Determination of incubated activity. Irradiating plasmid DNA in a cell-free system leads to DNA strand breaks. The plasmid formation changed from its native supercoiled configuration to either circular or linear indicating SSB or DSB, respectively. These configurations were quantified following separation by agarose gel electrophoresis. To determine suitable activity to induce plasmid damage, a series of “rangefinder” 67GaCl3 and 111InCl3 activities were tested including 0.1 MBq, 0.5 MBq and 1 MBq on 125 ng of pBR322 plasmid. With 67GaCl3, following 4 hours incubation, the presence of supercoiled plasmid reduced with increasing activity of 67GaCl3 (0.58 ± 0.04, 0.48 ± 0.08 and 0.10 ± 0.12 respectively with 0.1, 0.5 and 1 MBq) (Figure 2-2). Further damage can be seen at 24 hours with supercoiled fraction reduced to 0.36 ± 0.02, 0.06 ± 0.02 and 0.06 ± 0.07 at 0.1, 0.5 and 1 MBq treatment respectively. Supercoiled plasmid in the untreated group also reduced following prolonged treatment from 0.80 ± 0.08 at 4 hours to 0.65 ± 0.01 at 24 hours. At 4 hours of plasmid incubation, the fraction of circular plasmid formed with incubation of 0.5 MBq and 1 MBq of 111InCl3 were 0.37 ± 0.02 and 0.42 ± 0.02, higher (p<0.05) than untreated group at 0.16 ± 0.04. Prolonged incubation (24 hours) increased the fraction of circular plasmid formed at all activities tested (0.34 ± 0.02, 0.61 ± 0.03 and 0.84 ± 0.05 respectively for 0.1, 0.5 and 1 MBq of 111In) while the circular plasmid (SSB) fraction in untreated group remained at 0.17 ± 0.04 (Figure 2-3). It can be seen that between 4 to 24 hours of incubation damage to plasmid was highest with 1 MBq of both 67Ga and 111In with damage from 67Ga treatment is more extensive than 111In. 1 MBq treatment was chosen for subsequent studies as it was deemed as suitable activity to be used in the following experiment. Figure 2-2 Plasmid damage assessment following incubation with 0.1, 0.5 and 1 MBq of 67GaCl3 incubation at 4 and 24 hours. A is the image from gel electrophoresis; B is a bar graph of fraction of supercoiled (i.e. undamaged) plasmid following treatment. (Untreated supercoiled plasmid at 4h and 24h were 0.80 ± 0.08 and 0.65 ± 0.01 respectively) (n=1, run in triplicate, error bar indicates SD) A 4h 24h Fraction of Supercoiled Plasmid DNA B Untreated 0.1 MBq 0.5 MBq 1 MBq * ** **
Determination of incubated activity. Determination of suitable activity to produce a measurable range of observable effects on plasmids over the period of the experiment was necessary in this study. Too low activity would render the effect to be insufficient while too high would produce extensive damage to plasmid (saturation) making comparison impossible. In order to achieve this, a series of activity levels was used starting from 0.1
