<p>The present research was designed to investigate the mechanisms of cellular transport, metabolism and toxicity of selenium [inorganic (selenite) and organic (selenomethionine)] in a model teleost, rainbow trout (<i>Oncorhynchus mykiss</i>), using both <i>in vitro</i> and <i>in vivo</i> experimental approaches. The transport properties of selenite and its thiol (glutathione and cysteine) reduced forms were examined in isolated enterocytes and hepatocytes. The kinetics of selenite uptake revealed a linear profile in both cell types, suggesting a low affinity transport process. However, the uptake kinetics was different between the two cell types in the presence of extracellular glutathione, since a concentration-dependent Hill uptake kinetics was recorded in enterocytes, while a linear kinetics persisted in hepatocytes. Both cysteine and glutathione augmented cellular selenium accumulation in these cells. The selenium transport was found to be energy independent, but sensitive to the extracellular pH and inorganic mercury. The pharmacological examination suggested that the cellular transport of selenite is primarily mediated by anion transport systems (e.g., sulphite transporters and/or bicarbonate transporters), although cell-specific differences in transport efficiency was apparent. The metabolism of selenite, selenate and selenomethionine in hepatocytes was examined using X-ray absorption near edge structure spectroscopy (XANES). Inorganic and organic forms of selenium appeared to be metabolized <i>via</i> different cellular pathways, as both selenite and selenate were found to be metabolized into elemental selenium, whereas selenocystine constituted the primary metabolite of selenomethionine. My findings also suggested direct enzymatic transformation of selenomethionine into methylselenol at high exposure level, a process that leads to enhanced intracellular reactive oxygen species generation because of the redox-reactive properties of methylselenol. To validate the metabolite profile of selenium observed in <i>in vitro</i> studies, the tissue-specific differences in selenium metabolism <i>in vivo</i> was analyzed in fish exposed to elevated dietary selenomethionine for two weeks. Similar to the observation in hepatocytes, selenocystine and selenomethionine were found to be the major selenium species across tissues, although there were differences in their relative proportion in different tissues. In addition, a good correlation between the total selenium burden and selenocystine fraction was recorded among all the major tissues except gonads. To understand the role of oxidative stress in cellular toxicity of selenium, isolated trout hepatocytes were exposed to increasing dosage of selenite and selenomethionine over a period of 24h. Selenite was found to be 10 times more toxic than selenomethionine to the hepatocytes. Both selenite and selenomethionine induced rapid generation of reactive oxygen species, which subsequently triggered an upregulation of enzymatic antioxidants. Interestingly, a sharp dose-dependent decrease in intracellular thiol redox (reduced to oxidized glutathione ratio) was recorded with exposure to both selenite and selenomethionine, indicating that glutathione plays an important role in mediating selenium toxicity. At the high exposure dosage, both selenium compounds compromised membrane and DNA integrity, disrupted intracellular calcium homeostasis, and induced enzymatic apoptosis pathway, ultimately leading to cell death <i>via</i> aponecrosis. These findings suggested that high selenium exposure causes cellular toxicity by inducing a rapid loss of the intracellular reducing milieu. Overall, the findings from the present study provided novel information on the transport, metabolism and toxicity of selenium in fish. This fundamental information will be useful in understanding the chemical species-specific toxicity of selenium in fish, and may help in identifying cellular biomarkers for assessing the health of selenium-impacted natural fish populations.

The "n-like layer" is important in multilayer layer amorphous selenium (a-Se) based Xray detector structures because it blocks the injection of holes from the positive electrode. The dark current in these devices is controlled primarily by hole injection,and the introduction of the n-like layer to block hole injection was a key development in the commercialization of a-Se X-ray detectors. An n-like a-Se layer is defined as a layer in which the electron range is much greater than the hole range, ¦Ìe¦Óe >> ¦Ìh¦Óh, where ¦Ó and ¦Ì are the lifetime and drift mobility of the charge carriers and the subscript e and h represent electrons and holes.<p> This thesis examines the effect of doping a-Se with Group II elements (in particular Mg) towards finding a better n-like layer ¨C that with relatively long electron range (drift mobility ¡Á lifetime) , trap limited hole transport and which is stable against crystallization. Conventional Time of Flight (TOF) and Interrupted Field Time of Flight (IFTOF) transient photoconductivity measurements were used to characterize the electron and hole transport in various Group II doped a-Se layers. The dependence of the electron and hole lifetimes and drift mobilities on the composition of the n-like layer was examined. The addition of Group II materials converts the a-Se starting material from p-like into n-like. It was found that increasing the concentration of Mg increases the electron range while limiting the hole range by modifying the population of deep traps. The addition of As further limits the hole transport but does not alter the electron range. The clear reproducibility of the thermal properties obtained from the Differential Scanning Calorimetry (DSC) implies that small amounts of Mg can be used as a suitable n-type dopant.

Graduation date: 1992

<p>The present research was designed to investigate the mechanisms of cellular transport, metabolism and toxicity of selenium [inorganic (selenite) and organic (selenomethionine)] in a model teleost, rainbow trout (<i>Oncorhynchus mykiss</i>), using both <i>in vitro</i> and <i>in vivo</i> experimental approaches. The transport properties of selenite and its thiol (glutathione and cysteine) reduced forms were examined in isolated enterocytes and hepatocytes. The kinetics of selenite uptake revealed a linear profile in both cell types, suggesting a low affinity transport process. However, the uptake kinetics was different between the two cell types in the presence of extracellular glutathione, since a concentration-dependent Hill uptake kinetics was recorded in enterocytes, while a linear kinetics persisted in hepatocytes. Both cysteine and glutathione augmented cellular selenium accumulation in these cells. The selenium transport was found to be energy independent, but sensitive to the extracellular pH and inorganic mercury. The pharmacological examination suggested that the cellular transport of selenite is primarily mediated by anion transport systems (e.g., sulphite transporters and/or bicarbonate transporters), although cell-specific differences in transport efficiency was apparent. The metabolism of selenite, selenate and selenomethionine in hepatocytes was examined using X-ray absorption near edge structure spectroscopy (XANES). Inorganic and organic forms of selenium appeared to be metabolized <i>via</i> different cellular pathways, as both selenite and selenate were found to be metabolized into elemental selenium, whereas selenocystine constituted the primary metabolite of selenomethionine. My findings also suggested direct enzymatic transformation of selenomethionine into methylselenol at high exposure level, a process that leads to enhanced intracellular reactive oxygen species generation because of the redox-reactive properties of methylselenol. To validate the metabolite profile of selenium observed in <i>in vitro</i> studies, the tissue-specific differences in selenium metabolism <i>in vivo</i> was analyzed in fish exposed to elevated dietary selenomethionine for two weeks. Similar to the observation in hepatocytes, selenocystine and selenomethionine were found to be the major selenium species across tissues, although there were differences in their relative proportion in different tissues. In addition, a good correlation between the total selenium burden and selenocystine fraction was recorded among all the major tissues except gonads. To understand the role of oxidative stress in cellular toxicity of selenium, isolated trout hepatocytes were exposed to increasing dosage of selenite and selenomethionine over a period of 24h. Selenite was found to be 10 times more toxic than selenomethionine to the hepatocytes. Both selenite and selenomethionine induced rapid generation of reactive oxygen species, which subsequently triggered an upregulation of enzymatic antioxidants. Interestingly, a sharp dose-dependent decrease in intracellular thiol redox (reduced to oxidized glutathione ratio) was recorded with exposure to both selenite and selenomethionine, indicating that glutathione plays an important role in mediating selenium toxicity. At the high exposure dosage, both selenium compounds compromised membrane and DNA integrity, disrupted intracellular calcium homeostasis, and induced enzymatic apoptosis pathway, ultimately leading to cell death <i>via</i> aponecrosis. These findings suggested that high selenium exposure causes cellular toxicity by inducing a rapid loss of the intracellular reducing milieu. Overall, the findings from the present study provided novel information on the transport, metabolism and toxicity of selenium in fish. This fundamental information will be useful in understanding the chemical species-specific toxicity of selenium in fish, and may help in identifying cellular biomarkers for assessing the health of selenium-impacted natural fish populations.

The electronic properties of amorphous materials are greatly affected by the density of localized states in the mobility gap of these materials. The exact shape of the density of states (DOS) distribution in amorphous selenium (a-Se) is still unresolved despite decades of research. One of the most commonly employed methods to investigate charge transport properties in high resistivity materials is time-of-flight (TOF) transient photoconductivity experiment. The TOF transient photoconductivity technique is used to measure the induced photocurrent in the external circuit when the sample is photoexcited. Information pertaining to carrier mobility and other carrier parameters are deduced from the shape of the photocurrent. The investigation of the charge transport phenomenon is well known to be a complicated task. Monte Carlo (MC) simulation method has become a standard method for carrier transport studies in amorphous materials. The purpose of this research work is to develop a Monte Carlo simulation model for charge transport in typical TOF transient photoconductivity experiment to investigate the DOS distribution in a-Se. The MC simulations were first performed for relatively simpler models for which theoretical and analytical solutions were available. The MC model developed here is based on simulating the drift of carriers resulting from photogeneration, subject to the influence of an applied electric field and multiple trapping events. The free drift time of photocarriers and their dwell time in the traps are stochastic in nature, in accordance with the probabilities of these events. Electron time-of-flight transient photocurrents were calculated in amorphous selenium as a function of the electric field. The distribution of localized states (DOS) in a-Se has been investigated by comparing the experimentally measured and calculated transient photocurrents. The analysis of multiple-trapping transport has been done by the discretization of a continuous DOS. The DOS distribution has been optimized to produce the best agreement between the calculated and measured transient photocurrents. The resulting DOS has distinct features: A first peak at ~0.30 eV below Ec with an amplitude ~1017 eV1 cm3, a second small peak (or shoulder) at 0.450.50 eV below Ec with an amplitude 10141015 eV1 cm3, and deep states with an integral concentration 10111014 cm3 lying below 0.65 eV, whose exact distribution could not be resolved because of the limitations of the available experimental data. The density of states (DOS) distribution in the vicinity of the valence band mobility edge in vacuum coated a-Se films has been investigated by calculating the MC hole transient photocurrents at different temperatures, and also the dependence of the drift mobility on the temperature and field. The calculated TOF transient photocurrents were compared with experimental data published elsewhere. It is shown that, analogous to electron transport in a-Si:H, the DOS near Ev is a featureless, monotonically decreasing distribution in energy up to Ev + 0.4 eV, without the 0.28 eV peak near the valence band which was thought to control the hole drift mobility. Such a DOS was able to account for hole TOF data reported previously by several authors to date.

The "n-like layer" is important in multilayer layer amorphous selenium (a-Se) based Xray detector structures because it blocks the injection of holes from the positive electrode. The dark current in these devices is controlled primarily by hole injection,and the introduction of the n-like layer to block hole injection was a key development in the commercialization of a-Se X-ray detectors. An n-like a-Se layer is defined as a layer in which the electron range is much greater than the hole range, ¦Ìe¦Óe >> ¦Ìh¦Óh, where ¦Ó and ¦Ì are the lifetime and drift mobility of the charge carriers and the subscript e and h represent electrons and holes.<p> This thesis examines the effect of doping a-Se with Group II elements (in particular Mg) towards finding a better n-like layer ¨C that with relatively long electron range (drift mobility ¡Á lifetime) , trap limited hole transport and which is stable against crystallization. Conventional Time of Flight (TOF) and Interrupted Field Time of Flight (IFTOF) transient photoconductivity measurements were used to characterize the electron and hole transport in various Group II doped a-Se layers. The dependence of the electron and hole lifetimes and drift mobilities on the composition of the n-like layer was examined. The addition of Group II materials converts the a-Se starting material from p-like into n-like. It was found that increasing the concentration of Mg increases the electron range while limiting the hole range by modifying the population of deep traps. The addition of As further limits the hole transport but does not alter the electron range. The clear reproducibility of the thermal properties obtained from the Differential Scanning Calorimetry (DSC) implies that small amounts of Mg can be used as a suitable n-type dopant.

An overview of synchrotrons and synchrotron radiation is presented, along with the theory and practical considerations behind several types of X-ray spectroscopy. The theory and practical considerations of density functional theory are also given, with direct reference to some specific software packages.<p> Some synchrotron-excited X-ray spectroscopy measurements and density functional theory calculations of selenium and arsenic-doped selenium films are then outlined. The physical structure of crystalline and amorphous selenium and the electronic structure of amorphous selenium are discussed and comparison is made to the experimental results.<p> A weak feature in the conduction band is identified as a "fingerprint" of the degree of crystallization in amorphous selenium from X-ray absorption measurements. Similarly, a weak feature corresponding to lone-pairs in the valence band is identified as a "fingerprint" of the arsenic concentration from X-ray emission measurements.<p> Finally a detailed model of the structure of amorphous selenium is explained, and compared to experiment. This model is tested both by direct calculations and by a reverse Monte Carlo approach. The implications of this model with respect to the structure of amorphous and arsenic-doped amorphous selenium are discussed. Calculations suggest that simply randomizing the arrangement of "perfect" trigonal selenium is unable to reproduce the measurements of amorphous selenium; a moderate variation in the bond angle of "perfect" trigonal selenium is also necessary.

Chronic arsenic poisoning due to arsenic contamination of groundwater is a serious public health problem in Bangladesh and neighboring countries. Severe health effects associated with chronic exposure to arsenic include melanosis and several kinds of cancer. It is now generally agreed that the arsenic contamination of groundwater in Bangladesh is of geological origin. Arsenic naturally present in aquifers may be mobilized into drinking water by microbial action.<p> The formation of a novel arsenic-selenium compound: seleno-bis (S-glutathionyl) arsinium ion, [(GS)2AsSe]-, and its subsequent excretion in rabbit bile has been demonstrated previously. This molecular basis for the in vivo antagonism between arsenic and selenium was discovered using X-ray absorption spectroscopy. There is growing evidence that, in Bangladeshi people who are suffering long term chronic lowlevel arsenic poisoning, this antagonism is causing a selenium deficiency. Administering selenium supplements might provide a simple but highly effective treatment of the Bangladeshi arsenic poisoning.<p> In order to examine the disposition of [(GS)2AsSe]-, a set of rabbits were intravenously injected with selenite, arsenite or both. Whole blood, red blood cell and plasma samples were collected at different time intervals within 2hrs after injection and cecotrope samples 24hr after injection. Samples were examined using X-ray absorption spectroscopy and both arsenic and selenium K-near edge spectra were recorded.<p> iii Speciation of arsenic and selenium will be discussed in this thesis. Results indicate that [(GS)2AsSe]- is formed in blood very rapidly after injection of both arsenite and selenite, and then is removed from blood stream within 2hrs post injection. Results also show that [(GS)2AsSe]- is assembled in red blood cells, with no [(GS)2AsSe]- detected in plasma samples. [(GS)2AsSe]- is also found in cecotrope samples after injection of both arsenite and selenite.<p> The results of this study in rabbits will contribute to the understanding of chronic arsenic poisoning in humans.

The main objective of this study was to investigate small-bodied fish caging as an approach to evaluate selenium (Se) bioaccumulation and speciation in native fish species inhabiting lakes influenced by uranium (U) milling effluent in northern Saskatchewan, Canada. In contaminated environments freshwater fish show a high propensity to accumulate Se beyond levels needed for normal physiological function. Maternal transfer of elevated Se concentrations to offspring can cause deformities and reduced survival in fry, and in certain cases negatively impact the sustainability of native fish populations. This research included a caging validation study using wild, naïve (i.e., collected from a reference lake) lake chub (Couesius plumbeus) and spottail shiner (Notropis hudsonius), and three field based 21-day caging studies to investigate the dominance of the feeding pathway with respect to Se uptake and speciation in wild populations of northern small-bodied fish exposed to a gradient of Se. Three feeding regimes were used: an in situ benthic diet, a basal Se diet of Chironomus dilutus (1.5 µg Se/g dry weight) and a Se-spiked diet of C. dilutus (5.5 µg Se/g dry weight). Lake chub were identified as more suitable candidates for caging due to higher survival and condition factor at the completion of the in situ 21-day trial. The resulting Se bioaccumulation was compared among treatments as well as to wild small-bodied fish populations from the study area. Results from the caging experiments showed that caged lake chub exposed to natural and controlled diets with elevated Se had significantly greater whole-body Se concentrations after 21 days compared to fish caged in the reference lake. The results also showed that whole-body Se concentrations exceeded conservative Se thresholds, and approached the currently proposed USEPA regulatory threshold (7.91 µg/g dry weight) designed to protect fish species in only three weeks. The use of stable carbon (C), nitrogen (N), and sulphur (S) isotope ratios indicated that alternate benthic food sources native to the exposure lake were consumed in conjunction with the controlled diets. Stable isotope analysis of both wild and caged lake chub indicated that the N and S isotopic signatures decreased with increasing Se exposure, representing differences in isotopic signatures of the food sources. Speciation results from caged lake chub indicated that Se substituted for S in methionine (i.e. selenomethionine) was the dominant Se species found in caged lake chub exposed to dietary sources of elevated Se. Overall, this research demonstrates that using caged native lake chub represents a useful biomonitoring approach to investigate patterns of Se bioaccumulation and speciation in fish.

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