This thesis describes research done to investigate the effect of plasma on the integrity of cellular DNA. Nonthermal plasma is a state of matter, which can be described as an ionized gas containing reactive particles such as ions, electrons and radicals. A low power (<200 mW) atmospheric pressure plasma has been used to treat cultures of 3T3 and HCT 116 cells. The plasma is sustained in helium and contains a small admixture of air (<1%). DNA damage has been assessed by analysis of the cell cycle and by looking at the repair mechanism of the cell. The plasma is generated at the tip of a sharp needle. This needle is powered at a radiofrequency of 13.56 MHz. The tip of the needle is located at the end of a Teflon tube through which helium flows at a rate of typically 2 l/min. Temperature measurements show that the plasma is nonthermal and does therefore not thermally damage the cells. Flow cytometry was used to analyze the cell cycle. Arrest in the G2/M phase of the cell cycle was observed, which shows that the plasma is able to induce DNA damage. Furthermore immunofluorescence was utilized to detect Double Strand Breaks (DSBs) in DNA. When DSBs are formed, histone proteins H2AX surrounding the site of the fracture are phosphorylated to H2AX. Recently an antibody for H2AX has been produced. This allows detection of DSBs within minutes after formation. DNA damage induced by the plasma is comparable to damage induced by UV irradiation with an irradiance on the order of 10 mJ/cm2. The amount of damage is linearly dependent on plasma power and increases with duration of treatment. Furthermore the amount of damage increases when the needle is brought close to the sample (<3mm). Measurements on H2O2 concentrations show that the plasma increases these concentrations to physiological damaging levels (0.1 mM). It is hypothesized that the formation of H2O2 work as a sensitizing agent that acts in conjunction with UV to damage the DNA.
|Date of Award||31 Oct 2008|
|Supervisor||Gerrit M.W. Kroesen (Supervisor 1), E. Stoffels - Adamowicz (Supervisor 2) & J.L.V. Broers (External coach)|