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| Undergraduate Research Abstract |
Interaction of Heavy Metals and Thiol- Reducing Agents and May Anne Fernandez, Liu Lab, Rutgers UniversityThe primary goal of Dr. Alice Liu’s laboratory in Nelson Biological Laboratories is to clarify the mechanism of the stress response via activation of heat shock transcriptional response and induction of heat shock proteins (Hsps) in mammalian cells. This particular topic is of important relevance because of the role of Heat Shock Proteins (Hsps) as molecular chaperones in regulating cellular homeostasis and promoting survival, including apoptosis during severe stress.Initially, my particular research involved the effect of arsenic (As) as a strong inducer of the heat shock transcriptional response and the possibility that this pathway may have a role in the carcinogenic activity of arsenites. Arsenic is found to increase production of Hsps, thus making the cell stronger and more resilient against stress. However, there is lack of an appropriate experimental animal model and no consensus in the exact mechanism of action of arsenic [1]. My research this semester was extended to include other heavy metals as well, such as mercury (Hg) and cadmium (Cd), since the heat shock response is activated in response to heavy metals as stress inducers. Since ions of heavy metals such as mercury and arsenic have high affinities for thiol groups, thiol reducing agents were utilized such as dithiothreitol (DTT), glutathione (GSH), and N-Acetyl– L- Cysteine (NAC). Protein labeling to examine protein synthesis in cells, Hsp-70 reporter gene activity, and soft agar assay were conducted to quantify the effect of metals on cells alone and the effect of DTT, NAC, and GSH in As, Cd, or Hg exposed cells. [35S] methionine labeling of protein synthesis in cells and HSP-70 reporter gene assay were first conducted in neuroblastoma cell lines, then extended to JB6 cells, 3T3 cells, and NG108 cells. 96- well plate was used with cells exposed to various conditions such as control with no heavy metal exposure, heat shock alone, arsenic/mercury/cadmium alone at various concentrations (from 0 – 200 μM), or thiol reducing agents plus heavy metal. Bright Glo luciferase reagent system was utilized to assay the gene expression and used to determine the amount of heat shock proteins. PE Victor 2 plate reader quantified the luminescence produced by the reporter transfected in the cell in order to detect reporter gene expression. Dose response curves were then graphed based on the luminescence exhibited by the reporter gene. Results showed a sharp dose response curve for arsenic, indicating an abrupt decrease in cellular protein synthesis once the peak was reached at a particular As concentration. This was consistent with previous evidence emphasizing arsenic as an inducer of cell death. A shift in the dose response curve to higher As concentrations was observed for GSH ad NAC, supporting the hypothesis that thiol reducing reagents inhibited the effect of arsenite on protein synthesis [2]. Other results showed flat and short peaks for DTT and higher ones for GSH in the arsenic dose response curve. This indicated a greater inhibition produced by DTT in comparison to GSH. Furthermore, since DTT was a dithiol and GSH was a monothiol, it was expected that DTT should have a greater inhibitory effect, and results were consistent with previous studies [3]. Currently, studies are underway extending arsenic studies to other heavy metals such as mercury and cadmium. By investigating the effects of metal ions not only with regard to the heat shock response but with cell toxicity, prevention of the detrimental effects of metals in the body would be greatly improvised.
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last updated Aug 24, 2005 |
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