Cellular transport, metabolism and toxicity of selenium in rainbow trout (Oncorhynchus mykiss)
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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 (Oncorhynchus mykiss), using both in vitro and in vivo 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 via 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 in vitro studies, the tissue-specific differences in selenium metabolism in vivo 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 via 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.
DegreeDoctor of Philosophy (Ph.D.)
CommitteeJack Gray; David M. Janz; Derek Peak
Copyright DateJanuary 2011