Single extreme low dose/low dose rate irradiation causes alteration in lifespan and genome instability in primary human cells

Abstract

To investigate the long-term biological effect of extreme low dose ionising radiation, we irradiated normal human fibroblasts (HFLIII) with carbon ions (290 MeV u(-1), 70 keV microm(-1)) and gamma-rays at 1 mGy (total dose) once at a low dose rate (1 mGy 6-8 h(-1)), and observed the cell growth kinetics up to 5 months by continuous culturing. The growth of carbon-irradiated cells started to slow down considerably sooner than that of non-irradiated cells before reaching senescence. In contrast, cells irradiated with gamma-rays under similar conditions did not show significant deviation from the non-irradiated cells. A DNA double strand break (DSB) marker, gamma-H2AX foci, and a DSB repair marker, phosphorylated DNA-PKcs foci, increased in number when non-irradiated cells reached several passages before senescence. A single low dose/low dose rate carbon ion exposure further raised the numbers of these markers. Furthermore, the numbers of foci for these two markers were significantly reduced after the cells became fully senescent. Our results indicate that high linear energy transfer (LET) radiation (carbon ions) causes different effects than low LET radiation (gamma-rays) even at very low doses and that a single low dose of heavy ion irradiation can affect the stability of the genome many generations after irradiation.

Figure 1

HFLIII cells were irradiated with 1 mGy carbon ions (290 MeV u⁻¹ original energy, 70 keV μm⁻¹) at 1 mGy 6 h⁻¹ and the cell growth was compared with that of non-irradiated control cells. The numbers in the figure indicate cell passage numbers. Carbon ion irradiation induced accelerated senescence at passage number around 25. (^*P<0.05 compared to non-irradiated control cells by Student's t-test)

Figure 2

HFLIII cells were irradiated with 1 mGy carbon ions (290 MeV u⁻¹, 70 keV μm⁻¹) at 1 mGy 7.3 h⁻¹ and with 1 mGy γ-rays at 1 mGy 6 h⁻¹, and the cell growth was compared with that of non-irradiated control cells. The numbers in the figure indicate cell passage numbers. The cells irradiated with carbon ions senesced earlier than the non-irradiated control cells, while the cells with γ-irradiation showed delayed senescence when compared to control. (^*P<0.05 compared to non-irradiated control cells.) However, cells irradiated with γ-rays were not statistically significant (P=0.16) when compared with non-irradiated control.

Figure 3

(A) Yield of average numbers of γ-H2AX foci per cell as a function of cell passage number after irradiation. The number of foci at presenescence stage (p26) was increased in all the cells and further increased especially in carbon-irradiated cells. In the fully senescent (p30) cells, the number of foci was significantly reduced. (B) Yield of average numbers of phosphorylated DNA-PKcs foci per cell as a function of cell passage number after irradiation. Similar tendencies as in (A) can be observed (P=0.054 between carbon and control foci numbers).

Figure 4

(A) Representative images of γ-H2AX foci and phosphorylated DNA-PKcs (Thr 2609) foci in cells irradiated with carbon ions and γ-rays along with non-irradiated control cells at passage 22. The red and green dots indicate the γ-H2AX foci and phospholyrated DNA-PKcs foci, respectively. (B) Representative images of γ-H2AX foci and phosphorylated DNA-PKcs (Thr 2609) foci in cells irradiated with carbon ions and γ-rays along with non-irradiated control cells at passage 26. The red and green dots indicate the γ-H2AX foci and phosphorylated DNA-PKcs foci, respectively.