The overall goals of this research were to determine the risks of mutation induction and the spectra of mutations induced by energetic protons and iron ions at two loci in human lymphoid cells. During the three year grant period the research has focused in three major areas: (1) the acquisition of sufficient statistics for human TK6 cell mutation experiments using Fe ions (400 MeV/amu), Fe ions (600 MeV/amu) and protons (250 MeV/amu); (2) collection of thymidine kinase- deficient (tk) mutants or hypoxanthine phosphoribosyltransferase deficient (hprt) mutants induced by either Fe 400 MeV/amu, Fe 600 MeV/amu, or H 250 MeV/amu for subsequent molecular analysis; and (3) molecular characterization of mutants isolated after exposure to Fe ions (600 MeV/amu). As a result of the shutdown of the BEVALAC heavy ion accelerator in December 1992, efforts were rearranged somewhat in time to complete our dose-response studies and to complete mutant collections in particular for the Fe ion beams prior to the shutdown. These goals have been achieved. A major effort was placed on collection, re-screening, and archiving of 3 different series of mutants for the various particle beam exposures: tk-ng mutants, tk-sg mutants, and hprt-deficient mutants. Where possible, groups of mutants were isolated for several particle fluences. Comparative analysis of mutation spectra has occured with characterization of the mutation spectrum for hprt-deficient mutants obtained after exposure of TK6 cells to Fe ions (600 MeV/amu) and a series of spontaneous mutants

Kronenberg, Amy

English

NASA Technical Reports Server

NAsA-CR-198S99
Title of Grant -
Type of Report -
//. / tJ /+ ,.--
Mutagenesis in Human Cells with Accelerated H & Fe Ions
Final Summary of Research Progress
/ ,/ /-
!
Principal Investigator - Dr. Amy Kronenberg
Period Covered by Report- May 1991-April 1994
Name and Address of Institition Receiving Grant -
Regents of theUniversity of California
Lawrence Berkeley Laboratory
Mailstop 936A
Berkeley, CA 94720
Contract / Grant Number - T9309R
(NASA-CR-198599) MUTAGENESIS IN
HUMAN CELLS WITH ACCELERATED H ANO
Fe IONS Final Report t May 1991 -
Apr. 199k (LgL) 12 p
N95-28177
Unclas
G3/51 0049940
https://ntrs.nasa.gov/search.jsp?R=19950021756 2020-06-16T08:00:36+00:00Z
Summary
The overall goals of this research were to determine the risks of mutation
induction and the spectra of mutations induced by energetic protons and iron ions
at two loci in human lymphoid cells. During the three year grant period the
research has focused in three major areas: 1) the acquisition of sufficient statistics
for human TK6 cell mutation experiments using Fe ions (400 MeV/amu), Fe ions
(600 MeV/amu) and protons (250 MeV/amu), and 2) collection of thymidine
kinase- deficient (tk) mutants or hypoxanthine phosphoribosyltransferase-deficient
(hprt) mutants induced by either Fe 400 MeV/amu, Fe 600 MeV/amu, or H 250
MeV/amu for subsequent molecular analysis, and 3) molecular characterization of
mutants isolated after exposure to Fe ions (600 MeV/amu). As a result of the
shutdown of the BEVALAC heavy ion accelerator in December 1992, efforts were re-
arranged somewhat in time to complete our dose-response studies and to complete
mutant collections in particular for the Fe ion beams prior to the shutdown. These
goals have been achieved. A major effort was placed on collection, re-screening, and
archiving of 3 different series of mutants for the various particle beam exposures:
tk-ng mutants, tk-sg mutants, and hprt-deficient mutants. Where possible, groups
of mutants were isolated for several particle fluences. Comparative analysis of
mutation spectra has occured with characterization of the mutation spectrum for
hprt-deficient mutants obtained after exposure of TK6 cells to Fe ions (600
MeV/amu) and a series of spontaneous mutants. Molecular analysis of the
remaining tk-deficient or hprt-deficient mutants is underway in the laboratory. In
addition, we have initiated a series of investigations using the WIL2NS cell line, to
examine the effect of differences in DNA damage responsiveness to sparsely
ionizing X-rays on cell killing and mutagenesis by densely ionizing radiations.
Cytotoxicity Results for TK6 Lymphoblasts:
Data are presented for two Fe ion beams (400 MeV/amu and 600 MeV/amu
initial energy), two proton beams (55 MeV/amu and 250 MeV/amu), and for X-rays.
We would note that no difference was observed between the results obtained for 160
kVp x-rays from our present Pantak x-ray generator as compared with results
obtained with a Phillips 100 kVp x-ray generator used for previous studies. The
results reported for the 400 MeV/amu (initial energy) and 600 MeV/amu (initial
energy) beams come from a minimum of 3 replicate dose-response curves exposed
at the BEVALAC heavy ion accelerator at Lawrence Berkeley Laboratory. The data
reported for the 250 MeV proton beam (initial energy) is a compilation of three
replicate dose-response curves exposed at the Loma Linda University Proton
Accelerator. The data reported for the 55 MeV proton beam (initial energy) is
preliminary data from two runs at the 88 inch cyclotron at Lawrence Berkeley
Laboratory.
LET values for BEVALAC beams were determined based on the residual
range of the extracted beam as measured in a water phantom. We can place these
data into context by comparison with results obtained for several additional beams
obtained at the BEVALAC. In certain instances, the LET values were also measured
directly using a compound LET spectrometer. For all charged particle irradiations,
cells were positioned in the plateau region of the Bragg ionization curve. The
results are summarized in Table I. Eachof the dose-response functions for survival
of TK6 cells exposed to charged particle beams is best fitted by a simple exponential
curve with the exception of the Fe 400 MeV/amu beam. A small shoulder is
apparent on this survival curve (intercept = 1.3). This shoulder is most likely
artificial and due to the fact that at the lower doses,not every cell is traversed by an
Fe ion. The DO (terminal slope) is 57 cGy. We can also calculate the inactivation
cross section using the formula 16x LET (keV/_m) x l/D0. The inactivation cross
section for 400 MeV/amu Fe ions is 65.96 _tm2, which is still smaller than the
average cross sectional area of the TK6 cell nucleus (85.5_tm2). Thus, single particle
traversals are not uniformly lethal to these cells.
TABLE I
Radiation LET DO
(keV/_m)
Inactivation Cross Section
Lu_w_2)
X-rays
H 250 MeV / amu
Ne 425 MeV/amu
Si 670 MeV/amu
Si 456 MeV/amu
Si 330 MeV/amu
Ar 470 MeV/amu
Fe 600 MeV/amu
Fe 400 MeV/amu
.... 77 .....
0.4 75 00.09
32 48 10.67
50 47 16.91
61 28 34.86
80 38 33.33
95 34 44.71
190 51 59.60
235 57 65.96
The cytotoxicity of 250 MeV protons is compared with the results obtained for
100 kVp X-rays in Figure 1 below. In all cases, cell were irradiated in suspension
culture in T-25 flasks or in larger T-75 flasks. Dosimetry was performed with a
Victoreen ionization chamber for the appropriate irradiation geometry.
O-F,4
¢D
FIGURE 1
Cytotoxicity of H 250 MeV/amu or 100 kVp X-rays - TK6 cells
.1
.01
k__ :r • X-rays
mtl
I I I I
0 50 100 150 200
Dose in cGy
Results are shown (Figure 2) for cytotoxicity of 55 MeV protons (initial
energy) and 160 kVp x-rays for cells irradiated in Eppendorf tubes. This irradiation
geometry is designed to minimize changes in LET through the sample irradiated
with the proton beam.
FIGURE 2
Cytotoxicity of 55 MeV protons or 160 kVp x-rays - TK6 cells in Eppendorf tubes
_o .1 • []
-¢.
•01 I I i I I I i I
0 50 100 150 200
I
250
[] Proton A
• Proton B
A X-ray C
• X-ray D
• X-ray E
Dose
Additional experiments are planned to extend the present results obtained with 55
MeV protons. Initial results suggest that protons in this energy range do not exhibit
markedly different cytotoxic effects than the 250 MeV protons or X-rays.
Mutation Induction at the hprt and tk loci in TK6 cells
Mutation experiments were carried out with 250 MeV protons at the Loma
Linda University Medical Center, 55 MeV protons at the 88 inch cyclotron at
Lawrence Berkeley Laboratory, and with 600 MeV iron ions and 400 MeV/amu iron
ions at the Bevalac accelerator at Lawrence Berkeley Laboratory.
The results of a comparison between mutation induction by 250 MeV protons
and mutation induction following exposure to 160 kVp X-rays is shown in Figure 3.
FIGURE 3
Comparison of mutation induction by 250MeV protons or x-rays - TK6 cells
O
o1,,4
VQ
X
O
U
,4..a
8
"O
6O F
I. [] Proton hpFl ." 4
A Proton tk-total oO" •
n @ x-ray hprt ,o,* o'_Y
.^ [ • x-ray tk T .*°- .f
4u
r ._.." ./
0 50 100 150
I
200
Dose in cGy
The shape and magnitude of the dose response curves is quite similar for the two
different types of radiations. The data were analyzed by zero-intercept linear
regression and represent the results of a minimum of 3 independent experiments
for each type of radiation. The results are displayed in Table II below. We also
initiated a series of mutation experiments using 55 MeV protons (initial energy)
extracted from the 88 inch cyclotron at Lawrence Berkeley Laboratory. Preliminary
results are presented for the first two experiments using this new beam line. Cells
are irradiated in suspension Costar T-25 flasks positioned so that the 1.7 cm
thickness of the flasks is maximum thickness of water-equivalent material through
which the beam passes. LET will vary through the flask thickness and the range was
verified on film exposures through defined thickneses of polyethylene sheets.
Energy measurements are in process. Absorbed dose is estimated at the center of the
flask position. The results of the first experiments are displayed below in Figure 4
and results of the second experiments are shown in Figure 5.
FIGURE 4
50
v
o
"_ 40
X
O
3o
._ 20
10u
55 MeV protons experiment #1
[]
@
@
• ! I !
0 100 200 300
Dose
• Tk-ng
[] Hprt
• Tk-total
v
o
Ir--I
0
t_
u
8
FIGURE 5
Mutation induction by 55 MeV protons in TK6 cells
60
,0!
0
0
• Tk-ng
0 Hprt
• Tk-total
50
0 0
oI
I , I i I
100 150 200
Dose
!
250
We have quantitated mutations induced at the hprt and tk loci for Fe ions
with an initial energy of 600 MeV/amu (190 keV/Bm), and Fe ions with an initial
energy of 400 MeV/amu (235 keV/Bm) in TK6 cells. The results are compared with
results obtained for x-rays and other several other charged particle beams (Table II).
The results are presented in as a function of dose or as action cross-sections A
graphical representation of the dose-response for Fe ions is shown in Figure 6.
TABLE II
Radiation LET Induced Mutant Action Cross
(keV/l_m) Locus Fraction x (10-7/cGy) Section xl0 "4
100 kVp X-ray
H 250 MeV/amu
Ne 425 MeV/amu
Si 670 MeV/amu
Si 456 MeV / amu
Si 330 MeV/amu
Ar 470 MeV/amu
Fe 600 MeV/amu
Fe 400 MeV/amu
hprt 0.6+0.1 N/A
tk-total 3.0 ± 0.3 N / A
0.4 hprt 0.6 ± 0.1 0.004
tk-total 3.5 + 0.3 0.022
32 hprt 2.5 ± 0.1 1.3
tk-total 4.5 ± 0.2 2.3
50 hprt 2.1 ± 0.1 1.7
tk-total 4.7 ± 0.3 3.8
61 hprt 4.2 +0.4 4.1
tk-total 16.1 0.9 15.8
80 hprt 4.3 ± 0.3 3.4
tk-total 8.2 + 0.5 10.5
95 hprt 3.0 + 0.3 4.60
tk-total 6.5 _ 0.5 9.8
190 hprt 1.2 ± 0.1 3.7
tk-total 2.3 ± 0.3 7.0
235 hprt 0.6 + 0.1 1.5
tk-total 1.1 + 0.1 2.7
o¢arJ
v
o
0
-4:1
U
_kJ
8
HGURE 6
Comparison of Fe-ion-induced mutation in TK6 cells:
600 MeV/amu initial energy or 400 MeV/amu initial energy
30 T s [] Fe600hprt
it A Fe 600 tk-total
25 T/fT • Fe 400 hprt
/ I Fe 400tk-ng20
0 50 I00 150 200
Dose in cGy
The results obtained for TK6 cells exposed to 400 MeV/amu Fe ions were
somewhat surprising as there was no demonstrable dose response for the induction
of slowly growing tk-deficient mutants. Although some tk-sg mutants were
observed in the irradiated cultures, the frequencies were so low as to be
indistinguishable from the background. If we place these results into context, it is
known that the slowly growing tk-deficient mutants appeared to be preferrentially
induced by particles with LET's up to about 61 keV/#m, and that for more densely
ionizing particles the efficiency of induction of the slow growth variants declines
rapidly. The data obtained for this very densely ionizing beam (235 keV/_m)
demonstrates that we cannot reliably detect tk slow-growth variants above the
spontaneous incidence. Further studies on the maximum deletion size associated
with chromosome 17 mutations inclusive of the tk locus may tell us whether the
failure to induce slow growth mutants is due to the placement of essential genes
along chromosome 17 or whether the processing of the initial damage arising after
the most densely ionizing radiation exposures differs from that arising after sparsely
ionizing radiation. We would note that a similar difficulty in recovering slow-
growth tk-deficient mutants has been reported by Metting, et al., (Radiation
Research 1992) in the case of TK6 cells exposed to 212Bi alpha particles
(approximately 60-240 keV/_tm).
In addition, we have carried out preliminary experiments to compare
mutation induction at the hprt locus in TK6 cells with that obtained for WIL2NS
cells (Figure 7). WIL2NS and TK6 cells are derived from the same initial donor
(Levy, et al., 1968). WIL2NS cells are the direct parent of WTK1 cells and differ from
TK6 cells in their ability to carry out recombination. WIL2NS cells are not
heterozygous at the tk locus, and further studies will utilize WTK1 cells for
comparison against TK6 cells.
Figure 7
Hprt mutation yields WIL2 NS and TK6 cells- Fe 600 MeV/amu (190 keV/_tm)
25
A
'9
o
20
X
O
"0 15
lO
5
o
N 0
TK6 hprt
• • • • i m i i • iI !
0 50 100 150
Dose in cGy
Molecular characterization of hprt-deficient mutants isolated after Fe ion exposure
We have completed the analysis of intragenic alterations in a series of hprt-
deficient mutants isolated from cultures exposed to 100 cGy of Fe ions (600
MeV/amu, LET=190 keV/_tm). This exposure level was chosen as it represents a 5-
7X increase over the spontaneous incidence, and we were particularly interested to
compare the effects of particle exposures of the same fluence but different ionization
densities in order to determine whether the nature of the mutational spectrum was
similar or different as a function of LET. We had earlier examined the mutation
spectrum for a series of hprt-deficient mutants isolated after exposures to 50 cGy of
Ar ions (470 Mev/amu, LET=95 keV/_m), and the comparative results are given
below. These two particle exposures were of equal fluence (slightly less than 3
particles/cell nucleus on average). The absorbed dose for the Fe ion exposure is
twice that for the Ar ion exposure as each Fe ion traversal is twice as densely
ionizing as each Ar ion exposure. The results are also compared with results
obtained for our own series of spontaneous hprt-deficient mutants and a larger
series of spontaneous hprt-deficient mutants assembled from the TK6 cell literature.
The level of induction in the case of the Fe ion exposed cells was 1.2 x 10 -5 while for
the Ar ion exposed cells it was 1.5 x 10 -5. Background mutant frequencies ranged
from 2-3 x 10 -6.
TABLE III
SUMMARY OF HPRT MUTANT SPECTRA
Mutant Type Ar ions Fe ions Spontaneous
50 cG_ 100 cGy Mutants
Total deletion 9 32 2 13
Partial deletion
rearrangement 11 30 6 48
No detectable
alteration 20 8 22 99
TOTAL 40 60 301 1602
1 Spontaneous hprt mutants from parallel control cultures.
2. Spontaneous hprt mutants from this series and other published TK6 data
(Gennett and Thilly, 1990; Whaley and Little, 1990).
The pairwise comparisons are summarized in Table IV:
TABLE IV
Statistical Analysis of HPRT mutant spectra
Mutants Compared _2 (2 d.f.) p-value
Ar ions vs. 160 spontaneous 6.84
Fe ions vs. 160 spontaneous 64.17
Ar ions vs. Fe ions 20.17
30 spontaneous vs. 1.47
160 spontaneous
p <0.05
p<0.001
p<0.001
p>0.25
The spectrum of mutants isolated after exposure to the more densely ionizing Fe
ions was strongly biased towards large scale alterations (total gene loss and partial
deletion) and was distinct from the spectra obtained for either Ar ion-induced
mutants or spontaneous mutants. The maximum deletion size tolerated in the
vicinity of the hprt locus may be quite large (up to a few Mb) and it is likely that the
maximum observed deletion size will not be governed by the ionization density of
the incident particle but by the position of essential genes along the X chromosome.
Very large deletion mutations (in excess of 50 Mb) have been isolated along a largely
non-essential human chromosome following exposure of AL cells to a 2X higher
fluence of the same particles in our collaborative studies with Dr. Charles Waldren
funded under the NSCORT in Radiation Health. In that study, the frequency of hprt
mutations was 50-fold lower than mutations for the $1 locus on the non-essential
chromosome. In addition to DNA polymerase 0_ on the short arm of the X
chromosome, there are likely additional essential genes on the long arm of the X
that are required for viability of both human and hamster cells.
Table V summarizes the collection of TK6 derived mutants archived and in
the process of analysis:
Radiation Type
H 250 MeV
Fe 600 Mev/amu
Fe 400 MeV/amu
Locus Doses
TABLE V
Number of Mutants Archived
hprt 150 cGy 90
tk-ng 150 cGy 90
tk-sg 150 cGy 90
hprt 100 cGy 80
tk-ng 50, 100 cGy 61 at 50 cGy, 66 at 100 cGy
tk-sg 100 cGy 39
hprt 150 cGy 78
tk-ng 100, 150 cGy 73 at 100 cGy, 54 at 150 cGy
We have also isolated 68 hprt-deficient mutants of WIL2NS cells for comparative
analysis.
Early disintegration of DNA in TK6 and WIL2NS cells following exposure to Fe
ions
Cell counts in the first several days following exposure of TK6 cells to most
types of ionizing radiations show a dose-dependent lag in growth. This may be a
function of cell cycle delay or cell loss or a combination thereof. In collaboration
with Dr. Bjorn Rydberg and Dr. Priscilla Cooper (under the auspices of the NSCORT
in Radiation Health), we performed one preliminary experiment with irradiated
TK6 and WIL2NS cells with 50 Gy of 600 MeV/amu Fe ions to examine the
rejoining of double strand breaks using quantitative pulsed-field electrophoresis
techniques over the period from 0-8 hours post irradiation. We observed two major
bands in each of the lanes below the position of the wells. These represent limits of
resolution for the pulse-sequence used in the electrophoresis with the lower band
corresponding to molecular weight of about 150kb. In contrast to what Dr. Rydberg
has observed for fibroblasts, there was evidence of degradation of DNA within 1-2
hours to sizes below 150kb (the resolution in this area of the gel is such that these
pieces may be quite small). These changes are consistent with observations of
apoptosis. Apoptosis is a common feature of irradiated lymphoid cells, and has been
associated with the presence of wild-type p53 (Livingstone, et al, 1992).
Literature Cited:
Gennett, I. N. and Thilly, W. G. Mapping large spontaneous deletion endpoints in
the human HPRT gene. Mutation Res. 201, 149-160, 1990.
Livingstone, L. R., White, A., Sprouse, J., Livanos, E., Jacks, T., and Tlsty, T. Altered
cell ccle arrest and gene amplification potential accompany loss of wild-type
p53. Cell 70, 923-935, 1992
Metting, N. F., Payaloor, S. T., Macklis,R. W, Atcher, R. W., Liber, H.L., and
Little, J.B. Induction of mutations by bismuth-212 alpha particles at two
genetic loci in human B-lymphoblasts. Radiation Res. 132, 339-345, 1992.
Whaley, J. M. and Little, J. B. Molecular characterization of hprt mutants induced by
low-and high-LET radiations in human cells. Mutation Res. 243, 35-45, 1990.
Publications associated with this work:
Kronenberg, A. Mutation induction in human lymphoid cells by energetic heavy
ions. Advances in Space Research, 14:339-346 (1994).
Kronenberg, A., Criddle, K., Gauny, S., Vannais, D., Ueno, A. Kraemer, S., Waldren,
C. Comparative analy-sis of mutations induced by densely ionizing particles in
human and hamster/human hybrid cells. In Molecular Mechanisms of Ionizing
Radiation-Induced Mutations, (eds. K. Chadwick and H. Leenhouts),
Vol.EUR15294, Commission of European Communities Press, Luxembourg, pp.
191-196, (1994).
Kraemer, S., Ueno, A., Vannais, D., Hanks, T., Tavakolian, M., Craven, P., Hei, T.,
Kronenberg, A., Waldren, C. Molecular analysis of mutant spectra induced in AL
cells by high and low dose rates of 137Cs gamma rays. In Molecular Mechanisms
of Ionizing Radiation-Induced Mutations, (eds. K.Chadwick and H. Leenhouts),
Vol. EUR15294, Commission of European Communities Press, Luxembourg, pp.
29-34, (1994).
Kronenberg, A., Gauny, S., Criddle, K., Vannais, D., Ueno, A., Kraemer, S. and
Waldren, C.A. Heavy ion mutagenesis: LET effects and genetic linkage. In press
in Radiat. Environ. Biophysics (1995).


Mutagenesis in human cells with accelerated H and Fe ions

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