This thesis is directed towards the development of the diffusive gradients in thin films (DGT) technique for the measurement of total dissolved As, and for As speciation measurements. In addition, a preliminary investigation of a novel laboratory-based method for measuring labile metal species was carried out; this method involved the coupling of a microcolumn of adsorbent with a standard electrothermal atomic absorption spectrometer. An iron-oxide adsorbent was utilized for As measurements by DGT. The diffusion coefficients of inorganic Asv and AsIII> were measured through the polyacrylamide diffusive gel using both a diffusion cell and DGT devices. A variety of factors that may affect the measurement of total As by DGT were investigated. These factors, which included pH, anions, cations, fulvic acid, FeIII-fulvic acid complexes, and colloidal Fe, may affect the adsorption of the As species to the iron-oxide, or may affect the diffusion coefficients of the individual As species. The DGT method was further developed to selectively accumulate the AsIII species in the presence of Asv. This was achieved by the placement of a negatively charged Nafion membrane at the front of the DGT device which slowed the diffusion of the negatively charged Asv species (H₂AsO₄₂ ⁻) considerably, relative to the uncharged AsIII species (H₃AsO₃). The effect that pH, anions, and cations may have on the selective accumulation of AsIII, in the presence of Asv, was investigated. DGT devices without a Nafion membrane and with a Nafion membrane were deployed in natural waters to determine the total inorganic As and AsIII> concentrations, and to evaluate its performance. A preliminary investigation of the coupling of a microcolumn of Chelex-100 resin with a standard electrothermal atomic absorption spectrometer was undertaken to establish its value as a laboratory-based speciation method. This involved the examination of various microcolumn materials to accommodate the Chelex-100 resin, and finding an appropriate buffer that could be used to buffer the Chelex-100 resin without interfering with the ETAAS measurement. Furthermore, factors that may affect the uptake of metal by the Chelex-100 resin, such as concentration of buffer in solution, ionic strength, and conditioning of the Chelex-100 resin, were investigated.

This thesis is directed towards the development of the diffusive gradients in thin films (DGT) technique for the measurement of total dissolved As, and for As speciation measurements. In addition, a preliminary investigation of a novel laboratory-based method for measuring labile metal species was carried out; this method involved the coupling of a microcolumn of adsorbent with a standard electrothermal atomic absorption spectrometer. An iron-oxide adsorbent was utilized for As measurements by DGT. The diffusion coefficients of inorganic Asv and AsIII> were measured through the polyacrylamide diffusive gel using both a diffusion cell and DGT devices. A variety of factors that may affect the measurement of total As by DGT were investigated. These factors, which included pH, anions, cations, fulvic acid, FeIII-fulvic acid complexes, and colloidal Fe, may affect the adsorption of the As species to the iron-oxide, or may affect the diffusion coefficients of the individual As species. The DGT method was further developed to selectively accumulate the AsIII species in the presence of Asv. This was achieved by the placement of a negatively charged Nafion membrane at the front of the DGT device which slowed the diffusion of the negatively charged Asv species (H₂AsO₄₂ ⁻) considerably, relative to the uncharged AsIII species (H₃AsO₃). The effect that pH, anions, and cations may have on the selective accumulation of AsIII, in the presence of Asv, was investigated. DGT devices without a Nafion membrane and with a Nafion membrane were deployed in natural waters to determine the total inorganic As and AsIII> concentrations, and to evaluate its performance. A preliminary investigation of the coupling of a microcolumn of Chelex-100 resin with a standard electrothermal atomic absorption spectrometer was undertaken to establish its value as a laboratory-based speciation method. This involved the examination of various microcolumn materials to accommodate the Chelex-100 resin, and finding an appropriate buffer that could be used to buffer the Chelex-100 resin without interfering with the ETAAS measurement. Furthermore, factors that may affect the uptake of metal by the Chelex-100 resin, such as concentration of buffer in solution, ionic strength, and conditioning of the Chelex-100 resin, were investigated.

Arsenical-induced carcinogenesis in human bladder has been established through epidemiological evidence, but unfortunately, no mode of action had been determined for this phenomenon. UROtsa cells, a normal, immortalized cell culture model of human urothelium does not form tumors when injected into immuno-compromised mice nor does it have anchorage-independent growth. UROtsa cells were shown to be malignantly transformed following low-level exposure to both arsenite [As(III)] and its more toxic metabolite, monomethylarsonous acid [MMA(III)] providing additional models for studying arsenical-induced carcinogenesis of the bladder. These transformed cell lines allow researchers the ability to investigate the process of urothelial tumorigenesis at multiple time points of arsenical exposure. In the studies discussed here in, environmentally relevant levels of As(III) and MMA(III) were chosen. UROtsa cells were exposed to As(III) and MMA(III) both acutely and chronically to begin investigations into signaling pathway alterations that can lead to carcinogenesis in the human bladder upon exposure to arsenicals. In acute studies, it was shown that As(III) and MMA(III) generate oxidative stress response in UROtsa at low, environmentally relevant levels. The ROS generated by MMA(III) led to an increased 8-oxo-dG formation after 30 min, supporting the importance of MMA(III) in damage caused in the bladder by arsenicals. Because ROS has been linked to MAPK signaling, it was shown that 50 nM MMA(III) and 1 µM As(III) induce MAPK signaling following acute exposures and this increase is dependent on the production of ROS.Next, it was necessary to begin to look at changes that occur during transformation of UROtsa with MMA(III). Chronic exposure to 50 nM MMA(III) constitutively increases the amounts of EGFR, activated Ras, and COX-2 protein in MSC cells. Chronic upregulation of COX-2 in MSC52 cells is due to increased levels of ROS. Phenotypic changes seen in MSC52 cells (hyperproliferation and anchorage independent growth) are dependent on the secondary generation of excess ROS in MSC52 cells. These data clearly present evidence supporting a role for ROS in both acute and chronic toxicities associated with low-level arsenical exposure, and gives evidence that ROS are important in cellular transformation following MMA(III) exposure.

Chromosomally determined arsenate resistance in Escherichia coli is well characterized. Little, however, is known about chromosomally determined arsenite resistance in E. coli. Accordingly, spontaneous arsenite resistant mutants were selected in a plasmid free strain of E. coli. One mutant strain was analyzed in detail, genetically and physiologically. The mutation confering arsenite resistance was shown to be a single gene mutation. Genetic mapping studies using conjugation and transduction showed that the mutation was closely linked to the ilv region of the E. coli map.

Although arsenic is ubiquitous in the environment, certain forms of arsenic are added deliberately in ever increasing quantities. The addition of large quantities of the trivalent inorganic form (arsenite) could pose a serious hazard because of the toxicity of this form explicable in terms of its action of sulfhydryl containing enzymes and/or coenzymes. In the environment B.rsenite is rapidly converted to arsenate (the pentavalent inorganic form). Since a biological explanation for the oxidation of arsenite to arsenate was available only for cattle dips (a unique environment), this study was undertaken to examine sewage for the presence of arsenite oxidizing bacteria. The isolation of bacteria capable of arsenite oxidation would allow a study of cultural and physiological properties and thus provide information useful in making rational decisions regarding the continued use of inorganic arsenicals as pesticides; as well as elucidating one segment of the arsenic cycle. Thirty-four different strains of arsenite oxidizing pseudomonads were isolated from sewage and classified into two major groups, Pseudomonas fluorescens-arsenoxydans and Pseudomonas acidovorans-arsenoxydans, with three and four ecotypes in each group. One particularily rapid arsenite oxidizing strain (designated Pseudomonas acidovorans-arsenoxydans YE56) was extensively studied. A minimal, defined medium was used to study the relationship of temperature, pH, aeration and nutrition to growth of this organism versus the ability of the organism to convert arsenite to arsenate under the same conditions. The conversion of arsenite to arsenate in YE56 was shown to be due to the appearance at stationary phase of an enzyme and/or component of the electron transport system when the organism is grown in chemically defined medium. In contrast, induction of the arsenite oxidizing system occurs in late exponential growth phase when the organism is grown in a rich medium. Thus, even though cell populations may never reach stationary phase in sewage, the ability of organisms to oxidize arsenite is probably constant. No energy could be detected from the oxidation of arsenite as measured by increase in growth rate or total cell yield. The adaptability of this organism to the changing environmental conditions of a sewage plant helps explain the findings that pseudomonads are the predominant species in sewage. A simple, unambiguous method for the separation of arsenite, arsenate and phosphate, using a combination of thin layer chromatography and paper chromatography, is described which makes it possible for investigators to define arsenic determinations to include the form (organic or inorganic), oxidation state (trivalent or pentavalent) and the quantity present in the samples.

As aquaporinas são proteínas de membrana presentes em quase todos os órgãos e tecidos de animais e plantas, onde desempenham funções que vão além do transporte de água, transportando também moléculas como ureia, ácido bórico, ácido silícico, amônia, dióxido de carbono e arsênio. Em plantas, as aquaporinas podem ser classificadas de acordo com suas sequências de aminoácidos em cinco subfamílias: proteínas intrínsecas da membrana plasmática (PIPs), proteínas intrínsecas de tonoplastos (TIPs), proteínas intrínsecas do tipo nodulina 26 (NIPs), proteínas intrínsecas pequenas (SIPs) e proteínas intrínsecas não caracterizadas (XIPs). Dados genômicos determinam o número de genes de aquaporinas em 33 para arroz, 35 para Arabidopsis, 71 para algodão e 66 para soja. Dentre as principais culturas utilizadas como alimento, o arroz é particularmente eficiente no acúmulo do semimetal altamente tóxico e carcinogênico arsênio (As), representando um risco significativo para a saúde humana Assim, o principal objetivo deste trabalho é elucidar o papel das aquaporinas no transporte de As em arroz. Utilizando cultivares que apresentam susceptibilidade diferencial ao arsênio, foi analisada a expressão dos genes de aquaporinas em resposta ao tratamento com arsenito em diferentes condições. Para a caracterização dos genes de aquaporinas diferencialmente expressos, foram realizados ensaios de complementação funcional em leveduras. Nossos resultados indicam que membros das subfamílias NIP, TIP, PIP e SIP podem estar envolvidos no transporte e metabolismo de As em arroz, dentre estes, quatro podem estar envolvidos no transporte de As para dentro da célula e seis membros podem estar envolvidos no transporte de As para os vacúolos, fazendo com que essas proteínas sejam candidatas a estratégias de melhoramento genético e fitorremediação. / Aquaporins are membrane proteins present in almost all organs and tissues of animals and plants, where they perform functions that go beyond water transport, also transporting molecules such as urea, boric acid, silicic acid, ammonia, carbon dioxide and arsenic. In plants, aquaporins can be classified according to their ami-no acid sequences into five subfamilies: plasma membrane intrinsic proteins (PIPs), tonoplast intrinsic proteins (TIPs), nodulin-26 like intrinsic proteins (NIPs), small intrinsic proteins (SIPs) and uncharacterized intrinsic proteins (XIPs). Genomic data set the number of aquaporin genes in 33 for rice, 35 for Arabidopsis, 71 for cotton and 66 for soybean. Among the main crops used as food, rice is particularly effi-cient in the accumulation of the highly toxic and carcinogenic metalloid arsenic, thus representing a significant risk to human health. Therefore, the main goal of this work is to elucidate the role of aquaporins in the transport of arsenic in rice. Using cultivars with differential susceptibility to arsenic, the expression of aquaporin genes in response to the arsenite treatment under different conditions was ana-lyzed. For the characterization of differentially expressed aquaporin genes, func-tional complementation assays were performed in yeast cells. Our results indicate that members of the subfamilies NIP, TIP, PIP and SIP may be involved in the transport and metabolism of arsenic in rice, of these, four may be involved in the transport of As into the cell and six members may be involved in transporting As to the vacuoles, making these proteins candidates to genetic improvement strategies and phytoremediation.

As aquaporinas são proteínas de membrana presentes em quase todos os órgãos e tecidos de animais e plantas, onde desempenham funções que vão além do transporte de água, transportando também moléculas como ureia, ácido bórico, ácido silícico, amônia, dióxido de carbono e arsênio. Em plantas, as aquaporinas podem ser classificadas de acordo com suas sequências de aminoácidos em cinco subfamílias: proteínas intrínsecas da membrana plasmática (PIPs), proteínas intrínsecas de tonoplastos (TIPs), proteínas intrínsecas do tipo nodulina 26 (NIPs), proteínas intrínsecas pequenas (SIPs) e proteínas intrínsecas não caracterizadas (XIPs). Dados genômicos determinam o número de genes de aquaporinas em 33 para arroz, 35 para Arabidopsis, 71 para algodão e 66 para soja. Dentre as principais culturas utilizadas como alimento, o arroz é particularmente eficiente no acúmulo do semimetal altamente tóxico e carcinogênico arsênio (As), representando um risco significativo para a saúde humana Assim, o principal objetivo deste trabalho é elucidar o papel das aquaporinas no transporte de As em arroz. Utilizando cultivares que apresentam susceptibilidade diferencial ao arsênio, foi analisada a expressão dos genes de aquaporinas em resposta ao tratamento com arsenito em diferentes condições. Para a caracterização dos genes de aquaporinas diferencialmente expressos, foram realizados ensaios de complementação funcional em leveduras. Nossos resultados indicam que membros das subfamílias NIP, TIP, PIP e SIP podem estar envolvidos no transporte e metabolismo de As em arroz, dentre estes, quatro podem estar envolvidos no transporte de As para dentro da célula e seis membros podem estar envolvidos no transporte de As para os vacúolos, fazendo com que essas proteínas sejam candidatas a estratégias de melhoramento genético e fitorremediação. / Aquaporins are membrane proteins present in almost all organs and tissues of animals and plants, where they perform functions that go beyond water transport, also transporting molecules such as urea, boric acid, silicic acid, ammonia, carbon dioxide and arsenic. In plants, aquaporins can be classified according to their ami-no acid sequences into five subfamilies: plasma membrane intrinsic proteins (PIPs), tonoplast intrinsic proteins (TIPs), nodulin-26 like intrinsic proteins (NIPs), small intrinsic proteins (SIPs) and uncharacterized intrinsic proteins (XIPs). Genomic data set the number of aquaporin genes in 33 for rice, 35 for Arabidopsis, 71 for cotton and 66 for soybean. Among the main crops used as food, rice is particularly effi-cient in the accumulation of the highly toxic and carcinogenic metalloid arsenic, thus representing a significant risk to human health. Therefore, the main goal of this work is to elucidate the role of aquaporins in the transport of arsenic in rice. Using cultivars with differential susceptibility to arsenic, the expression of aquaporin genes in response to the arsenite treatment under different conditions was ana-lyzed. For the characterization of differentially expressed aquaporin genes, func-tional complementation assays were performed in yeast cells. Our results indicate that members of the subfamilies NIP, TIP, PIP and SIP may be involved in the transport and metabolism of arsenic in rice, of these, four may be involved in the transport of As into the cell and six members may be involved in transporting As to the vacuoles, making these proteins candidates to genetic improvement strategies and phytoremediation.

As aquaporinas são proteínas de membrana presentes em quase todos os órgãos e tecidos de animais e plantas, onde desempenham funções que vão além do transporte de água, transportando também moléculas como ureia, ácido bórico, ácido silícico, amônia, dióxido de carbono e arsênio. Em plantas, as aquaporinas podem ser classificadas de acordo com suas sequências de aminoácidos em cinco subfamílias: proteínas intrínsecas da membrana plasmática (PIPs), proteínas intrínsecas de tonoplastos (TIPs), proteínas intrínsecas do tipo nodulina 26 (NIPs), proteínas intrínsecas pequenas (SIPs) e proteínas intrínsecas não caracterizadas (XIPs). Dados genômicos determinam o número de genes de aquaporinas em 33 para arroz, 35 para Arabidopsis, 71 para algodão e 66 para soja. Dentre as principais culturas utilizadas como alimento, o arroz é particularmente eficiente no acúmulo do semimetal altamente tóxico e carcinogênico arsênio (As), representando um risco significativo para a saúde humana Assim, o principal objetivo deste trabalho é elucidar o papel das aquaporinas no transporte de As em arroz. Utilizando cultivares que apresentam susceptibilidade diferencial ao arsênio, foi analisada a expressão dos genes de aquaporinas em resposta ao tratamento com arsenito em diferentes condições. Para a caracterização dos genes de aquaporinas diferencialmente expressos, foram realizados ensaios de complementação funcional em leveduras. Nossos resultados indicam que membros das subfamílias NIP, TIP, PIP e SIP podem estar envolvidos no transporte e metabolismo de As em arroz, dentre estes, quatro podem estar envolvidos no transporte de As para dentro da célula e seis membros podem estar envolvidos no transporte de As para os vacúolos, fazendo com que essas proteínas sejam candidatas a estratégias de melhoramento genético e fitorremediação. / Aquaporins are membrane proteins present in almost all organs and tissues of animals and plants, where they perform functions that go beyond water transport, also transporting molecules such as urea, boric acid, silicic acid, ammonia, carbon dioxide and arsenic. In plants, aquaporins can be classified according to their ami-no acid sequences into five subfamilies: plasma membrane intrinsic proteins (PIPs), tonoplast intrinsic proteins (TIPs), nodulin-26 like intrinsic proteins (NIPs), small intrinsic proteins (SIPs) and uncharacterized intrinsic proteins (XIPs). Genomic data set the number of aquaporin genes in 33 for rice, 35 for Arabidopsis, 71 for cotton and 66 for soybean. Among the main crops used as food, rice is particularly effi-cient in the accumulation of the highly toxic and carcinogenic metalloid arsenic, thus representing a significant risk to human health. Therefore, the main goal of this work is to elucidate the role of aquaporins in the transport of arsenic in rice. Using cultivars with differential susceptibility to arsenic, the expression of aquaporin genes in response to the arsenite treatment under different conditions was ana-lyzed. For the characterization of differentially expressed aquaporin genes, func-tional complementation assays were performed in yeast cells. Our results indicate that members of the subfamilies NIP, TIP, PIP and SIP may be involved in the transport and metabolism of arsenic in rice, of these, four may be involved in the transport of As into the cell and six members may be involved in transporting As to the vacuoles, making these proteins candidates to genetic improvement strategies and phytoremediation.

Bacterial strains resistant to arsenite, arsenate (or both) are readily isolated from nature. The resistance, in many cases, can be shown to be determined by conjugal plasmids. The mechanisms by which these plasmids confer resistance to arsenic compounds are entirely unknown. This study was undertaken to shed light on some of the physiological and genetic aspects of this resistance using plasmid-bearing strains of Escherichia coli which are highly resistant to both arsenite and arsenate. Most studies were carried out using E. coli J53 (R773) or the plasmid-free strain J53.

Arsenic contamination in drinking water has been a cause of serious concerns across the United States as well as throughout the world. Over 70 million people in Eastern India, Bangladesh, Vietnam, Taiwan, and Northern China have been victims of arsenic poisoning. The USEPA has classified arsenic as a Class A carcinogen and recently reduced the Maximum Contaminant Level (MCL) in drinking water from 50ppb to 10ppb. The deadline for all the water utilities to meet this level is 23rd January 2006. To meet those drinking water standards, small water utilities need low cost and effective arsenic removal techniques. Natural zeolites such as Chabazite are excellent sorbents for several metallic and radioactive cations. Modifying the zeolite structure can effectively enhance the adsorption capacities of these zeolites for removal of heavy metals. The present work investigates the adsorption capacities of Cuprous and Ferrous treated Chabazite for removal of arsenic. This investigation is a part of a broader project directed at developing an effective pretreatment process that uses modified Chabazite in conjugation with Microfiltration (MF) or Ultrafiltration (UF) for removal of organic and inorganic contaminants. The goal of this research is to determine how well Cuprous and Ferrous treated Chabazite sorbs arsenic in its trivalent and pentavalent state. The other objectives of this research are to examine which modification of the chabazite has the higher removal efficiency of arsenic. This study will also compare arsenic adsorption on the modified zeolites in response to competitive adsorption of various anions present in natural source waters such as sulfates, hydroxides, and chlorides. The potential benefit of this study is to find the most effective treatment of for removal of arsenic species from aqueous solutions. This investigation may provide small water utilities, with a cost effective way for removal of arsenic and thus meet the recommended new regulatory maximum contaminant level (MCL).