted. The first one is a simplified batch, containing PbC12 and CdCl2 only. The second one contains also also sodium chloride and potassium chloride and the batch compositions are choosen so that they correspond to an average secondary ash composition. Finally, some results about the chemical durability of the phosphate glasses obtained by the conversion process are presented and the improvement of the chemical durability of these phosphate glasses with iron oxide addition is discussed

Etude rpe de (ch::(3))::(4) nmn::(1-x) cd::(x) cl::(5) entre 20 et 300 k, pour x = 0,02; 0,08 et 0,2; mesure des temps de relaxation spin-reseau. Mise en evidence de variations importantes en fonction de la composition. Interpretation des resultats au moyen du formalisme de la fonction memoire et d'un modele de la fonction de correlation de spins des chaines finies

碩士
國立清華大學
輻射生物研究所
85
Cadmium was classified as a carcinogen by international agency on Research in 1993. However, the molecular mechanisms of Cd- induced carcinogenesis are not understood. It is believed that multiple mutations occur simultaneously in mutations occur simultaneously in mutagen ecposed mammalian cells and finally induce cancers. Whether Cd-induced cancers are correlated with the increased mutations frequency on more than one gene in cells remains to be elucidated.

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Tereza Zahradníková Supervisor: Prof. PharmDr. František Štaud, Ph.D. Consultant: Alexander Zaremba, Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Germany Title of diploma thesis: Effect of cadmium chloride on P-glycoprotein in the blood-brain barrier The blood-brain barrier (BBB) separates the central nervous system (CNS) and the peripheral blood circulation. It regulates the material transport between these compartments due to its specialised structure and cellular constitution. The endothelial cells forming the BBB are characterized by the expression of different multidrug resistance proteins which belong to the ATP-binding cassette (ABC) transporter family. These transmembranous ABC proteins actively transport molecules out of the BBB endothelia into the bloodstream and protect the brain against harmful xenobiotics, toxins and metabolites. On the other hand, ABC export proteins constitute obstacles to the delivery of many therapeutic drugs across the BBB into the CNS, thus the efficacy of CNS pharmacotherapy is limited. One of the most important efflux transporters is P-glycoprotein (P-gp). Cadmium is a heavy metal that is dangerous to human health....

碩士
國立清華大學
生命科學系
90
HSP86 and HSP84 are mammalian heat shock proteins identified to be the inducible and constitutive isoform of HSP90, respectively. However, the regulatory mechanism of hsp86 upon heavy metal and antitumor drugs stress are still unclear. In this study, rat hsp86 promoter region was used to investigate the mechanism of hsp86 expression induced under stress. The full length promoter region of rat hsp86 was used to search the putative transcription elements in hsp86 promoter from transcription element database. We also conducted sequence analysis between promoters of several hsp86 in mammals. The sequence comparison results showed that the -270 to +1 region of mammalian hsp86 promoters is almost identical, suggesting the importance of functional conservation. To test whether the -270 to +1 region is sufficient and to characterize the individual cis-acting elements, full length and serial deletions of the predicted hsp86 promoter isolated from genomic library were constructed into firefly luciferase reporter plasmid. The functional analyses of these promoter constructs were first verified under normal condition and then tested under the stimulation of cadmium chloride, geldanamycin, 17-allylamino, 17-demethoxygeldanamycin, and radicicol in 9L rat brain tumor (RBT) cells. The same minimal essential promoter region of rat hsp86 located within the —471 to +1 region is identified under normal condition, and stimulation of the heavy metal cadmium, and three anticancer drugs. Our experimental results identified two more regulatory regions than the hypothetic result from conserved sequence analysis data of the -270 to +1 region. The functional analysis indicated, consistently, that within the hypothetic -270 to +1 region, the heat shock element (HSE) containing region from —137 to +1 plays the main transactivation role, and the GC containing region (from —265 to —137) exhibits a positive regulatory activity. However, the nuclear factor kappa chain transcription B cells (NF-kappaB) containing region (from -471 to —265) has a newly found negative regulatory activity and a distal region containing cAMP responsive element (CRE) from —1418 to —1177 is identified as a possible cadmium-specific, responsive region. To detect the secondary important elements, HSE was specifically mutated from the deletion constructs and the results identified NF-kappaB as the secondary effective element during cadmium treatment. No obvious secondary element was found under normal condition indicating the NF-kappaB element is required but not sufficient for the basal level expression of hsp86.

碩士
國立清華大學
輻射生物研究所
86
Arsenic and cadmium are well-reconized human carcinogens, but their mechanismsare not entirely clear. Recent studies suggested that reactive oxygen species(ROS) and/or reactive nitrogen species (RNS) are involved in the clastogenici-ty of arsenic and cadmium. Since chromosome aberrations mainly come from DNA damage, and DNA damages may require several cellular steps to become chromoso-me aberrations, a more simple question to be asked is: how DNA damages are in-duced by arsenic and cadmium? The aim of this invesgation is to find out whatkinds of ROS and RNS are involved in arsenic- and cadmium-induced DNA strand breaks. DNA strand breaks in sodium arsenite (SA) and cadmium chloride (Cd) treated bovine aortic endothelial cellswere analyzed by single-cell alkaline electrophoresis. The results indicate that inhibitors of nitric oxide syntha-se, Nw- nitro-L-arginine methyl ester (NAME) and S-methyl-L- thiocitrulline (MTC),scavengers of hydroxyl radicals, dimethyl sulfoxide and D-mannitol, could decrease DNA strand breaks in SA- but not in Cd-treated cells. On the contray, catalase, a scavenger of hydrogen peroxide, could decrease DNA strand breaks in Cd- but not in SA-treated cells. However, superoxide dimutase,a scavenger of superoxide, uric acid and Trolox, scavengers of peroxynitrite, could decrea-sse DNA strand breaks breaks in both SA- and in Cd-treated cells. These rsults suggest that SA may induce DNAstrand breaks via nitric oxideand peroxynitrite; and Cd induce DNA strand breaks via superoxide, hydrogenperoxide,and hydroxyl radicals. Moreover, calcium seems to be involved in both SA- and Cd-induced DNA breaks, since calcium chelators, ethylene glycol-bis (beta-ami-ethyl ether)-N, N,N',N'-tetraacetic acid (EGTA), Quin 2, 1,2-Bis(2-aminophe- noxy)ethane-N,N,N',N'-tetra-acetic acid (BAPTA), supressed SA- as well as Cd- induced DNA strand breaks. calcium ionophore, A23187, increased SA- but not Cd-induced comet. Inorganic trivalent arsenic compounds are generally recognized to be more toxic than their metabolite, methylated arsenic compounds. However, SA has recently been shown not to induce DNA strand breaks in human diploid pulmonary epithelial cells, but its metabolite, dimethylarsinic acid (DMA) does. We have compared the induction of DNA strand breaks by SA and DMA in a human embryo lung cell line, MRC-5, and in bovine aortic endothelial cells by single-cell alkaline electrophoresis. The results indicate that SA could induce DNA strand breaks in both cell lines with or wihtout the blockage of DNA repair synthesis by hydroxyurea (Hu) plus cytosine-beta-D-arabinofuranoside (AraC). Whereas, DMA did not induce DNA strand breaks without Hu/AraC. Induction of DNA strand breaks by SA plus Hu/AraC, reached a plateau by 6 h of treatment, wheras, DNA strand breaks induced by DMA plus Hu/AraC contiously increased beyound 10 h. The SA- induced DNA strand breaks were also repaired in a faster rate than that of DMA. Moreover, while the SA-induced DNA strand breaks were mediated by the nitric oxide production and were sensitive to calcium modulation, the DMA-induced DNA strand breaks were insensitive to nitric oxide synthase inhibitors and calcium modulators. These results indicate that SA and DMA induce DNA strand breaks through distinct different mechanisms.

碩士
國立清華大學
生命科學系
90
Cadmium (Cd) is a ubiquitous environmental heavy metal that has been classified as a human carcinogen. Deregulation of cell cycle progression is an important issue to cause cancer. Previous studies indicate that Cd causes mitotic arrest and decreases replicative DNA synthesis. However, the effect of Cd on molecules regulating G1 cell cycle progression remains unclear. In this thesis, we explore whether Cd affects G1 cells entering S phase at molecular levels using synchronized human non-small lung carcinoma cells, CL3. CdCl2 (40 mM, 2 h) markedly delayed S phase progression of synchronous cells at the G1/S border derived from aphidicolin procedure. Cd also delayed G1 progression of cells synchronized at G1 phase derived from either the nocodazole release procedure or the counterflow centrifugal cell elutriation system. Using western blotting and immunocomplex kinase activity analysis techniques, we have observed that Cd significantly delayed RB phosphorylation, down-regulated the protein levels of Cdk4, and decreased cyclin D1- and cyclin E-associated kinase activity in elutriated G1 cells; however, the protein levels of cyclin D1 did not altered. Furthermore, exposure these elutriated G1 cells to Cd also markedly delayed the induction of cyclin A expression, and cyclin A-associated kinase activity; however, the protein levels of Cdk2 were unchanged. On the other hand, Cd induced p38 MAPK phosphorylation and p21WAP1 protein levels, but not phospho-p53 (Ser15) in elutriated-G1 cells. SB202190, an inhibitor of p38 MAPK, delayed G1 progression of untreated or Cd-treated elutriated-G1 cells, elevated phospho-p53 (Ser15) while decreased p21WAP1 levels. Taken together, these results suggest that Cd delays RB phosphorylation by decrease Cdk4 protein levels, and cyclin D1- and cyclin E- associated kinase activities, thereby further slow-down the expression of cyclin A and cyclin A associated kinase kinase activity to delay G1 to S progression. Additionally, the activated-p38 MAPK may result in decreased phospho-p53 (Ser15), which may allow damaged cells bypass G1 checkpoint.

碩士
國立清華大學
輻射生物研究所
82
Cisplatin-resistant cells, Pt4 and Pt8 cells, were obtainedom Chinese hamster ovary (CHO-K1) cells after long-term culturecisplatin containing medium. Pt8 cells were about 1.3-foldesistant to cadmium chloride (CdCl2) than CHO-K1 cells. SH content in Pt4 cells was 1.3-fold more than that in4 cells were about 2.9-fold more sensitive to CdCl2after examination with clonogenic survival.20 uM of CdCl2, Pt4 cells were induced aboutmicronucleus than K1 cells were. But Pt8 cellsss micronucleus (about 0.9-fold) than K1 cells were. From nuclear division index, nuclear division in4 cells was inhibited more than that in K1 cells was. Theite results were observed in Pt8 cells (CdCl2 concentration50: K1=20 uM; Pt4=11 uM; Pt8=21.5 uM). In addition,from nucleoid sedimantation assay indicated that the DNAon was significant changed in Pt4 cells after highns of CdCl2 exposure. Also, K1 cells recovered by time dependence after incubatedth fresh medium for 4 or 8 h when treated with CdCl2 for 2 h.Pt4 cells did not recover for 4 or 8 h when treated withfor 2 h. So it seems that Pt4 cells did not recover damagediciently. The detoxfied enzymes, such as catalase,ne S-transferase, and heme oxygenase were nottly different in K1, Pt4 and Pt8 cells. These suggestls were more resistant than K1 cells after exposedmore GSH contents, and Pt4 cells were moreK1 cells after CdCl2 exposure for less reapirmore cadmium accumulation.

Sodium arsenite and cadmium chloride are two widespread environmental toxicants which have deleterious effects on living organisms. At the cellular level, sodium arsenite and cadmium chloride cause oxidative stress, dysregulation of gene expression, apoptosis, and the unfolding of protein. Furthermore, both chemical stressors individually have the ability to induce heat shock protein (HSP) accumulation. HSPs are molecular chaperones that aid in protein folding, translocation and in preventing stress-induced protein aggregation. Previously, our laboratory demonstrated that treatment of A6 kidney epithelial cells of the frog Xenopus laevis, with either cadmium chloride or sodium arsenite plus a concurrent mild heat shock resulted in an enhanced accumulation of HSPs that was greater than found with the sum of the individual stressors. To the best of our knowledge, no information is available to date on the effect that these two chemical stressors have in combination on HSP accumulation in aquatic organisms. The present study examined the effect of simultaneous sodium arsenite and cadmium chloride treatment on the pattern of HSP30 and HSP70 accumulation in Xenopus A6 cells. Immunoblot analysis revealed that the relative levels of HSP30 and HSP70 accumulation in A6 cells treated concurrently with sodium arsenite and cadmium chloride for 12 h were significantly higher than the sum of HSP30 or HSP70 accumulation from cells subjected to the treatments individually. For instance, the combined 10 µM sodium arsenite plus 100 µM cadmium chloride treatment resulted in a 3.5 fold increase in HSP30 accumulation and a 2.5 fold increase in HSP70 accumulation compared to the sum of the stressors individually. This finding suggested a synergistic action between the two stressors. Pretreatment of cells with KNK437, an HSF1 inhibitor, inhibited the combined sodium arsenite- and cadmium chloride-induced accumulation of HSP30 and HSP70 suggesting that this accumulation of HSPs may be regulated, at least in part, at the level of transcription. Immunocytochemical analysis employing the use of laser scanning confocal microscopy (LSCM) revealed that simultaneous treatment of cells with the two stressors induced HSP30 accumulation primarily in the cytoplasm in a punctate pattern with some dysregulation of F-actin structure. Increased ubiquitinated protein accumulation was observed with combined 10 µM sodium arsenite and 10, 50 or 100 µM cadmium chloride treatment compared to individual stressors suggesting an impairment of the ubiquitin-proteasome degradation system. Finally, while incubation of A6 cells with 1 µM sodium arsenite plus 10 µM cadmium chloride did not induce a detectable accumulation of HSPs, the addition of a 30 °C mild heat shock resulted in a strong accumulation of HSP30 and HSP70. This study has demonstrated that concurrent sodium arsenite and cadmium chloride treatment can enhance HSP accumulation. Since HSP accumulation is triggered by proteotoxic stress, these findings are relevant given the fact that aquatic amphibians in their natural habitat may be exposed to multiple chemical stressors simultaneously.

碩士
國立臺灣大學
園藝學研究所
91
Summary Turfgrass is important for landscaping. Effect of CdCl2 on germination and growth of turfgrass was to studied to determine the tolerance of turfgrass under Cd environment and the ability to absorb Cd to evaluate the potential of phytoremediation . The objectives of the research were to study the improvement of germination in turfgrass by priming. Seeds of bermudagrass , centipedegrass,carpetgrass and tall fescue were placed in petri-dishes with 0,1,10,20,40,60,80,100ppm CdCl2 solution, and then mesure the germination.The result showed that germination rate was lower when concentration was increasing. Besides carpetgrass, other turfgrass still maintain more than 50% germination percentage under 100ppm CdCl2. This shows that they had tolerance to Cd then Bermudagrass and centipedegrass had the best result. Germination rate of bermudagrass and tall fescue was improved after treated by lower concentration of CdCl2. Effect of CdCl2 on the growth of turfgrass seedlings and cuttings, all kinds of turfgrass had the situation that dry weight ,fresh weight, water content, and cholophyll content were decreased ,and more significant with increasing concentration of CdCl2. Decreasing ratio as the index for Cd tolerance, the tolerance of seedlings from high to low is tall fescue,perennial ryegrass, centipedegrass and carpetgrass. Concentration of CdCl2 in plant tissue from high to low is carpetgrass, centipedegrass, tall fescue and perennial ryegrass, manila grass, St. Augustinegrass and cool season turfgrass: tall fescue were considered as greener plants of contaminated place. Bermudagrass (Cynodon dactylon) and seashore paspalum (Paspalum vaginatum Swartz) are two of the most saline-tolerant warm season turfgrasses. A whole plant microculture (WPMC) system has been developed which permits intact root growth observation through the gelrite solidified culture medium. To efficiently screen for potential whole plant level salt tolerance, root morphology was studied using scanning electron microscopy. The experimental results demonstrated significant differences among stress mediums in terms of root number, root length, shoot length, nodal number, and ratio of root length to shoot length. Moreover, SEM examination of regenerated WPMC roots under Cd-free, 1, ,10and 100 ppm of CdCl2 stress media displayed obvious morphological and adaptation differences. In this investigation microculture at the whole plant level in vitro enables intact root growth observations through the culture medium and vessel, and offers a potential pre-screening method for putative heavy metal tolerant turfgrasses before commercialization and use in phytoremediation to clean up contaminated soils. Seeds of Centipedegrass were treated with different concentration of PEG（polyethylene glycol,-0.1,-0.5MPa）and different temperature （15 and 20℃）for four days ,and then mesure the germination under warm and cool temperatures .Primed seeds had shorten MGT ( Mean germination time) and T50. MGT of unprimed treatment was 8.6 days, and T50 was 7.5 days. Maximum germination for priming was recorded at PEG-0.5MPa under 20℃, which resulted in higher than 20℃, and thus the promotion of priming at 30℃ was less beneficial than seeds were exposed to 20℃.The germination rate of stored seeds was lower than that of fresh seeds. Reducing osmotic potential and increasing priming time could shorten germination time and increase the regularity of germination .

Sodium arsenite (NA) and cadmium chloride (CdCl2) are two relatively abundant environmental toxicants that have numerous detrimental effects on living organisms. At the cellular level, NA and CdCl2 produce reactive oxygen species which cause protein damage. Recent studies, in mammalian systems, have suggested that NA and CdCl2 can inhibit the ubiquitin-proteasome system (UPS). The UPS is the major degradation route for the elimination of damaged protein. This process involves two successive steps: the addition of ubiquitin to damaged protein and subsequent degradation by the 26S proteasome. Previously our laboratory determined that inhibition of the UPS can induce the accumulation of heat shock protein (HSPs) in Xenopus laevis A6 cells. HSPs are molecular chaperones, which bind to unfolded proteins to prevent aggregation and assist in protein refolding. The goal of this study was to examine the effect of NA and CdCl2 on the UPS of Xenopus laevis cells and to investigate any possible association between HSP accumulation and proteasomal activity. In the present study, treatment of A6 cells with NA or CdCl2 caused an increase in HSP30 and HSP70 accumulation, as well as in protein ubiquitination. In fact, treatment with 30 μM NA or 200 μM CdCl2 resulted in a 1.7- and 2-fold increase, respectively, in the relative levels of ubiquitinated protein compared to control. Examination of the relative levels of ubiquitinated protein over time revealed significant increases in cells treated up to at least 24 h after exposure of A6 cells with 30 μM NA or 200 μM CdCl2. To further examine the effect of NA and CdCl2 on proteasome activity, a cell-based assay measuring proteasomal chymotrypsin (CT)-like activity was employed. Treatment with 20 or 30 μM NA caused a 40 % decrease in the relative levels of chymotrypsin (CT)-like activity in A6 cells compared to control cells. The CT-like activity of A6 cells treated with 100 or 200 μM CdCl2 decreased by 40 % and 75 %, respectively, compared to control. The increase in ubiquitinated protein and a decrease in CT-like activity suggest that NA and CdCl2 can inhibit the UPS in A6 cells. In order to examine any possible association between HSP accumulation and proteasome activity an inhibitor of HSP accumulation, KNK437, was employed. In A6 cells pretreated with KNK437 followed by NA or CdCl2 exposure a decrease in the relative levels of HSP30 and HSP70 and ubiquitinated protein was noted compared to cells treated with NA alone. However, the CT-like activity of cells pretreated with KNK437 prior to NA or CdCl2 showed no significant difference compared to cells treated with NA or CdCl2 without pretreatment. The findings of this study suggest that NA and CdCl2 inhibit proteasomal activity in A6 cells and that there is a possible association between HSP accumulation and the mechanism by which damaged proteins are ubiquitinated.

碩士
國立清華大學
生命科學系
89
The heat-shock proteins (HSPs) or chaperones can protect and stabilize the folding and functions of normal proteins in cells. Chaperones are generally composed of large, evolutionarily conserved families within which the members are highly related, yet might differ in subcellular localization or gene regulation. We used semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) to analyze the expression profile of HSP70 isoforms in human non-small cell lung cancer H460 cells under CdCl2 stress. The expressions of hsp70-1, hsp70-2, hsp70-hom, hsp70-3, grp78, hsp70B, and hsp70B’ were monitored by amplifying from a conserved, common primer set and then differentiated by isoform-specific restriction enzymes. It is known that CdCl2 could elicit different cellular responses and gene expressions in a concentration-dependent and treatment time-dependent manner. Consistent with which, in our system, the expression level or pattern of chaperones varied low concentration (5 uM) and high concentration (40 uM to 60 uM) of CdCl2. We compared the changes of the newly synthesized chaperones by metabolic labeling with [35S]methionine and of the total chaperones by Western blotting analysis in CdCl2 treatment. We further analyzed changes of gene expression of individual isoforms by RT-PCR. From our study, this novel method can be performed to differentiate the members of HSP70 family under stress. Here, it has been found that the main members of HSP70 family in CdCl2-treated cells include hsp70-1, hsp70-2, and grp78. Furthermore, other four members of HSP70 family (hsp70-hom, hsp70-3, hsp70B, and hsp70B’) can be analyzed by this novel method. These four members don’t take part in stress conditions with CdCl2-treated. 二、英文附錄 1. ABSTRACT....................................2 2. INTRODUCTION................................3 3. EXPERIMENTAL PROCEDURES.....................7 4. RESULTS....................................11 5. DISCUSSION.................................18 6. REFERENCES.................................23 7. FIGURE LEGENDS.............................30 8. FIGURES....................................34 9. TABLES.....................................45

Prokaryotic and eukaryotic organisms respond to various stresses with the production of heat shock proteins (HSPs). HSPs are molecular chaperones that bind to unfolded proteins and inhibit their aggregation as well as maintaining their solubility until they can be refolded to their original conformation. Stress-inducible hsp gene transcription is mediated by the heat shock element (HSE), which interacts with heat shock transcription factor (HSF). In this study, we examined the effect of KNK437 (N-formyl-3,4-methylenedioxy-benzylidene-g-butyrolactam), a benzylidene lactam compound, on heat shock, sodium arsenite, cadmium chloride and herbimycin A-induced hsp gene expression in Xenopus laevis A6 kidney epithelial cells. In studies limited to mammalian cultured cells, KNK437 has been shown to inhibit HSE-HSF1 binding activity and stress-induced hsp gene expression. In the present study, western and northern blot analysis revealed that exposure of A6 cells to heat shock, sodium arsenite, cadmium chloride and herbimycin A induced the accumulation of HSP30 protein and hsp30 mRNA, respectively. Western blot analysis also determined that exposure of A6 cells to heat shock, sodium arsenite, cadmium chloride and herbimycin A induced the accumulation of HSP70 protein. Pre-treatment of A6 cells with 100 µM KNK437 inhibited stress-induced hsp30 mRNA as well as HSP30 and HSP70 protein accumulation. Immunocytochemistry and confocal microscopy were used to confirm the results gained from western blot analysis as well as determine the localization of HSP30 accumulation in A6 cells. A 2 h heat shock at 33°C resulted in the accumulation of HSP30 in the mostly in the cytoplasm with a small amount in the nucleus. Heat shock at 35°C resulted in substantial HSP30 accumulation in the nucleus. This is in contrast with A6 cells treated for 14 h with 10 µM sodium arsenite, 100 µM cadmium chloride and 1 µg/mL herbimycin A, where HSP30 accumulation was found only in the cytoplasm and not in the nucleus. A 6 h pre-treatment with 100 µM KNK437 completely inhibited the accumulation of HSP30 in A6 cells heat shocked at 33 or 35°C as well as cells treated with 1 µg/mL herbimycin A. The same pre-treatment with KNK437 resulted in a 97-100% decrease in HSP30 accumulation in A6 cells treated with 10 µM sodium arsenite or 100 µM cadmium chloride. These results show that KNK437 is effective at inhibiting both heat shock and chemical induced hsp gene expression in amphibian cells.

La présente étude visait d'abord à caractériser la ou les protéines impliquées dans le métabolisme du cadmium lors d'une intoxication subaiguë chez la truite mouchetée, Salvelinus fontinalis. Mitchill. Trente et un jours après l?injection hebdomadaire intrapéritonéale de chlorure de cadmium, le tissu hépatique principalement, présente diverses protéines associées au cadmium. On distingue trois ordres de poids moléculaires, soit des protéines à très hauts poids moléculaires (THPM % 45 OOO) correspondant aux molécules de complexation primaire, des protéines à poids moléculaires élevés (PME * 28 000) pouvant être des apo-métallothionéines et des protéines à faibles poids moléculaires (FPM s 15 000) possiblement des métallothionéines ou des protéines similaires. On observe que le cadmium sature des sites autres que la métallothionéine ou le ligand de zinc lors d'intoxication sous-létale. Le cadmium cohabite alors avec le zinc. Lorsqu'il y a un déplacement métallique du zinc par le cadmium en situation de toxicité aigüe, le zinc se combine à d'autres particules et on constate un certain niveau de saturation du tissu hépatique en cadmium. Ceci est appuyé par les valeurs du facteur de bioconcentration. Un deuxième objectif consistait à déterminer le niveau de contamination du cadmium et des métalloprotéines dans le tissu hépatique de la truite après un séjour de cinq mois dans les eaux du fjord du Saguenay où la concentration en métaux lourds et plus particulièrement en cadmium dépasse fréquemment la norme. Nous avons noté un taux extrêmement faible de cadmium dans le tissu hépatique, et des valeurs plus élevées au niveau du rein et des branchies. Nous retrouvons aussi, quoiqu'en concentration plus faible, la présence des trois ordres de poids moléculaires associés au métal. Comme dans la première expérience, nous n'observons aucun effet biotique de la contamination, confirmant ainsi l'aspect chronique de cette expérience. Des aspects d'antagonisme métallique y sont discutés. Finalement une troisième expérience visait à déterminer les caractéristiques métaboliques du tissu hépatique de la truite mouchetée dans une situation d'intoxication aiguë (dosage de CdC12 élevé, courte période déposition), plus particulièrement en relation avec les types moléculaires de métalloprotéines déjà identifiées. Nous observons ici une dominance des protéines à poids moléculaires élevés (PME) avec le cadmium prépondérant dans les protéines à très hauts poids moléculaires (THPM). Nous estimons ici que dans le cas d'une contamination massive au cadmium effectuée dans un très bref délai où cette même contamination dépasse la capacité de la cellule à s"acclimater au métal, la thionéine n'aurait pas suffisamment de temps pour être synthétisée avant que la cellule ne soit intoxiquée. De plus on sait que des protéines telle l'albumine sérique pourrait jouer un rôle important dans la complexation des ions métalliques et que les taux de dégradation et de resynthétisation des protéines s'effectuent rapidement. Enfin du point de vue morphologique, on peut souligner que les cellules hépatiques présentent un réarrangement des membranes internes et que plusieurs altérations du noyau, des mitochondries et du réticulum endoplasmique rugueux, sont observables.