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UNCLASSIFIED
ORNL P
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ORNUP-750
CHIEP
CONF-72008
*"STER COPY
DEC 301964
LUTHAL, MUTAGENIC AND CYT XENETIC EFFECTS OF FAST, CHARGED PARTICLES
ON VARIOUS BIOLOGICAL MATERIALS*
G. E. Stapleton
Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee
Presented at Second Symposium on Protection
Against Radiations in Space
Gatlinburg, Tennessee, October 12-14 (1961)

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*Research sponsored by the U.S. Atomic Energy Commission under contract
with the Union Carbide Corporation.
Running head:
Send Proof to: tr. George E. Stapleton
Biology Division
Oak Ridge National Laboratory
P. O. Box Y
Oak Ridge, Tennessee 37831
Members of the Biology Division have participated in two general types
of investigations related to the biological effects of space radiations ·
(1) Attempts to detect the possible eynergistic action of radiations and
other flight parameters such as vibration, and weightlessness by use of
sensitive systems in orbiting or probe vehicles and (2) groundbaseå
investigations of cellular response to high-energy protons and heavy
particles, ranging in energy from 20 to hundreds of Mev. This paper will
report results obtained from the second category of experiments. The
concept that prompted these studies was that the changes in relative
biological effectiveness (RBE) with increasing linear energy transfer (LET)
of the particles might vary with different biological materials as well as
the effects studied. Therefore, several materials were chosen as test
objects, varying from microorganisms to human cells, and lethal, mutagenic
and cytogenetic responses were surveyed.
It was understood that similar work preceded and continued during our
survey, in other laboratories, notably at the Lawrence Radiation Laboratory
in Berkeley, California, and at Yale University. It was hoped that the
results of the many experiments with widely varied test materials and
end-points would fortify each other in helping us to understand this seemingly
complicated interrelationship.
My own work was concerneu with inactivation or lethality and induction
of mutations in the often used bacterium, Escherichia coli..?
The small
size of these cells permits the irradiation of 10% or more cells in a very
thin layer with any of the radiations of interest. Also, these cells respond
to the presence or absence of oxygen in the atmosphere by a several fola
change in their radiosensitivity. This change was studied as a function of
the LET of the various radiations. The ability of the chemical protector
B-mercaptoethylamine to reduce the radiosensitivity was also investigated in
a limited number of irradiations. Mutation frequencies were estimated by
use or a airferent strain of the same species.
The results of Drs. F. J. de Serres and B. B. Webber are basea cn
lethulity and forward mutations at specific loci in the fungus Neuros pora
crassa.? The irradiated samples were asexual spores that contained two
genetically different nuclei. 'This material allows the estimations of the
types of genetic effects displayed by diploid cells. The techniques used
permit the easy identification of all mutations at a specific locus by a
color change from white to purple, therefore tremendous numbers of irradiated
and unirradiated cells can be screened for the mutations induced. Subsequent
genetic analysis with well marked test strains allows the investigator to
classify the genetic alterations ranging from single nucleotide changes to
loss of entire chromosomes.
Dr. E. F. Dakberg's experiments are concerned with Jethal effects on
spermatogonia and oocytes in irradiated mice."
The dynamics of gametogenesis
in mice is now well enough understood to make a study of degeneration of
certuin cell types in the mouse ovaries and testes a reliable and sensitive
measure of radiation damage incurred by wnold body irradiation of these
animals. The technique permits the amount of degeneration produced to be
measured in the absence of any apparent repair by cell replacement. Since
whole animals are irradiated, these experiments could not be perfornied with
the short range protons and heavy-ions.
Dr. M. A Bender's experiments on production of chromosome aberrations
in human cells) are carried out with samples of the blood of the investigator
and as one would guess are "in vitro" experiments. These samples of whole
heparini.zed blood are irradiated, the leukocytes are separated, by
centrifugation, from the rest of the blood elements and cultured for three
days in a tissue culture medium. The cells are stopped from further division,
by addition of colchicine, in their first postirradiation cell division,
fixed, stained and the number of aberrations scored microscopically. Two
easily identifiable types of chromosomal aberrations are scored.
The various radiations used are described in Table 1. The LET's quoted
are taken from the literature and track average values. As Randolph has
suggested the composite average LET for all particles, primaries and secondaries,
should be considered in critical LET calculations. This has not been done
for the data reported here.
The types of dosimetry for the various radiations differea. The X-ray
doses were measured in air with Victoreen "r" meter, with the thimble chambers
placed in the position occupied by the biological sample. These dosimeters
Standards. The wou gamma ray doses were measured by the system described
by Conger et al.': The proton doses were determined either by measurement of
the proton flux incident or the biological sample by activation of thin
copper foils, or by activation of solid organic scintillators, or by
lon-chambers interposed in the beam just upstream of the biological sample.
The dosimetry used for the heavy particle irradiations was that adequately
described by Brustad et al.lu
.
6
When possible, we irradiated the different materials in sequence on
the same day, after the beam had been characterized, and the dosimetry
performed. This was done in an effort to eliminate possible physical errors ,
Figure 1 shows the type of inactivation data obtained for Escherichia
coll B/r. It is clear that survival of this strain is not an exponential
function of dose of X rays or prctons. This organism was chosen because of
this characteristic, to determine if the shape of the curve would change,
as a function of LET as is found for mammalian and other cells.
It is clear
that aerobic cells are equally sensitive to Xizys and protons of the
indicated energies. The protection afforded by anaercbiosis is also similar
for X rays, 130 and 450 Mev protons. The uppermost curve indicates that
B-mercaptoethylamine protects equally well against damage by X rays and
130 lev protons.
Table 2 shows the summary of data available for inactivation and
mutation of E. coli.
The inactivation coefficient for aerobic cells changes
significantly only for the high LET carbon-ions. Anaerobic cells show a
significantly higher RBE for 22 Mev protons and the heavy carbon ions thay
for the other radiations. Brustadº and others have shorn similar data for
Shigella, a closely related bacterium. The data for change in the ratio of
sensitivities in aerobic and anaerobic conditions with radiations of various
LET's are similar also to those of Brustad. The data for mutation frequencies
are far less complete than those for lethality but within experimental error
do not indicate any difference in mutagenetic efficiency of protons of 100
to 750 Mev as compared with X rays.
Table 3 is a compilation of the available data on inactivation and
mutation of Neurospora crassa. The inactivation coefficients are the
reciprocals of the median lethal dose (e-) in kilorads. The RBE's for the
various radiations increase as a function of increased LET as indicated
by the coefficients for 39 Mev helium ions and 101 Mev carbon ions. The
RBE'G estimated for two different types of mutations are shown. They can
be seen to increase likewise over the same range of LET, as does inactivation.
The highest values of RBE, measured with carbon ion irradiation ranged from
6 to 9.
The data obtained with 750 Mev protons indicate an RBE significantly
higher than 1 for the effects studied. It is not clear why the low LET,
750 Mev protons, yielded RBE's significantly greater than 1. The data
obtained with the high LET radiations, however, indicate a very high RBE
for heavy charged particles as high as 6 to 9 for the several effects studied.
Such heavy particles can be produced at low frequency with chese high-energy
protons. Whether secondary particles with very high LET can account for
this result cannot be juiged on the basis of these experiments.
As far as they can be compared the data shown here are in accord with
the recent data on lethal and mutagenic effects of radiations on a diploid
strain of yeast, reported by Mortimer.l.
Dr. Cakberg's investigations of relative biological effectiveness of
different radiations on gametogenesis in the mouse are less complete than
the others as indicated in Table 4. The RBE's for lethali-;' are shown only
for 130 and 750 Mev pratons and for 14.1 Mev neutrons. Although the
confidence intervals for RBE are quite large, the values for. RBE of protons
are not above l as compared with 250 Kvp X rays. It is clear that the values
for 14.) Mev neutrons are significantly greater than 1. Preliminary data
from experiments with fission neutrons indicate an RBE of about 5. The peak
or saturation RBE as a function of LET was not determined in these experiments,
but the available data compare favorably with the data on cultured human
cells of Barendsen2 and Todd, ts which show the initial increase in RB2 in
the LET region of 100-200 Mev cm/gm (10-20 Kev/y of tissue). Dr. Bender's
data on RBE for production of chromosome aberrations on humeun leukocytes
can logically be considered together with Dakberg's.
Bender's data,
sumarizeå in Table 5 Por proton and neutron irradiations include a large
range of LET. The aberration frequencies include deletions as well as rings
and dicentrics.
The frequencies of the former type increase linearly with
dose for low and high LET radiations and are presumed to result from one-hit
events, while the frequencies of the latter type increase approximately with
the square of the dose with low LET radiations and linearly with dose with
high LET radiations.
The protons yield values for RBE not significantly above 1 as was found
for all of the test systems used. This is the case for either type of
aberration scored. With increasing LET above 60 Mev cm/gm the coefficient
of deletion production increases, the RBE increasing to 5 or greater with
the 1 Mev neutrons. It is of some interest that the change in kinetics of
production of two-hit aberrations (rings and dicentrics) occurs in the same
region of LET (~ 100 Mev cm/gm) where overall efficiency in aberration
production of two-hit aberrations per particle is reached, b . A
more meaningful analysis could be made, however, if the complete LET response
were known. Such data can only be obtained with very high LET particles
such as those to be described by Dr. Todd in a later paper in this session.
It is clear from the data presented here for four cellular systems that
each system responds to increasing LET with a change in RBE, and within the
errors of the estimation of RBE and LET it seems the increase occurs within
the same LET range about, 100-200 Mev cm/gm.
This is in good agreement
with the data of Barendsent and Todas and others for lethality in mammalian
cells. No attempt has been made in these studies to assess the effect of
dose rate on the RBE versus LET relationship as has been investigated by
the aforementioned authors.
The preliminary data obtained for inactivation and mutation of
Could
Neurospora conidia might suggest that high LET secondary radiation to be
detectable with this system. We will need to know more about tlie maximum
RBE as a function of LET to make any further statements about this phenomenon.
It is clear from the results discussed that large gaps exist in our
present assessment of the role of LET in relative biological effectiveness
of protons as well as other types of radiation. Although the different
systems show different RBE's with the same radiation, there are reasonable
consistencies among the responses of the various systems. Our data indicate
that the maximum or peak RBE has not been obtaineci in any of the experiments
so far performed. This is in apparent contrast to the data reported by
Conger et al.' for production of chromosomal aberrations in Tradescantia,
which show maximam RBE in the range of LET produced by 1.3 Mev neutrons.
The data are not inconsistent however with those presented by Barendsen
nor with those of Tobias and Toda-3, Brustad, 10 and Mortimer, 12 which show
maximum effectiveness per particle in the range of 2000 Mev cm /gm for a
number of effects on various types of living cells. We hope that more complete
investigations with some of these systems will allow us to make more positive
statements about this complicated interrelationship and ultimately about the
hazards expected from space flights which will involve the encounter with
radiations of the types studied here.
.
10
Footnotes
1. A. Hollaender, G. E. Stapleton and F. L. Martin. Nature 167: 103-104
(1951).
2. A. Hollaender and G. E. Stapleton. Peaceful Uses of Atomic Energy
(1955), Vol. 2. United Nations, N. Y., pp. 206-113.
3. F. j. de Serres and B. B. Webber.
4. E. F. Dakberg and E. Clark. Jour. Cell Comp. Physiol. 58 (Suppl. 1)
173-182 (1962).
5. M. A Bender and P. C. Gooch. Proc. Natl. Acad. Sci. (U.S.) 48: 522-532
(1962).
6. M. L. Randolph. Ann. N. Y. Acad. Sci. 114: (1) 85-95 (1964).
7. A. D. Conger, M. L. Randolph, C. W. Sheppard and H. J. Luippola.
Rad. Res. 2: 525-547 (1958).
(1957). Bi. G. J. Hines and C. L. Brownell. Acad. Press, New York.
Ch. 14, pp. :3-665.
9. W. A. Gibson, G. E. Stapleton and B. BWebber. ORNL-TM 924 (June 30,
196!+).
10.
T. Brustad. Adv. in Biol. Med. Physics 8: .161-220 (1962).
11. R. K. Mortimer, T. Brustad, and D. V. Cormack. Univ. of Calif. Report
UCRL-11387, 35-53 (1964).
12. G. W. Barendsen. Ann. N. Y. Acad. Sci. 114 (1): 96-413 (1964).
13. C. A. Tobias and P. W. Todd. Univ. of Calif. Report UCRL 21387, 25-34
(1964).
Table 1. Radiation Facilities Used
Facility
Radiation
LET
Mev cm²/sm
Track Average
University of California -
184-in. Synchrocyclotron
HIIAC Accelerator
2.50
750-Mev protons
Carbon ions
Helium ions
~ 20000
130
University of Chicago -
170-in. Synchrocyclotron
450-Mev protons
2.50
Harvard University -
260-in. Synchrocyclotron
50-Mev - 100-Mev protons
12 - 70
ORNL - 86-in. Cyclotron
22-Mev protons
2.50
ORNI. - Maxitron 250 X-ray Machine
250-Kvp X rays
~
258
C060 Gamma Source
1.2 - 1.4-Mev photons
~
2.68
ORNL - Health Physics Reactor
Fssion neutrons
~ 3008
ORNL - Cockcroft-Walton Accelerator
14.1-Mev neutrons
2.5-Mev neutrons
~
3008
conger et al. See Ref. 7.
PT. Brustad. See Ref. 10.
CA. C. Birge et al. See Ref. 8.
Table 2. Bacterial Inactivation and Mutation
Mutation Cocfficient
Revertants per Survivor
per Kilorad
Inactivation coefficienta
Ratio
Radiation
Aerobic
Anaerobic
Aer./Anaer.
Proline
Galactose
0.14
0.043
0.15
0.050
10 x 10-10
10 x 10-10
10 x 10-10
12 x 10-10
20 x 10-20
30 x 10-10
20 x 10-10
0.13
750-Mev protons
430- Mev protonso
130-Mev protons
250-Kvp X rays
22-Mev protons
100-Mev carbon ions
0.042
0.125
0.010
20 x 10-20
0.13
0.062
0.11
0.078
The inactivation coefficient is the reciprocal of the et dose (LD-37) determined from the exponential
slope of the survival curves.
The data obtained with the 430-Mev proton bcam. are somewhat less reliable than the others because the
uniform beam area was smaller and the dosimeter system was not cross-calibrated with the other
radiations
Tuble 3. RBE's for Cellular Inactivation and putation in Neuros pora
Mitation
Radiations
Cellular Inactivation
RBE for ad-3A
Mutation
(one-hit)
RBE for ad-3 IR
Mutation
(two-hit)
RBE
750-Mev protons
0.1145
1.77
1.36
1.147
1447-Mev protons
0.0675
1.02
1.00
0.87
1.214
2142-Mev protons
0.0839
2.30
1.30
250 Kvp X ray
0.0648*
1.00
1.00
1.00
39-Mev helium ions
0.105
2.37
1.62
6.1
1.81
4.38
101-Mev carbon ions
0.396
9.10
*Average of inactivation constants from four experincnts is used for 250-Kvp X-ray inactivation
constant.
co y Rays
Table 4. RBE of Protons to X Rays and 14.1-Mev Neutrons to
for Spermatogonial and Cocyte Killing
RBE
Lower 95%
Confidence Limit
Upper 95%
Confidence Limit
Radiation
Cell Type
Point Estimate
Spermatogonia:
14.1-Mev Neutrons*
A
1.76
1.41
2.19
Late A
2.52
2.38
2.76
2.89
2.69
Late A + In
2.11
Spermatogonia:
130-Mev protons
0.28
0.47
0.64
0.70
0.95
Late A
0.41
Late A + In
0.68
1.40
0.27
0.00
Oocytes
0.28
0.73
Spermatononia:
750-Mev protons
A
0.84
1.10
0.77
1.11
Late A
Late A + In
Oocytes
0.64
0.52
0.69
0.20
0.96
1.34
0.66
1.53
*From Oakberg and Clark, 1961
Table 5. Coefficients of Chromosomal Aberration Production
for Proton Irradiation of Human Leukocytes
Radiation
Coefficient of Aberration Production
Deletions Rings and Dicentrics RBE
(103 x) (106 x)
0.6
6.0
0.7
750-Mev protons
450-Mev protons
0.9
1.0
100-Mev protons
0.7
0.8
50-Mev protons
0.4
250-Kvp X rays
6.0 + 0.5
14-Mev neutrons
0.4
0.9 1 0.03
2.3 0.2
2.8 + 0.2
5.0
N
w
2.5-Mev neutrons
1-Mev neutron
5.6
From Y = a + bd; the coefficient 18 b, expressed in aberrations/cell/rad.
From Y = cd"; the coefficient is e, expressed in aberrations/cell/rada.
calculated from deletion coefficient only.
By definition.
For purposes of comparison, these coefficients would be meaningless
because the kinetics of two-hit aberration production change in this
LET range, becoming approximately linear for 2.5 Mev neutrons.
UNCLASSIFIED
ORNL - DWG 64.7792

AMINE
-
-- ANAEROBIC -. te
SURVIVING FRACTION
..-AEROBIC
I X-RAY
V ORNL-22 Mov PROTONS
• HARVARD - 130 MON PROTONS -
CHICAGO -450 Mov PHOTONS ----
CALIFORNIA - 750 Mov PROTONS
20
40
60
RADIATION DOSE (kllorods)
Fig. 3. Surviving Fraction of Escherichia coli B/r as a Function
of Dose (Kilorads) for Protons of Various Energies. Survival curves
are for cells irradiated aerobically, anaerobically, and in the
presence of cysteamine (0.12 M).
...
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DATE FILMED
4 / 13) 65





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