LIFE01200604005 Shri Somnath Ghosh - Homi Bhabha National ...

CHAPTER 3
RESULTS
in all the cells. The average numbers of foci after 15min of 2Gy gamma irradiation were 24±5,
while after 2Gy oxygen irradiation; the foci were 28±4.7 (Fig. 3.3.2.2 A & C). Thus unlike the
cell survival, the difference in induction of γ-H2AX foci 15 min after 2Gy of either radiation was
not as profound. Activation of signaling molecules at dose of 2Gy of gamma radiation was
studied because the initial response manifested as double strand breaks, as indicated by γ-H2AX
foci, was found to be almost the same at 2Gy of either radiation. Since the RBE of oxygen ions
was nearly 3, it would be more logical to compare 2Gy of oxygen ion with 6Gy of gamma ray.
The average numbers of foci after 15min of 6Gy gamma irradiation were 35±3.5 (Fig. 3.3.2.2 A
& C). Thus, 6Gy gamma ray induced 1.25 times more foci than 2Gy oxygen ions.
The γ-H2AX foci were also counted 4 hours after irradiation to compare the repair status at that
time. 53% of 2Gy gamma irradiated cells still showed γ-H2AX foci but the average foci per cell
had reduced to only 4.3±2.2, where as 100% of 6Gy gamma irradiated cells showed γ-H2AX
foci and the average foci per cell had reduced to only 8±2.5. In oxygen ion irradiated cells,
although bright green γ-H2AX intensity was observed till 4 hours in 60% of cells, there was a
significant reduction in appearance of distinct foci like structures (10±2) (Fig. 3.3.2.2 B & C).
Disappearance of γ-H2AX foci indicates DNA repair/rejoining. However, from the survival data
of A549 cells it was clear that in oxygen irradiated cells the repair of DNA had not taken place.
The result was so intriguing that it was essential to measure the total intensity of γ-H2AX in
irradiated cells. Instead of counting number of foci, the intensity of γ-H2AX in cells showing the
foci was estimated by ImageJ software in the same images. In case of 2Gy or 6Gy gamma
irradiation, both γ-H2AX foci and its total intensity levels had reduced at 4 hours (Fig. 3.3.2.2
D). However, in case of oxygen irradiation, the total intensity of γ-H2AX at 4 hours was yet very
high (Fig. 3.3.2.2D) although foci were not properly visible. This could indicate diffusion of γ-
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