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Analysis of Selenium Toxicity on Reduced Thiol Content

Kulkarni, Samatha
http://rave.ohiolink.edu/etdc/view?acc_num=ysu1300301099

Abstract Details

2010, Master of Science in Chemistry, Youngstown State University, Department of Chemistry.
Enterobacter sp. YSU a multimetal resistant strain, grew when exposed to 40 mM selenite by reducing it to elemental selenium and thus detoxifying it. Growth curves established in M-9 minimal medium showed that Enterobacter sp. YSU grew in the presence of 40 mM selenite only when supplemented with L-cysteine and died when exposed to selenite in the absence of L-cysteine. A previous study involving M-9 minimal medium suggested that when selenite concentration was higher than the sulfate concentration selenite could enter the Enterobacter sp. YSU cells through two pathways: a specific pathway and a non specific pathway (sulfate permease channel). The presence of L-cysteine prevents the entrance of selenite through non specific pathway. The actual toxicity mechanism of the selenium oxyanion, selenite (SeO32-), is unknown. To determine if the interaction of selenite with the reduced thiols is involved in the toxicity mechanism and the reason for oxidative stress, the RSH content of Enterobacter sp. YSU was assayed. This strain was grown under sensitive (no L-cysteine) and resistant (presence of L-cysteine) conditions. Selenite was added during the early log-phase. Cells were harvested before and after the exposure to selenite, lysed and reacted with RSH specific derivatizing agent 5, 5' – dithiobis (2-nitrobenzoic acid) for thiol analysis. HPLC was performed for specific quantification of reduced glutathione. The derivative formed represented the level of oxidative stress. Cells were also harvested for Bradford assay to determine the cell protein concentration. It allowed for the RSH content to be normalized to total cell protein. Upon exposure of selenite, the RSH content of the cells grown under sensitive conditions decreased markedly. In the presence of L-cysteine, only a small fraction of RSH content became oxidized. Total protein concentration of the sensitive cells declined as well, as compared to the resistant cells. HPLC analysis showed that there was a decrease in reduced glutathione in sensitive cells. However, when the reduced thiol and glutathione levels were normalized to total cell protein, the results were not significant. This observation was probably caused by the rapid lysis of the sensitive cells in to the culture media. Using the data obtained by measurement of protein both in the cells and in the culture media could be significant.
Jonathan Caguiat, PhD (Advisor)
Nina Stourman, PhD (Committee Member)
Josef Simeonsson, PhD (Committee Member)
113 p.
Analytical Chemistry; Biochemistry; Biology
selenium toxicity; glutathione; thiol content; HPLC analysis

Recommended Citations

Citations

  • Kulkarni, S. (2010). Analysis of Selenium Toxicity on Reduced Thiol Content [Master's thesis, Youngstown State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=ysu1300301099

    APA Style (7th edition)

  • Kulkarni, Samatha. Analysis of Selenium Toxicity on Reduced Thiol Content. 2010. Youngstown State University, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=ysu1300301099.

    MLA Style (8th edition)

  • Kulkarni, Samatha. "Analysis of Selenium Toxicity on Reduced Thiol Content." Master's thesis, Youngstown State University, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=ysu1300301099

    Chicago Manual of Style (17th edition)

ysu1300301099
828
© 2010, all rights reserved.
This open access ETD is published by Youngstown State University and OhioLINK.