[CANCER RESEARCH 26 Part 1, 2045-2052,September I966|
The Molecular Basis for Radiation
FRANKLIN
HUTCHINSON
Department of Molecular Biophysics,
Yale University, New Haven, Connecticut
Summary
The reasons are pointed out for the generally accepted con
clusion that radiation inactivation of cells happens as the result
of only a few events, or even a single event, at the molecular
level. Nine reasons are then given for thinking that DXA is the
radiosensitive target. From the known mechanisms of radiation
damage to DNA, the inactivation of single-stranded and doublestranded viruses can be at least partly understood. A simple
hypothesis is that ionizing radiation acts by either breaking the
DNA molecule physically in 2, thus interfering with its con
tinuity, or by destroying bases on both complementary strands.
If these ideas are applied to the radiation inactivation of bac
terial or mammalian cells, it is difficult to see why they are so
radioresistant, even making allowance for the operation of
repair systems.
In my talk today I want to summarize our present under
standing of the molecular events by which high energy radiations
affect living cells. I will treat only the main features. Even so, it
will be necessary to limit the field if I am to project any kind of
a clear picture in the time at my disposal. I shall talk mainly of
the effects of ionizing radiations, although on occasion I will de
scribe results ol experiments with ultraviolet irradiation where
this supplies insights to an understanding of the events taking
Iliace with ionizing radiations. And, furthermore, I shall concen
trate my attention on the immediate physical and chemical
events which take place within a fraction of a second after the
absorption of the ionizing radiation. The long, complex bio
chemical changes which take place as a result of these primary
events will be ignored except in 1 or 2 specific instances.
Inactivation
Level
Effects on Cells'
Caused
by a Few Events
at the Molecular
First, let us discuss the general nature of the way in which
ionizing radiation acts on living cells. It has been accepted for
many years that ionizing radiation affects cells mainly through a
small number of primary events, perhaps in some cases only a
single event (20). This may be contrasted, for example, with the
possibility that ionizing radiation produces a generalized cell
poison.
A 1st reason for believing that ionizing radiation acts through
a relatively small number of events has to do with the manner
in which radiation energy is dissipated in matter. For incident
X-rays or 7-rays the absorbed energy sets in motion fast electrons
within the material. These fast electrons then lose their energy
1The writing of this paper was supported in part by contract
AT(30-1)-2053 from the U. S. Atomic Energy Commission.
by further interactions with the target atoms, releasing on the
average the order of 50 or 100 e. v. per interaction (28), at spacings ranging from thousands of A for very fast particles, to only
a few A apart for relatively slow ones. If charged particle beams,
such as fast electrons or a-particles, are used as the source of
irradiation, the same process takes place directly.
The energy lost in each energy loss event, 50-100 e. v., or the
order of 2000 kcal/mole, is so large that a considerable number
of chemical bonds are almost inevitably broken in the immediate
vicinity of the event. From such a picture, it is very easy to see
the origin of the "point heat" theory of Dessauer (7) for the
biologic action of ionizing radiation.
It is easy to show that if we are to ascribe biologic inactivation
to a single high energy event produced randomly within the
irradiated cell, a plot of the logarithm of the number of cells
surviving the irradiation versus the radiation dose should be a
straight line. Chart 1 shows schematically the types of survival
curves that are actually found on irradiating either bacterial cells
or mammalian cells. The straight line corresponds to the ex
ponential survival predicted for inactivation by a single event.
The "sigmoidal" curve, so called because it produces a sigmoidal
curve when the survival is plotted linearly against the dose, can
readily be ascribed to a situation in which 2 or more events must
take place before the cell is inactivated. Typical experimental
survival curves correspond to the requirement that between 2
and 6 events are necessary to produce inactivation. We shall see
later that the interpretation of the "shoulder" on the sigmoidal
survival curve can also be ascribed to possible repair and degradative systems within a cell. However, it is clear that the over-all
shape of the survival curves fits well with the idea that only a
few primary events are required to inactivate a cell. In particular
the straight exponential part of the sigmoidal curve at high
doses appears to have only 1 simple explanation. A cell (which
possibly may have accummulated a certain amount of sublethal
radiation damage) is, nevertheless, still capable of going through
its reproductive cycle until finally the production of a final high
energy event in the cell causes it to lose its reproductive ability.
A 3rd reason for believing that radiation inactivation is caused
by a few key events is the relative insensitivity of most survival
curves to the rate at which the radiation dose is delivered. When
intracellular systems are irradiated in vitro, or when cells are
irradiated under conditions in which they are not metabolizing,
the survival curves are nearly always essentially independent of
dose rate. When cells are metabolizing, it is true that there are
dose rate effects. However, the effect of changing the dose rate is
far less than would he expected on the theory of a radiationproduced cell poison, for example. In those cases which have been
carefully investigated it usually turns out that the dose rate
effects can be accounted for by repair or dcgradative mechanisms,
as I shall discuss later.
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f f CHART 1. The 2 typical types of survival curves for single cells
irradiated with ionizing radiations.
It is comforting to know that in certain special cases an effect
of a generalized radiation-produced poison such as hydrogen
peroxide produced on the irradiation of water can be identified,
and can readily be separated from the primary effects under
discussion here.
The Radiosensitive
Macromolecule
in Cells
We are now in the position to ask the question: what are the
radiosensitive macromolecules in living cells? Over the course of
years a number of different lines of evidence have built up that
this sensitive site is the cell DNA. I would like now to discuss
the major lines of evidence that the DNA is, in fact, the radio
sensitive material in the cell.
1. It is well established in most cells that the function of the
cell which is most sensitive to ionizing radiation is that of repro
duction. Cells which never reproduce themselves, such as nerve
cells, are usually almost totally unaffected by doses which will
stop reproduction in other cells. Since DNA is closely concerned
with the replicative function in cells, this is one connection
between radiosensitivity and DNA.
2. It has been clearly established that in cell culture the
irradiation of the nucleus readily inhibits cell reproduction,
whereas enormous doses of the cytoplasm seem to have relatively
little effect. This has been most clearly shown by the use of a
proton microbeam by Zirkle and Bloom (42). The same phenom
enon appears to be shown by insect eggs which have their
nuclei located close to one wall of the egg (4, 4a, 39) and fern
spores (41). Some results on marine organisms do not give as
clear-cut a picture, but do not give clear and unequivocal evidence
for another conclusion, either.
2046
3. In a number of cells, it is well established that a change of
ploidy will change the radiation sensitivity. In Chart 2 is shown
an interesting example of this in yeast cells, where the ploidy
may be changed from 1 to 6 by appropriate genetic technics (23).
Although it is usually not simple to predict whether increasing
the ploidy will increase or decrease the radiation sensitivity, it is
nonetheless usual for the radiation sensitivity to change.
4. In a more precise way, Sparrow and his collaborators have
shown a remarkable correlation between chromosome volume
and cell radiosensitivity in a number of plants (34). As shown in
Chart 3, the doses needed to produce a given biologic end effect
for a number of species with widely different nuclear sizes and
numbers of chromosomes fall on a common line when plotted
against chromosome volume. The line shown in Chart 3 is a least
squares fit, showing the slope of —0.93.If the slope were —1, it
would indicate that the radiosensitivity depends simply on the
volume per chromosome, and that the energy necessary to
produce a given radiobiologic effect would be characterized by a
fixed amount of energy deposited per chromosome.
The preceding reasons primarily concern a correlation between
radiosensitivity and the genetic apparatus in general. We can
now turn to the reasons for implicating DNA in particular as
the radiosensitive molecule.
5. Over the past few years molecular biologists have built up a
very detailed picture of the function of DNA, particularly in
bacterial systems. According to this picture, DNA stores the
information necessary to produce a new cell in the sequence of
the nucleotides along the double helical chains. It is clear that
X-rays can damage these bases and thus destroy information
which, as far as we know at present, is stored in no other way.
Thus, we would certainly expect that an effect of ionizing
radiation on DNA might alter the ability of a cell to duplicate
itself.
6. Very significant correlations have been pointed out by
Terzi (38) and by Kaplan and Moses (18) between radiosensi
tivity, the quantity of DNA, and the organization of the genetic
100%
DOSE, krad
CHART 2. Survival curves for related strains of yeast cells of
different ploidy. Strain 320 is diploid, 323 is tetrapolid, and 362 is
hexaploid. The haploid strain shows an exponential survival with
a slope about twice as steep as the hexaploid strain. Reproduced
from Mortimer (23).
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Radiation Effects on Cells
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ESTIMATED
INTERPHASE
CHROMOSOME
VOLUME (cu)
(AVERAGE NUCLEAR
VOLUME/CHROMOSOME
NUMBER)
CHART3. Relation between interphase chromosome volume and acute lethal exposure for 16 plant species. (Reproduced from A. H.
Sparrow, L. A. Schairer, and R. C. Sparrow, Science, 141: 163-66, 1963.)
apparatus. Chart 4 is from the paper of Kaplan and Moses. It is
seen that on the basis of radiosensitivity the organisms fall into 4
distinct classes. Within each class the sensitivity increases
approximately linearly with the DNA content. The most radio
sensitive class is that of viruses having single-stranded nucleic
acid as the carrier of genetic information. The next most radio
sensitive class is that of viruses having double-stranded DNA.
Since the radiosensitivity of these viruses can be greatly varied
by irradiation in solutions of different kinds, the radiosensitivities
which have been taken here are those in which the indirect effect
of radiation-produced radicals from the solution has been sup
pressed as completely as possible. The next class is that of
haploid microorganisms, and the most radioresistant class is
that of diploid cells, including mammalian cells.
7. The radiosensitivity of bacterial cells may be altered in a
number of ways—by using different kinds of ionizing radiation,
by varying the oxygen concentration, or by adding various
protective chemical compounds such as cysteamine. If DNA is
the radiosensitive material, its radiosensitivity must vary in the
same way.
It is known that the DNA of certain microorganisms can enter
into the process known as genetic transformation (29). That is
to say, DNA from certain strains of organisms containing,
perhaps, a mutation for resistance to a certain drug, possesses
the ability to transfer this mutation to the genome of a closely
related strain. This effect is known to be mediated by DNA
only (29). The transformation property can be destroyed by the
action of ionizing radiation. This gives one a biologic assay for a
certain kind of genetic competence of the DNA.
Transforming DNA can be irradiated within the cells, then
extracted and checked for transforming ability. An example of
what happens when different kinds of ionizing radiation are
used is shown in Chart 5. In this figure is plotted the relative
biologic efficiency (RBE) as a function of that property of the
radiation known as the linear energy transfer (LET). By LET
is meant the mean rate at which the charged particles created in
the tissue lose energy. If they lose energy at a very low rate, as is
the case for the electrons set in motion by high energy 7-rays,
the primary energy loss events in the tissue are far apart. The
common point on the graph refers to the radiation sensitivity
normalized to that for -y-rays. If the rate at which energy is
transmitted to the medium is increased, by going to a-particle
radiation for example, the average spacing between the primary
energy loss events gets smaller and smaller. For nearly all
biologic molecules which have been studied (27), the efficiency
of the radiation decreases as the spacing between events de
creases, as shown for the case of 4>X174 virus (31). This decrease
has a very simple explanation. As the primary events get closer
and closer together, there is a greater and greater chance of 2
or more events occurring within the same molecule. This is
wasting some of the absorbed energy, and there is a lower
efficiency for producing the biologic effect.
The sensitivity of typical biologic cells shows quite a different
trend with increasing LET, as shown for a haploid microorganism
(30) and a diploid mammalian cell (6). The significant thing is
that the transforming activity of DNA shows a very similar
curve (15). There is no other molecule known, except DNA,
which gives such a shape of curve.
It can also be shown that when the oxygen concentration is
lowered from the normal value to 0, the radiosensitivity of
transforming DNA decreases by a factor of about 3, in exactly
the same way that the radiosensitivity of bacterial cells changes
(17). Similarly, if the radioprotective drug cysteamine is added,
the radiosensitivity of the transforming DNA is decreased in a
very similar fashion to the radiation sensitivity of the ability of
the cell to replicate (17). Finally, the dry vegetative cells of
Bacillus subtilis are about 4 times as radiosensitive as are the dry
spores of the same organism. If the transforming activities of
DNA irradiated in the dry vegetative cells and in spores are
compared, the transforming activity is again found to be 4 times
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Franklin Hutchinson
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CHART4. A plot of DNA content versus the dose needed to reduce survival to 37% (Dav) for: single-stranded RNA and DNA viruses
(solid squares); double-stranded DNA viruses (solid circles); haploid bacteria and yeast cells (hatched circles); mammalian, avian, and
diploid yeast cells (hatched squares). The lines are for constant G-value, the lowest line representing a G-value ~1. (Reproduced from
Kaplan and Moses (18).)
as sensitive in the dry cells as in the spores (37). There is thus a
very close parallel between the radiosensitivity of transforming
DNA irradiated in cells and of cell radiosensitivity.
8. The compound bromouracil is very similar to thymine, one
of the naturally occurring bases in DNA. The only difference is
that a bromine atom is substituted for a methyl group on the
5-position of the pyrimidine ring. There are methods for in
corporating bromouracil instead of thymine in the DNA of a
variety of cells. Such a substitution leads to an increase of 2 or 3
in the radiosensitivity of these cells for ionizing radiation. This
increase has been shown in viruses (35), in bacterial cells (19),
and with mammalian cells in tissue culture (8). Furthermore, in
bacterial systems it has been demonstrated that the bromouracil
must actually be incorporated in the DNA to produce radio2048
sensitivity, and not merely present in the cell during the irradia
tion (2). And, most convincingly, it has been demonstrated
that transforming DXA containing bromouracil and irradiated
within the cell has its radiation sensitivity increased by the
same ratio as is the radiation sensitivity of the cell. This was
first shown by Szybalski and collaborators (26) in B. subtilis,
and has later been confirmed by us, in pneumococcus cells (16).
This again supplies very convincing evidence that DNA forms
the principal radiation-sensitive target in cells.
9. It has been shown that pretreatment of cells with ultraviolet
light can change the X-ray sensitivity of some cells (13). Since
the effectiveness of ultraviolet of different wave lengths varies in
close accord with the absoiption spectrum of nucleic acid (32),
this provides still another link between radiation action and the
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Radiation Effects on Cells
100
1000
10,000
LET, MEV(sqcm/gm)
CHART5. The variation of the relative biologic efficiency (RBE)
with linear energy transfer (LET). The data for HeLa cells is
from Deering and Rice (6); for diploid yeast cells, from Schambra
(30); for*X-174 virus, from Schambra and Hutchinson (31); for
transforming DNA, from Hutchinson (15). All data for all systems
was obtained using the same irradiation apparatus, and the same
dosimetry, except for HeLa cells. In this case, somewhat different
dosimetrj' was used, because the doses were so very much lower.
The common experimental arrangement precludes the possibility
of dosimetrie errors with the different radiations used.
DNA molecule. The most convincing argument is based on the
effect of photoreactivation of the ultraviolet damage. If light of
wave length 3500-4000 A is given to bacterial cells after irradia
tion with ultraviolet light, a large fraction of the ultraviolet
damage can be restored (13). It is well established in specific
cases that this photorestoration involves the breakage of thymine-thymine dimer formed in the DXA by the action of ultra
violet light (40). Such treatment, whose only known effect is the
removal of these thymine dimers in DNA, substantially changes
the sensitivity of such cells for a following X-ray dose. Thus,
since the radiation sensitivity appears to depend among other
things on the number of thymine dimers existing in the DNA,
DNA is implicated as the radiation-sensitive target in cells.
This is an impressive list of reasons for ascribing the radiation
sensitivity of cells to damage to the DNA within these cells.
There are many other experiments which implicate DNA as the
radiation-sensitive target, but the ones that I have given here
are those which seem most convincing to me.
At this point we must ask: What evidence is there to implicate
other macromolecules, or other cellular structures, as the radia
tion-sensitive target? I suspect it is safe to say that at some time
or another any type of molecule in the cell and every subcellular
structure has been suggested as the site of radiobiologic action.
In so far as I am aware, there seem at present to be only 3 struc
tures which might be seriously considered in this connection.
These are the chromosomes in larger cells, specifically including
the protein as well as the DXA of the structure, messenger RNA,
and the cell membrane. Let us deal with the case of the chromo
somes first.
The 1st 4 points listed above would apply equally to the
entire chromosomal structure, the protein as well as the DNA,
as the radiation-sensitive target. For diploid cells with large
chromosomes at least, one could not rule out the possibility that
much of the chromosomal protein represents a radiosensitive
target as well as the DNA. Unfortunately, much of the data
implicating DNA itself has been for bacterial systems only.
However, the radiosensitization produced by bromouracil and
the complex course of cell survival with varying LET have been
determined in mammalian cells, and imply that effects on the
surrounding protein are not very important. Conversely, there
as yet seem to be no clear data which would show that the
protein component of chromosomes is involved. Thus, the entire
chromosome as the radiation-sensitive target is not ruled out, but
is not supported by very convincing evidence.
The destruction of messenger RNA, particularly any long-lived
messenger RNA which might possibly exist in highly differenti
ated cells, might also be important. Not much can yet be said
about this possibility.
From an o priori point of view, the cell membrane represents
a very reasonable target for the action of ionizing radiation.
Aside from the information-carrying DNA molecule, it is the
one other major cell component, except for messenger RNA,
where a single localized event could well be imagined to do a
consideratile amount of damage. Furthermore, the membranes
contain a large lipid component, and lipids are the 1 biologicclass of molecules in which chain reactions arc known to occur
(22), even though the reports of such chain reactions within
living cells are presently viewed with suspicion.
A specific theory of cell membranes as the radiosensitive
element has been discussed at some detail by Haeq and Alexander
(1). In their version, radiation damage to membranes is assumed
to release various degradative enzymes within the cell. It is true
that extensive degradation of DNA is found in many cases after
irradiation (36), but a simpler interpretation would involve steps
in the operation of repair mechanisms, as we will discuss in a
moment. The relationship to repair mechanisms seems much more
realistic, since extensive degradation of cell components other
than DNA does not seem to occur, an objection to the enzyme
release hypothesis. Furthermore, the effects of massive doses of
radiation on such readily measured properties of cell membranes
as permeability to a variety of molecules is really surprisingly
small (1). At the moment, I believe that the kindest judgment
that can be made about the hypothesis that cell membranes are
the site of the primary radiation lesion is the verdict "not proven."
The identification of the radiosensitive target is confused by
the fact that both repair and degradative mechanisms act to
affect the radiosensitivity of the cell. These mechanisms can be
demonstrated in 2 ways. Firstly, over-all cell survival can be
shown to be greatly affected by treatment after irradiation (14).
Secondly, changes in survival occur if the dose rate is varied
over a large range (14), or if the dose is fractionated into 2 or
more fractions with a long time between fractions (9).
Although it is clear that repair mechanisms must be very
important for X-irradiation, the 1 well-established repair system
is for damage from ultraviolet light. Setlow and Carrier (33) have
showed that for bacteria which have received lethal doses of
ultraviolet light, there is an enzyme mechanism which excises the
radiation-produced thymine dimers from the DNA, and then
resynthesizes the single-strand which was affected, presumably
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Franklin Hutchinson
using the complementary strand of DNA as the template. This
work has been completely confirmed and extended by Boyce and
Howard-Flanders (5) and by Pettijohn and Hanawalt (26a). A
certain amount of progress has been made in demonstrating
changes in DNA synthesis after treatment with X-rays (3) and
with nitrogen mustards (12, 21). However, this work is still
fragmentary, and there is no definite evidence yet for repair of
DNA after X-irradiation. Evidence for the repair of X-rayinduced single strand breaks was reported by R. A. McGrath
and P. W. Williams at the Biophysical Society meeting, Febru
ary 1966, and has been confirmed by H. S. Kaplan (personal
communication).
As has been mentioned before, extensive degradation of DXA
following X-irradiation of bacterial systems has been demon
strated (36). The simplest explanation of this degradation would
be that it represents the action of an enzyme similar to the one
that excises the thymine dimers in the ultraviolet repair mecha
nism discovered by Setlow and Carrier, but this is a working
hypothesis only.
The "noise" introduced by repair and degradative mechanisms
hinders the identification of the radiosensitive target to a very
considerable degree. There is, however, the possibility of corre
lating the effect of these mechanisms on cell survival and on
particular cellular constituents to confirm the identification of
the primary radiation target.
Mechanisms
of Maeromolecular
Damage
We now need to consider the damage to DNA in irradiated
cells.
First consider what happens when we irradiate a sample
composed of molecules of a single kind. The primary loss events
created by the ionizing radiation release the order of 50 or 100
e. v. in a small localized region, perhaps 10 or 20 A in size. A
useful concept here is the number of molecules damaged by a
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10°
WEIGHT
CHART6. The G-value (molecules destroyed per 100 e.v. ab
sorbed) for a number of different molecules. Most of the low molec
ular weight data is taken from Hart andPlatzman (Ila); most of
the high molecular weight data from Pollard el al. (27). The low
G-value at low molecular weight is that for terphenyl (Ila), a
substance consisting of linked aromatic rings. The low G-value at
high molecular weight is that for the DNA of bacteriophage Tl
(31). The cross symbol at a molecular weight of about 200 includes
data for several dipeptides (27).
2050
given dose of radiation. The radiation chemist has adopted a
unit useful for this purpose, called the G-value (Ila). The G-value
is defined, in the context in which we are interested here, as the
number of molecules damaged per 100 e. v. of energy dissipated
in the substance. Since 100 e. v. is the order of magnitude of the
energy released in an average primary loss, the G-value can be
thought of in crude terms as measuring roughly the number of
molecules destroyed per primary event.
Chart 6 shows some G-values for the irradiation of a number of
pure materials ranging from water to the nucleic acids. Over a
very wide range of molecular weight the G-value for most mole
cules falls between the general limits of 1 and 10. Furthermore,
there is a general trend from rather higher G-values at the low
molecular weights to a G-value approaching unity at the highest
molecular weights. The reason can be seen in terms of a somewhat
oversimplified picture. A primary event in a material of low
molecular weight stands a reasonable chance of damaging
chemical bonds in several different molecules. As the size of the
molecule increases, there is a greater and greater chance that the
energy will be dissipated mainly within a single molecule.
One low G-value is shown, for the compound terphenyl. This is
characteristic of the yield for a number of compounds consisting
largely of aromatic rings. Apparently the delocalized electrons
allow the energy to be spread over a number of chemical bonds,
without damage.
Also, very high G-values, up to the thousands, can be measured
for some compounds in which chain reactions can occur (Ila).
Chain reactions initiated by ionizing radiation have been shown
for only 1 class of biologic macromolecules, the unsaturated
lipids (22), and there is no reliable evidence for the existence of
chain reactions, even in lipids, in cells.
According to the simple picture presented, the loss of molecules
on irradiation by this "direct" mechanism depends solely on the
size of the molecule and not on its surroundings.
A somewhat different situation arises when we consider
biologic molecules in an aqueous medium, as they are in the
living cell. A simple limiting case which can be readily studied is
that of biologic molecules in very dilute aqueous solutions.
Under these conditions, essentially all the energy is absorbed
by the water, and a negligible amount by the biologic molecules
themselves. As a result of the action of ionizing radiation, the
water molecule is decomposed into highly reactive free radicals,
which can readily diffuse to the molecules, and there react. A
summary of the damage produced in DNA and its constituents
under these conditions has recently l>een published by Scholes
(31a). Since the radicals are so highly reactive, they tend to
react within the 1st few collisions with organic molecules. The
relative importance of this mechanism thus depends on the
amount of free water immediately surrounding the molecules
being observed, DNA in this case.
Lastly, it is known that in mixtures of 2 or more kinds of
molecules there may be a transfer of electronic excitation energy
from one species to another (Ila). A particularly well-known and
clear example of this occurs in liquid scintillation solutions. In
these solutions energy adsorbed in the solvent, toluene for
example, is transferred with high efficiency to the scintillating
material, which radiates a fraction of the energy as visible light.
Such processes which occur with high efficiency in carefully
selected cases can also occur in other cases with much lower
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Radiation Effects on Cells
efficiency. Since the processes by which such energy transfer
takes place are not well known, their possible contributions, both
to enhance and decrease inactivation of specific molecules, must
always be watched. Nevertheless, the utility of classifying
radiation damage in the way we have just done depends on the
fact that in most cases these intermolecular energy transfer
mechanisms do not change the order of magnitude of the over-all
inactivation (27).
Correlation between Damage to DNA and Loss of Repro
ductive Ability
We would now like to take the information that we have on
the damage to the DXA in cells and viruses by ionizing radiation
and see if this can be correlated with the biologic loss of ability
to replicate. For this purpose, it will be convenient to use the
data classified in Chart 4.
The lowest line corresponds to viruses containing singlestranded nucleic acids, both RNA and DNA, and irradiated
under such conditions that the indirect action of water radicals
should be suppressed as much as possible. The line drawn
through the points corresponds to a G-value of about 1. To put
it another way, it would appear that a single energy loss event
anywhere within a single-stranded nucleic acid molecule removes
the ability of the virus to multiply. It might be mentioned that
for some of the viruses the protein coat may be removed, and the
nucleic acid alone used to infect the appropriate recipient cells.
If the radiosensitivity of the nucleic acid preparations is com
pared with that of the intact viruses, it is found that there is no
change in removing the protein coat (11, 25). In other words, the
presence or absence of the surrounding protein makes no change
in the radiation sensitivity of the nucleic acid.
From Chart 4 it is clear that viruses containing double-stranded
DNA are more radiation resistant by an order of magnitude.
Electron spin reasonance measurements (24) show that the
number of damage sites produced in the highly ordered array of
stacked bases in double-strand DNA is about the same as in
other materials, so that protection by delocalization of the energy
is not important. The best insight into the action of ionizing
radiation on the double-stranded DNA is given by recent
experiments by Freifelder (10) on the radiation inactivation of
T7 bacteriophage. He irradiated the phage under several different
conditions, and his results may be summarized as follows. When
T7 were irradiated under 2 experimental conditions (in phosphate
buffer with oxygen and in cysteine solution under nitrogen)
there was a 1-to-l correlation between the number of phage
inactivated and the number of broken DNA molecules found
after extraction from this phage. Thus, inactivation under these
conditions may be ascribed to a double-strand scission of the
DNA. When the phage were inactivated in a histidine solution,
equivalent to irradiating the cells in broth, only 4090 of the
inactivated phage had double-strand scissions. In both cases
something like 10-20 single-strand breaks were produced per
double-strand scission, but the single-strand breaks did not seem
to be responsible for loss of biologic activity. For irradiation in
the histidine or broth solution, Freifelder came to the conclusion
that the loss of infectivity could be caused by damage to pyrimidine bases.
Thus, the radioresistance of the double-stranded DNA viruses,
as compared with the single-stranded ones, could be ascribed to
the double-stranded structure. An extremely simple picture is
suggested. Radiation inactivation occurs when the DNA mole
cule is broken physically into 2 parts. In addition, inactivation
can also occur if bases on both complementary strands are
destroyed, thus removing information not available in any other
way. Single-strand breaks in double-stranded DNA, or damage
to bases on only 1 of the complementary strands, need not
inactivate.
The increased radioresistance of haploid microorganisms is at
present without any reasonable explanation. According to the
present ideas, the genome of a simple bacterial cell such as
Escherichia coli consists of a single DNA molecule replicated at 1
point (or perhaps a few points) which moves uniformly over the
length of the molecule (5a). It would be expected that any such
mechanism would be drastically affected by a break in the DNA
molecule. Yet at the doses required to inactivate bacterial cells
a number of breaks should occur, judging from the results of
Freifelder's experiments in phage (10). Even more damage
should arise from the attack of water radicals.
We must assume either of 3 alternatives, (a) Double-stranded
breaks, as opposed to other kinds of damage, are suppressed in
DNA in cells. (6) There is a mechanism which can realign the
broken ends of DNA in the cell genome and repair the breaks,
(c) Our simple-minded picture of DNA replication is wrong.
Unfortunately, we still do not have convincing evidence to tell
us which of these alternatives is correct.
It is interesting, in passing, that 1 original goal of racliobiology
was to explain why cells were so sensitive to ionizing radiation.
We now find ourselves trying to explain why they are so re
sistant! To look at the question in another way, it may be true
that there are other radiosensitive sites in the cell. But on the
basis of our present knowledge, the DNA more than accounts
for the observed sensitivities.
Naturally, the increased radioresistance of diploid cells cannot
be explained either. We know even less about such cells than
about bacteria. True, we do know that in mammalian cells the
DNA is embedded in protein, which could hold the broken DNA
strands together until the breaks are repaired. Also, we know
that some of the DNA in such cells is not necessary, since occa
sionally a chromosome, or a part of a chromosome, may be left
behind in an abnormal mitosis, and yet have a viable cell. And
lastly, we know that there are 2 copies of each piece of DNA in
the cell. These are all good arguments for expecting the cells to
be resistant to radiation. But to find the actual reason will very
probably require considerably more knowledge of the mechanics
of DNA replication and of cell division.
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CANCER RESEARCH VOL. 26
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The Molecular Basis for Radiation Effects on Cells
Franklin Hutchinson
Cancer Res 1966;26:2045-2052.
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