JAEA-Review-2010-065.pdf:15.99MB - 日本原子力研究開発機構

3-02
Mutagenic effects of He ion particles in Escherichia coli
T. Suzaka a, b) , H. Tauchi a) , N. Shikazono b) , K. Fujii b) and A. Yokoya b)
a) Department of Environmental Sciences, Faculty of Science,
b) Advanced Science Research Center, JAEA
Non-DSB type of clustered damage is induced by
ionizing radiation and is proposed to cause deleterious
1)
effects to cells . Their yields and configurations likely
depend on the radiation qualities, such as particle energy or
ionization density along the particle track characterized by
1,2)
LET (linear energy transfer) . To date, the biological
consequences of radiation of both low and high LETs have
been extensively studied in various organisms, including
bacteria, yeasts, higher plants, and mammalian cells. It is,
however, still not clear on the features of the induced DNA
damage in cells as well as how and to what extent the
damage is processed and thus related to biological
consequences, especially to mutation. In this project, we
aim to find out the significance of clustering of DNA
damage in mutagenesis. Here, we present the results of
lacZ reversion assay of wild type cells after irradiation of
soft X-rays and He ion particles with different LETs.
The cells of CC104 (wild type E. coli) were incubated in
0.2% glucose minimal medium at 37 C unless otherwise
8
stated. An order of 10 of cells were filtered through a
nitrocellulose membrane of a diameter of 1.3 cm. To
prevent cells from drying, the membrane was placed on a
filter paper pre-wetted with 300 μL of 0.15 M NaCl solution
with 20% glycerol. Subsequently, the membrane with the
filter paper was placed on 0.15 M NaCl agar in a Petri dish,
and then the dish was covered with a polyimide film
(Kapton film) of 7.5 m thickness. Cells on the membrane
were irradiated with soft X-rays (tungsten target, tube
voltage 150 kV, tube current 6 mA) at a dose rate of
0.47 Gy/s, and He ion particles at dose rates of 1 ~ 4 Gy/s.
LETs of He ions were altered by placing a Ni foil in front of
the sample. After irradiation, recovered cells were plated
and incubated overnight. The fraction of survived cells
was determined by dividing the number of survived cells by
that of unirradiated cells. In this study, the reverse
mutation frequency of lacZ was measured. G to T
transversion in the lacZ gene reverts the cells so that they
could grow on a medium with lactose as the only carbon
3)
source . In determining the mutation frequency, irradiated
cells were initially propagated in 0.2% glucose minimal
medium at 37 °C for 16 hours to fix mutations. Cells were
plated on a 0.2% lactose/glucose minimal medium, and were
incubated at 37 °C for 40 hours. The reverse mutation
frequency was determined by dividing the number of
colonies on the lactose plate by that on the glucose plate.
Lethal and mutagenic effects of radiations with different
qualities were assessed from dose response curves. Lethal
effects of soft X-rays and He ion particles at LETs of 18 and
89 keV/m did not show significant differences, indicating
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that the lethality of E.coli is largely independent on the LET
(data not shown). The result suggests that the amount of
unreparable lethal damage is similar after irradiation of
different qualities of radiation in wild type E.coli cells.
Dose response curves of mutation induction, however,
differed significantly for each type of radiation (Fig. 1).
Unexpectedly, the mutation frequencies increased and then
leveled off with increasing dose for all types of radiation.
The amount of dose to show the leveling off seemed to
depend on the LET and the dose appeared to get smaller
with increasing LET (Fig. 1). We do not have a clear
explanation for these dose responses of mutation induction
in E.coli at present, but similar dose response curves that
show leveling offs at higher doses are also found in yeasts
4, 5)
and in fungi . Studies with another target gene (lacI
gene) for mutation induction are now underway.
References
1) N. Shikazono et al., J. Radiat. Res. 50 (2009) 27.
2) H. Nikjoo et al., Radiat. Res. 156 (2001) 577.
3) C.G. Cupples and J.H. Miller, Proc. Natl. Acad. Sci.
USA 86 (1989) 9163.
4) Y. Matsuo et al., Mutat. Res. 602 (2006) 7.
5) Y. Toyoshima et al., JAEA Takasaki Ann. Rep. 2006
(2008) 93.
Mutation frequency
(number of mutants per 10 8 cells)
10
8
6
4
2
0
0 100 200 300 400 500 600
Dose(Gy)
Fig. 1 Reverse mutation frequencies of wild type E. coli
(CC104) at the lacZ gene. Cells were exposed to soft
X-rays (○), He ions with an LET of 18 keV/m (●),
and 89 keV/m (△).