1. Background Information
Investigating the Impact of Oxidative Stress on Tetrahymena thermophile Sirtuin, THD18
Emmanuel C. Dubuisson,New York City College of Technology
Ralph Alcendor PhD, Department of Biological Sciences, New York City College of Technology
Results
Background Information
Summary of Results
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Sirtuins are a family of enzymes that fulfill various important biological functions. Investigators have looked for the implication of Sirtuin genes in cell signaling mechanisms, in the formation and silencing of heterochromatin, in the regulation of ion channels, and in the modulation of the cellular redox reactions[2]. Different model organisms have been previously used to conduct these studies; among them, there are yeasts, nematodes, and fruit flies. Each one has made some valuable contribution into the vast body of knowledge related to this field.
However, gaps in the understanding of Sirtuins functions still remain to be filled. In this optic, the purpose of this experiment is to uncover the impact of oxidative stress on Tetrahymena thermophila Sirtuin, THD18. This protist offers several advantages. In addition, of being widespread, it is easy to culture and to reproduce in the laboratory environment, inexpensive to maintain, and have a short generation time. From a phylogenetic perspective, mammalian Sirtuins share some homology with those found in Tetrahymena. Therefore, results of scientific investigations conducted on Tetrahymena Sirtuins can have direct applications to other fields such as human medicine.
At a molecular level, oxidation reactions in living organisms are catalyzed by enzymes. In human beings for example, enzymes called SIRT3, and SIRT4, which are Sirtuins, participate in oxidative reactions. While SIRT3 protects against oxidative stress by acetylating and activating the Super Oxide Dismutase 2 (SOD2), which is an enzyme found in the mitochondria, SIRT4 on the other hand has the opposite effect, and tends to facilitate the oxidation of fatty acid in the liver and muscle cells[1]. Based on this fact, we hypothesized that the level of mRNA of T. thermophila sirtuin genes will increase in response to high levels of oxidative stress.
To determine the impact of oxidative stress on Tetrahymena thermophila, the cells were cultured, exposed to hydrogen peroxide, and then evaluated for their metabolic activity. mRNA was extracted from the cells using the TRizol protocol. cDNA was synthesized, amplified, and analyzed with a gel electrophoresis. The results show
Hydrogen peroxide induced oxidative stress in T. thermophila (figure1). The induction of oxidative stress was concentration dependent.
Hydrogen peroxide induced cell death. 2 and 4 mM showed the highest levels of cell death (figure 2).
Hydrogen peroxide lead to an increase in the mRNA expression levels of THH13 and THD-190. The highest level was in the presence of 2 mM of hydrogen peroxide. At 4 mM the mRNA levels decreased.
Figure 1. DCF stained cells. Cells exposed to 1 – 4 mM of hydrogen peroxide had high levels of oxidative stress. Green fluorescence indicates presence of oxidative stress.
Conclusion
In conclusion, induction of oxidative stress by hydrogen peroxide lead to high levels of cell death. The expression of sirtuin THD13 and THD-190 increased in the presence of hydrogen peroxide induced oxidative stress. Consistent with the hypothesis, these results suggest sirruins may be involved in oxidative stress protect and or regulation.
Materials and Methods
Cell culture
Cells were exposed to increasing concentration of hydrogen peroxide, for hours at 30 °C with constant shaking.
mRNA Isolation
mRNA was extracted using ml of TRIzol and 0.2 ml chloroform. The aqueous later was extracted and 500 µl of propanol was added and centrifuged. The pellet was washed with 1 ml of 75% ethanol and centrifuged for 5 minutes. The dried pellet was suspended in 100 µl of nuclease free water. Concentration of RNA was determined using a NanoDrop spectrophotometer.
DCF Stain
DCF ( 2',7'-dichlorofluorescein) was added to 200 µl of cells and incubated for 30 min., following which cells were viewed using a fluorescent microscope.
Synthesis of cDNA
This process was done using High Capacity cDNA Reverse Transcription kits. 10 µl of RNA was add to 10 µl of cDNA synthesizing mixture and incubated for 10 at 25 °C, 120 min at 56 ° C and 5 min at 85 ° C.
PCR Reaction
Reverse and forward primers, as well as dNTP and cDNA were combined for this reaction. Two µl of the primer were mixed with 20 µl of buffer (GoTAQ master mix). Samples were incubated at 95 ° C for 3 min., 95 ° C for 30 sec., 56 ° C for 30 sec and 72 ° C for 30 sec. The cycle was repeated 28 times. Final extension was set for 72 ° C for 10 min.
Future Experiments
Repeat the experiments
Examine the effects of hydrogen peroxide on oxidative stress genes and genes involved in cell death
Figure 2. Light microscope images of cells treated with hydrogen peroxide. 2 and 4 mM of hydrogen peroxide showed high number of cell death
Acknowledgement
We are very grateful to the STEM program at City Tech for giving students the possibility to get involved in scientific research at an early stage of their career.
We also extent our gratitude to Professor Alcendor for his mentorship, and for accompanying us along our journey as future scientists
References
[1] Marcus, J. M., & Andrabi, S. A. (2018). SIRT3 Regulation Under Cellular Stress: Making Sense of the Ups and Downs. Frontiers in neuroscience, 12, 799. doi: /fnins
[2] (2014). The controversial world of sirtuins. Drug discovery today. Technologies, 12, e9-e17.
Figure 3. mRNA expression of sirtuin genes. Both sirtuins mRNA increased in the presence of hydrogen peroxide. 18S is a control gene.