Date of Award

1-2014

Document Type

Thesis

Degree Name

Master of Science (MS)

College/School

College of Science and Mathematics

Department/Program

Biology

Thesis Sponsor/Dissertation Chair/Project Chair

Lee H. Lee

Committee Member

Ann Marie DiLorenzo

Committee Member

Sandra Adams

Abstract

The antitoxic effect of selenium on zinc has been evaluated in different levels of living organisms. The growth was observed under various concentrations of zinc chloride as well as zinc chloride (ZnCl2)/Selenium dioxide (SeO2) using direct count and turbidity method while morphology and DNA is studied by performing DAPI stain. In prokaryotic Synechococcus sp. IU 625 (SIU 625), the growth was very similar to the control at the concentrations of lOmg/L, but reduced at 25mg/L while 50mg/L ZnCl2 was lethal dosage and hence inhibited the growth completely. Morphological study indicated that the cells become longer and colorless at high concentration. Y and V shape (curved cells and cells with ectopic poles) DAPI stained DNA and some fragmentations were observed in 25mg/L and 50mg/L ZnCl2 treated SIU 625 at 24hours and day 3. SeO2 is able to reduce the availability of ZnCl2 thus reduces the growth at lOmg/L ZnCl2 which is the concentration that enhance the growth of SIU 625. At higher concentration of ZnCl2 (50mg/L), SeO2 is able to reduce the toxicity of ZnCl2 and the long filament shape was observed at day 3 in SIU 625.The eukaryotic model, Chlamydomonas reinhardtii (C. reinhardtii) was very resistant to ZnCl2 treatment. Lower concentrations (l0mg/L and 25mg/L) did not affect the growth of the cells. At the 50mg/L of ZnCl2, the inhibition of growth was observed suggesting that C. reinhardtii might have stronger heavy metal tolerant mechanism compare to SIU 625 which helps them to combat stress caused by zinc. As the concentration of ZnCl2 increase, some cells appear light green and the dead cells appear dark brown at 12 and 24 hours under higher concentration (25 and 50mg/L ZnCl2). At the concentration of lOmg/L and 25mg/L of ZnCl2 in combination with lmg/L of SeO2, better growth was observed. At higher concentration (50mg/L), no significant effect of SeO2 on the toxicity of ZnCl2 was observed. DNA demargination was also observed in DAPI stained DNA under ZnCl2 and ZnCl2/SeO2 treatment. In mammalian cells, SeO2 increased viability in CHO cells treated with 25mg/L SeO2 significantly (about 25.25%) and with 50mg/L ZnCl2 slightly (about 12.26%). SeO2 increased viability in MRC-5 cells treated with 25mg/L significantly (about 37.85%) and with 50mg/L ZnCl2 about 12.27%. SeO2 (0.125mg/L) with WTC dust at 1.25, 12.5, and 125mg/L increased viability about 3.59%, 26.16% and 67.76% respectively in CHO cells, but not in MRC-5. Apotox Glo Triplex assay result was inconclusive in mammalian cells. SIU 625 was used as a model to do bioinformatics and proteomic study. A membrane topology prediction indicated that four (st, groEL, hmtA and ShmtA) out of five genes (st, groEL, ct, hmtA and ShmtA) selected for proteomics study were membrane proteins. Identification of these genes and expression study using qPCR based assay suggested that three genes (st, groEL, ct) had similar expression pattern and showed increased expression immediately after heavy metal exposure (10 and 25mg/L of ZnCl2) while hmtA and ShmtA did not express immediately. Both hmtA and ShmtA contained similar cysteine rich domain indicating its high affinity to be able to bind heavy metal, but only expressed significantly under lower concentration of ZnCl2 (l0mg/L).

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