Thesis (Ph. D.)--University of Rhode Island, 2005. / Typescript. Includes bibliographical references (leaves 114-122).

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 112-120).

Cadmium (Cd) is a toxic metal known to accumulate in and have adverse effects on the olfactory systems of fish. The objective of this project was to investigate the effect of cadmium on zebrafish larvae, specifically the effects on the olfactory system at cellular and functional levels. Zebrafish larvae (72 hours post fertilization) were exposed to sublethal concentrations of cadmium (0.5, 1, 5, and 10 µM) for 96 h. Whole-body cadmium accumulation during this exposure period as quantified using GFAAS (graphite furnace atomic absorption spectroscopy) was found to increase with both exposure length and concentration. Using a transgenic strain of hsp70/eGFP (heat shock protein 70/enhanced green fluorescent protein reporter gene) zebrafish, dose-dependent induction of the heat shock response was observed in the olfactory epithelium. Expression of hsp70/eGFP in the olfactory epithelium was a highly sensitive biomarker for the effects of cadmium in the olfactory system with a lowest observed effects concentration (LOEC) of 0.5 µM Cd. Strong induction of the transgenic reporter gene correlated closely with cell death (LOEC of 5 µM Cd) and histological alterations (LOEC of 1 µM Cd) in the olfactory epithelium of zebrafish larvae following cadmium exposure. Additionally, loss of sensory cilia from the surface of the olfactory epithelium was observed in larvae exposed to 5 and 10 µM Cd. Furthermore, behaviour tests to assess olfactory function revealed sensory deficits, likely due in part to the cadmium-induced degeneration of the olfactory epithelium (p<0.05 for 1 µM; p<0.001 for 5 and 10 µM Cd). <p>To determine if cadmium was entering the cells of the olfactory epithelium by acting as a calcium (Ca) antagonist, zebrafish larvae were co-exposed to 1, 5, or 10 µM Cd with 1 or 5 mM Ca for 96 h. Whole-body cadmium accumulation as quantified using ICP-MS (inductively coupled plasma mass spectrometry) was decreased in larvae co-exposed to cadmium and calcium. Additionally, induction of the heat shock response was reduced in the presence of increasing calcium co-treatment. These ameliorating effects of calcium were further revealed in cell death and histological analyses of the olfactory epithelium. Also, larvae co-exposed to cadmium and calcium exhibited greater olfactory sensory function compared to larvae exposed to cadmium only. Significant increases in aversion response were observed in larvae exposed to 5 µM Cd with 1 and 5 mM Ca (p<0.05). These results indicate that cadmium gains entry to the olfactory epithelium via calcium uptake systems, wherein it causes damage to the olfactory system and can lead to sensory impairment.

L'absorption intestinale de Cd, un métal toxique auquel la population générale est exposée par l'alimentation, a été largement étudiée mais aucune étude n'a considéré l'épithélium intestinal comme organe excréteur de Cd. Lors d'intoxications chroniques, le Cd circulant dans le sang pourrait être transporté à l'intérieur de l'entérocyte du côté basolatéral (BL). L'excrétion du Cd pourrait se faire par efflux du côté apical (AP) de l'entérocyte ou par accumulation du Cd dans l'entérocyte jusqu'à sa mort par apoptose lors du renouvellement de l'épithélium intestinal. L'objectif de notre étude a été de caractériser le transport de Cd du côté BL vers le côté AP en utilisant comme modèle in vitro les cellules TC7 cultivées sur filtres, un clone hautement différencié de la lignée enterocytaire humaine Caco-2. Les hypothèses suivantes ont été testées: 1) un transporteur de cations organiques (OCT1) pourrait transporter le Cd du côté BL; 2) un système de type MDR ou MRP pourrait être impliqué dans l'efflux AP de Cd. Nos résultats démontrent une stimulation de l'accumulation BL à pH 8,5 en milieu chlorure mais pas en milieu nitrate suggérant une implication d'OCT1 dans le transport des CdCIn²¯ⁿ mais pas du Cd²⁺. La cimetidine, un inhibiteur d'OCT1, a diminué de 37 % le transport de ¹⁰⁹Cd après 1h d'exposition à 0,5 µM de traceur. L'efflux AP de 30 min était inhibé de 19 % en présence d'inhibiteurs de MDR1 et MRP2, soient le vérapamil et le probenecid. Nos résultats suggèrent qu'OCT1 à la membrane BL ainsi que MDR1 et MRP2 à la membrane AP sont impliqués dans le transport de Cd dans les cellules TC7. De plus, la présence de Cd non radioactif dans le milieu d'efflux a stimulé l'efflux AP de ¹⁰⁹Cd. Ces résultats suggèrent l'implication d'un mécanisme de contre-échange différent de MDR1 et MRP2 qui sont strictement des mécanismes d'efflux. Notre étude a donc démontré que l'épithélium intestinal, tout comme le foie et les reins, pourrait jouer un rôle dans l'excrétion de Cd lors d'intoxications chroniques. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : Transport membranaire, Cd, OCT1, MRP2, MDR1, Basolatéral, Épithélium intestinal, Caco-2.

L'exposition au cadmium (Cd) représente un facteur de risque pour la perte osseuse et le développement de l'ostéoporose. Jusqu'à récemment, les études portaient principalement sur les effets toxiques indirects du Cd sur l'os via un dysfonctionnement rénal et une perturbation du métabolisme du calcium (Ca). Des données récentes révèlent toutefois qu'une faible exposition chronique au Cd sans effet néphrotoxique entraîne des dommages directs aux os. Le premier objectif de cette étude visait à évaluer dans quelle mesure le Cd est accumulé dans les ostéoblastes et à déterminer sa cytotoxicité. Pour ce faire, nous avons mesuré l'accumulation cellulaire de 109Cd ainsi que la viabilité cellulaire par l'essai MTT. De plus afin de tenter d'identifier les voies d'entrée cellulaire du Cd, nous avons utilisé certains bloqueurs (nifédipine, vérapamil, 2-APB, SKF26365 et capsazépine) et activateurs (Bay-K8644, icilin et thapsigargine) de canaux. Le second objectif était de mieux comprendre comment la cellule ostéoblastique se protège contre les effets toxiques du Cd. Pour y répondre, nous avons étudié l'expression de certains gènes reconnus pour être impliqués dans la protection cellulaire par une technique de RT-PCR. Après avoir exposé les cellules MG-63 au Cd pendant 24h, nous avons obtenu une valeur de CL50 de 15,5 ± 1,1 µM. Nos résultats ont également montré la présence de transport spécifique permettant l'entrée de Cd dans les ostéoblastes MG-63. La spéciation du Cd est déterminante pour son entrée. Nous avons observé une accumulation cellulaire sous forme de Cd2+ et possiblement sous forme de CdCln*2-n. L'observation d'une inhibition de l'entrée de Cd par le Ca nous a mené à étudier les canaux calciques comme voies d'entrée du Cd. Afin de déterminer si les canaux calciques voltage-dépendants (VDCC) constituent une voie d'entrée du Cd dans les ostéoblastes humains, les cellules MG-63 ont été exposées à 0,5 µM 109Cd durant 3 minutes dans différents milieux inorganiques. Le milieu nitrate (dans lequel 80% du Cd total dissous est présent sous forme de Cd2+) favorise l'entrée de Cd par rapport au milieu chlorure (dans lequel 14% du Cd total dissous est présent sous forme de Cd2+), suggérant que le Cd peut pénétrer la cellule sous forme Cd2+. Lorsque la concentration en potassium (K) du milieu d'exposition est augmentée afin d'activer les VDCC, aucune augmentation de l'entrée de Cd n'est observée. L'utilisation de nifédipine et de vérapamil, deux bloqueurs de VDCC, n'a aucun effet sur l'entrée du Cd. Nos résultats n'indiquent donc aucune implication des VDCC dans l'entrée de Cd dans les ostéoblastes MG-63. Les résultats de transport membranaires concordent avec les essais MTT qui montrent l'absence d'effet de la nifédipine, du vérapamil et du Bay-K8644 sur la cytotoxicité du Cd. Les canaux TRPC ne semblent pas non plus être impliqués dans l'entrée de Cd tel que démontré par l'absence d'effet de la thapsigargine sur le transport de Cd utilisée seule ou combinée au SKF96365. Aussi, le SKF96365 ne semble pas modifier les résultats de MTT. L'effet protecteur du 2APB et de la capsazépine observé lors d'essais MTT suggère la participation respective des canaux TRPM7 et TRPM8 dans l'entrée de Cd. De plus, nous avons observé une différence dans la cytotoxicité du Cd (CL50 variant jusqu'à 3 fois) dans les lignées ostéoblastiques humaines MG-63, SaOS-2 et U2OS (respectivement 16 ± 2,30 ± 6 et 55 ± 4 µM). Nos résultats n'indiquent aucune différence dans les niveaux d'accumulation de Cd entre les trois lignées. Nous proposons donc l'hypothèse voulant qu'il existe des différences au niveau des mécanismes de gestion du Cd intracellulaire. Nos résultats préliminaires obtenus par RT-PCR montrent que chacune des lignées expriment les ARNm de MTs et de HSP70. Il est donc possible que l'induction de MTs et de HSP70 diffère entre les lignées cellulaires. En conclusion, le Cd entre dans les ostéoblastes MG-63 de façon spécifique en empruntant une ou des voies calciques, dont possiblement les canaux TRPM7 et TRPM8. Il est possible que la cytotoxicité qui s'en suit contribue aux déséquilibres oméostatiques observés aux niveaux des os. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : cadmium, calcium, spéciation, ostéoblastes, os, cellules MG-63, cellules SaOS-2, cellules U2OS

Cadmium (Cd) is a toxic metal known to accumulate in and have adverse effects on the olfactory systems of fish. The objective of this project was to investigate the effect of cadmium on zebrafish larvae, specifically the effects on the olfactory system at cellular and functional levels. Zebrafish larvae (72 hours post fertilization) were exposed to sublethal concentrations of cadmium (0.5, 1, 5, and 10 µM) for 96 h. Whole-body cadmium accumulation during this exposure period as quantified using GFAAS (graphite furnace atomic absorption spectroscopy) was found to increase with both exposure length and concentration. Using a transgenic strain of hsp70/eGFP (heat shock protein 70/enhanced green fluorescent protein reporter gene) zebrafish, dose-dependent induction of the heat shock response was observed in the olfactory epithelium. Expression of hsp70/eGFP in the olfactory epithelium was a highly sensitive biomarker for the effects of cadmium in the olfactory system with a lowest observed effects concentration (LOEC) of 0.5 µM Cd. Strong induction of the transgenic reporter gene correlated closely with cell death (LOEC of 5 µM Cd) and histological alterations (LOEC of 1 µM Cd) in the olfactory epithelium of zebrafish larvae following cadmium exposure. Additionally, loss of sensory cilia from the surface of the olfactory epithelium was observed in larvae exposed to 5 and 10 µM Cd. Furthermore, behaviour tests to assess olfactory function revealed sensory deficits, likely due in part to the cadmium-induced degeneration of the olfactory epithelium (p<0.05 for 1 µM; p<0.001 for 5 and 10 µM Cd). <p>To determine if cadmium was entering the cells of the olfactory epithelium by acting as a calcium (Ca) antagonist, zebrafish larvae were co-exposed to 1, 5, or 10 µM Cd with 1 or 5 mM Ca for 96 h. Whole-body cadmium accumulation as quantified using ICP-MS (inductively coupled plasma mass spectrometry) was decreased in larvae co-exposed to cadmium and calcium. Additionally, induction of the heat shock response was reduced in the presence of increasing calcium co-treatment. These ameliorating effects of calcium were further revealed in cell death and histological analyses of the olfactory epithelium. Also, larvae co-exposed to cadmium and calcium exhibited greater olfactory sensory function compared to larvae exposed to cadmium only. Significant increases in aversion response were observed in larvae exposed to 5 µM Cd with 1 and 5 mM Ca (p<0.05). These results indicate that cadmium gains entry to the olfactory epithelium via calcium uptake systems, wherein it causes damage to the olfactory system and can lead to sensory impairment.

Cadmium (Cd) is one of the major contaminants in agricultural soil, threatening agricultural production and human health. The objectives of this research work were to understand the tolerance mechanism in rice (Oryza sativa L.) genotype with more Cd-tolerance, and the relation between changes of peroxidases activities and peroxidase gene expression profiles after Cd-treated. For more, we analysis about cis-acting elements in the rice peroxidase genes promoter sequences region, gene structure of rice peroxidase genes and phylogenic relation among 9 peroxidase genes which were blasted from 6 Arabidopsis thaliana Cd-induced based on the peroxidase genes protein sequences. We used 9 upland and 32 varieties rice seeds as materials for germination and the growth of seedlings test with 50 or 500 £gM CdCl2 application, respectively. Rice seeds germination is a complex physiological and biochemical process, and is highly affected by cadmium. The results showed that Cd inhibited both the growth of radicles and coleoptiles. At germination stage, Cd highly inhibited the growth of upland rice. Among these rice varieties, Japonica type cultivars are more tolerant to Cd, but Indica type are more sensitive to Cd. Upland rice cultivars are the most sensitive to Cd. At seedling stage, Cd highly inhibited the growth of roots, but slightly inhibited the growth of shoots. To cope with Cd-induced stresses, plants adopt different strategies and posses a variety of defense mechanisms to prevent themselves from Cd damage. Peroxidase (POXs) is an important antioxidative enzyme for defense responses against Cd oxidative stress. The results suggested that different rice variety has a specific peroxidase gene expression pattern by the pI focusing electrophoresis after Cd-treated, and the peroxidase activities are significant differences when these rice varieties faced Cd stress. In this study, we searched the rice databases (japonica type) and cloned the promoter sequences of the indica type of the rice peroxidase genes, pI 4.5 POX and pI 5.1 POX genes. According to the search results about cis-acting elements from PLACE and PlantCARE databases, these cis-acting elements can be divided into three classes, showing as follow¡G1. Transcriptional related; 2. Light regulated related, and 3. Plant hormones- and stress-related. Based on all reported cis-acting elements, there are many types of transcription factors (TFs) involved in regulation of rice pI 4.5 POX and pI 5.1 POX genes expression. These TFs, which can recognize the specific cis-acting elements to regulate the gene expression, and will be induced by stresses and defense-related plant hormones. We found a cis-acting element (CURECORECR) which response to copper in both rice peroxidase genes promoter regions. There is a number of difference between Japonica type and Indica type peroxidase genes, japonica type peroxidase has more cis-acting elements than indica type. Plant class III peroxidases are present in all land plants. All land plant peroxidase genes are with the same putative ancestor of peroxidase genes and are orthologous genes, but they have specific functions of individual perxidase genes owing to their promoter sequences are very divergent. We used 6 Arabidopsis Cd-related peroxidases protein sequences as a starting point for rice peroxidase datas mining. We found 9 rice peroxidase genes have a closely relation among them. For more details about these rice peroxidase genes, searching each one of these rice peroxidases its gene structure on the Rice Genome Annotation Project (RGAP), comparison and their relation. The expressions of these peroxidase genes are very different among them after Cd treatment, and we also found the same cis-acting element (CURECORECR) which response to copper in all 9 rice peroxidase genes promoter regions. There is a number of difference among them.

Cd significantly inhibited the growth of both rice cultivars. The Tainung 67 cultivar is more tolerant to Cd than Taichung 1 cultivar after 48 h incubation in CdCl2 solution. The Cd tolerant cultivar¡XTainung 67¡¦s PODs in roots might synthesize more lignin in Cd-treatments. Meanwhile, the decrease of H2O2 levels is accompanied with the enhancement of POD activity in Cd-treated tissues. PODs here might also remove excess H2O2, thus serving detoxifying role and synthesizing more lignin for protection. In Taichung 1 cultivar, the accumulation of H2O2 in Cd-treated tissues could be due to the less amount of POD enhancement induced by Cd. In response to Cd treatment, the Taichung 1 cultivar also synthesizes little lignin, and therefore is Cd-sensitive.

In this study we attempt to use phytoremediation techniques to treat the contaminated soil of cadmium. The experiment is divided into two stages. In the first stage, we selected three different species of plants which could tolerate heavy-metals: vetiver (Vetiveria zizanioides), Pteris ensiformis cv. 'Victoriae' according to the past records, and Alternanthera philoxeroides (Mart.) Griseb, which were sampled from the metal contaminated site in Hunei, Kaohsiung county. These three species were planted in three pots with 10, 20 and 30 mg Cd kg-1 in soil respectively. After 9 weeks of the growth, the vetiver was found accumulating the highest Cd and grew better than the other two species. Therefore, we selected the species of vetiver in the second stage of experiment. First, the species of vetiver was planted in the pots with concentrations of 30 and 50 mg Cd kg-1 in soil respectively. Then the pots were put in the greenhouse for incubation. After the test was run for 210 days, we found that the species of vetiver was helpful in the increasing the number of species and amounts of each species of microbe ( total bacteria, fungi and actinomycete ), as well as dehydrogenase activity. Meanwhile, it was effective to decrease the bioavailability of cadmium. In addition, the infection rate of mycorrhizal fungi was increased , which showed that the species of vetiver could resist the cadmium stress in soils and stimulate the soil fertility. Finally, we use molecular biotechniques of PCR-DGGE to observe the microbial diversity in the contaminated soil. We found that the pots with 30 mg Cd kg-1 in soil had more number of bands than the pots with different Cd concentrations in soil, while the pots without vegetation was found more fruitful than vegetated pots. These experimental results indicated that the pots planted with the species of vetiver under this situation would help some special microorganisms to grow, and thus that the microbial diversity was reduced. The results also showed that the pots planted with vetiver with initial cadmium concentrations of 30 and 50 mg Cd kg-1 respectively, in soil exhibited the degradation rate of about 30 percent for both. It was not satisfied to this result in this study. However, the phytoextraction rates of cadmium were measured equal to 7.8 and 8.9 percent, respectively. According to these results, we suggested that the plant, which could hyperaccumulate heavy metals, might be used to increase the removable ability of cadmium in the future.

Cadmium-treated rice seeding (Oryza sativa L. cv. Taichung Native 1 and O. sativa L. cv. Tainung 67) showed inhibition in the growth of rice roots or leaves, and an enhancement in POX activity. In Tainung 67 cultivar, Cd treatment may have influence over cis-regulatory elements in POX promoter region and enhanced transcription of POX or enhance glycosylation of POX. The increase in POX activity induced by Cd might remove excess hydrogen peroxide serving a detoxifying role and synthesizing more lignin for protection. In Taichung Native 1 cultivar, high amounts of H2O2 accumulated in Cd-treated tissues could be due to the less amounts of POX induced by Cd. In response to Cd treatment, the Taichung Native 1 cultivar also synthesizes a little lignin, and is more Cd-sensitive. Therefore, the Tainung 67 cultivar is more tolerant to Cd than Taichung Native 1.