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MICROCOPY RESOLUTION TEST CHART
NATIONAL BUREAU OF STANDARDS - 1963
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Cont 670709.-2
216
MASTER
AUG 23 1967
Symposium on the
"Pacific Uses of
Atomic Radiation;"
Rio de Janeiro,
July 9-15, 1967
CREAZ. DICTS
21.0 22.00; . 65
EFFECTS OF RADIATION ON CHROMOSOMES
Michael A Bender
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Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, U.S.A.
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operated by Union Carbide Corporation for the U. S. Atomic
Energy Commission.
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LEGAL NOTICE
This report was prepared as an account of Government sponoured work. Noither the United
States, nor the Commission, nor any person acting on behalf of the Commission:
A. Makes any warranty or representation, expressed or implied, with respect to the accu-
racy, completeness, or usefulne88 of the inforniation contained in this report, or that the use
of any information, apparatus, metuod, or process disclosed in this report may not Infringe
privately owned rights; or
B. Assumes any liabilities with respect to the use of, ur fur damages resulting from the
use of any information, apparatus, method, or process disclosed in this report.
As used in the above, "person acting on behalf of the Commission" includes any on-
ployee or contractor of the Commission, or employee of such contractor, to the extent that
Buch employee or contractor of the Commission, or employee of such contractor prepares,
disseminates, or provides access to, any information pursuant to his employment or contract
with the Commission, or his employment with such contractor.
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DISTRIBUTION OF THIS DOCUMENT IS UNLIMITED
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Running head:
Chromosomes
Send proof to: Michael A Bender
Biology Division
Oak Ridge Nationai Laboratory
P. 0. Box Y
Oak Ridge, Tennessee 37830, 0,5.A.
Radiation-induced chromosome breakage and recombination
have been extensively studied for over 30 years, starting
with the work of Sax (1938). During that time not only
has a very detailed body of information been accumulated
by radiation cytogenetics, but the study of radiation effects
on chromosomes has contributed heavily to our understanding
of their normal structure and behavior as well. Naturally,
there are many areas where our knowledge is still incomplete
ce
$
T
and active research is still going on. Nevertheless, our
knowledge of dose-effect kinetics, factors modifying aberration
yields, recovery and repair processes, and the fates of.
aberrations and of the cells containing them is extensive.
It is patently impossible for me to review the whole field
of radiation cytogenetics for this Symposium. Furthermore,
an excellent, complete review has recently been published
by Evans (1962). So in spite of the more general title
assigned me, I shall take the 1lberty of restricting myself
to a much narrower topic which has been of special interest
to me personally, that of radiation-induced chromosomal
aberrations in man.
In spite of a certain practical Interest, the study
of chromosomal aberrations in human cells was begun only
21
d.
1
about ten years ago. The reason lies in the technical
ma
difficulty of making useful cytological preparations from
mammalian material by classical techniques. During the
early 1950's the use of clssue cultures and of hypotonin,
prefixation treatments changed this.
The start of modern
human cytogenetics may be dated from the discovery in
1956 by Tjio and Levan that the human diploid chromosome
number was really 46 inscead of the 48 ont still sees
Solie
quoted. Human chromosome work received grea: impetus a
few years later when it was discovered that some clinical
disorders, such as mongolian idiocy and various sexual
abnormalities, have chromosomal bases (Lejeune, Gautier
and Turpin, 1959; Ford, et al., 1959a, 1959b). The
development during the next year of a practical sethod
for obtaining chromosome preparations from peripheral
blood samples (Moorhcad, et al., 1960) contributed to a
remarkably explosive growth of the new field of human
cytogenetics. Not only did a great number of people join
the search for new clinical chromosomal abnormalities,
but many laboratories started to work with radiation-induced
aberrations in hunan cells as well..
Unfortunately, the result, from the point of view
i of the radiation cytologist, has been disappointing, and
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sometimes even distressing. It was probably inevitable
that enthusiasm for work with human chromosomes would
lead some workers to enter the field without benefit of
the information already accumulated from studies with
other organisms. Many already well known (frequently
even trivial) facts have appeared in the literature as new
and important discoveries. Some authors have added to
the confusion by using non-standard nomenclature of their
orm invention. Worst of all; data is frequently obtained
or presented in such a way as to be obscure, and obviously
false conclusions are some times drawn.
In short, the
field of human radiation Cytogenetics has become much
more confused (and confusing) than there seems any real
excuse for.
It is my hope that by cutlining some of the
most basic facts in radiation cytogenetics, and then
examining the human chromosome work in their li.ght, I can
perhaps put human radiation cytology in somewhat better
perspective. Furthermore, I hope that some of the valid
reasons for studying radiation-induced human chromosonie
aberrations will be clarified, and that the conditions
which must be met by studies aimed at particular problems
will be evident.
Chromosomal Aberrations
Eynes
Before considering specific studies of radiation-
Induced abezrations in human chromosomes, then, I would
like to review a few of the basic phenomena in adiacion
cytogenetics. Those I shall cover here are all very well
known, and have been for many years. Details may be
found in almost any text on radiation effects. While I
will use human chromosomes as examples, the phenomena
described were actually discovered in non-human, usually
plant, material. When examined at the metaphase of a
cell division, each chromosome is composed of two parallel
strands, the chromatids, still joined together at the
centromere region, which is the point of attachment of
the chromosome to the spindle apparatus, and thus responsibia
for the movement of the daughter chromosomes to the poles
during the anaphase. These features may be seen in the
first slide (Fig. la) which shows a metaphase in a tissue-
cultured human peripheral leukocyte. A particular chromosome
F-la
number is, of course, characteristic of each species, and
a somatic cell, such as the leukocyte, is diploid and
contains two homologous chromosomes of each type (except
A
SC
mo
ON
DA
for the heteromorphic sex chromosome pair). The next
slide (Fig. 2) shows the chromosomes from the previous
F-2
slide (Fig. la) rearranged by pairs in what is known as
a karyotype.
The relative length and centromere position
of each of the chromosomes is constant. Some chromosomal
aberrations change the size or shape of one or more
chromosomes and may thus sometimes be discovered by
preparing a karyotype, although they may not be at all
obvious when one simply observes the original metaphase
figure.
Chromosonal aberrations are generally believed to
arise through the breakage of a chromosomal strand. If
S are
several breaks exist in the same cell at the same time,
the broken ends of the strands may recombine to give
various types of rearranged chromosomes. If the breakage
is induced early in the cell cycle, before the synthesis
of new chromosomal material begins, the chromosomes behave
as single strands and the aberrations seen are of what
is called, somewhat confusingly, the chromosome type.
If the breakage is induced later in the cell cycle, however,
ve
the chromosomes behave as two-stranded structures, and
no.
breakage in one of the strands is more or less independent.
of that in the other. Such aberrations are known as
1
chromatid-type aberrations. With one exception, which
we will consider later, analogous aberration types occur
within the chromosome-type and the chromatid-type classes.
A single chromosome break results in a fragment
without a centromere, plus the shortened centric chromosome
(provided, of course, that the break stays open and does
not "heal"). This sort of aberration is called a chromosome
deletion. An example from a human leukocyte is shown in
the next slide (Fig. 1b).
The aberration is recognized
F-lb
by the presence of tire paired acentric fragments. The
cell was irradiated, and the break induced, while the
chronosomes were still single stranded; when the
chromosomes replicated themselves, the fragment replicated
as well, producing the characteristic identical pair.
If two or more chronosome breaks occur in the same
cell at the same time "hey may, of course, simply yield
two or more deletions
on the other hand, they may recombine
to form new aberration types.
Two breaks could be either
in the same or in different chromosomes.
In either case,
the recombinations can be either symmetrical or asymmetrical.
By symmetrical is meant the case where a broken end on the
centric part of a broken chromosome is joined to the
-
broken end on an acentric part; in an asymmetrical rearrangement
one centric end united with the other centric end and an
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acentric one with the other acentric end. Symmetrical
rearrangement in a chromosome with two breaks yields an
inversion, while asymmetrical rearrangement yields a ring
chromosome, together with acentric fragments. Symmetrical
rearrangement between two breaks in different chromosomes,
on the other hand, yields a translocation, while agyınmatrical
rearrangement yields a dicentric chromosome along with
acentric fragments. The next slide (Fig. 1.c) shows an
example of such a dicentric chromosome induced by X zay
f-1c
treatmen" of a human blood sample. Similar, but more
complex, aberrations such as dicentric rings or tricentrics
may occur if three or more breaks ara available in a cell.
As already mentioned, the chromatid-type aberrations
are, with one exception, analogous to the chromosome types.
Thus one finds rings, dicuntrics, etc., but they involve
one chromatid only, instead of both. The exception is
known as the isochromatid deletion. In this type of deletion
both chromatide are broken at the same level. Such
aberrations are believed to arise as a result of breakage
of both strands by a single event, during the time while
the strands still lie in very close proximity. Unlike
the broken ends from a chromosome-type deletion, the ends
in an isochromatid deletion may recombine with each other
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in a sister chromatid-to-sister chromatid fashion.
These
chromosome-type deletions. Some isochromatid deletions,
nosom
however, involve no sister union, and these are indistinguishable
from chromosome-type deletions.
Kinetics
Chromosome breaks induced in cells by sparsely
ionizing radiations such as X or y rays are distributed
at random. Consequently, wiile one expects linear dose-
effect kinetics for the induction of single-break aberrations,
acute doses of these radiations are expected to induce two-
break zverrations with second order dose-effect kinetics,
three-break aberrations with third order kinetics, and
so on.
This follows from the random nature of the breakage,
because if the breaks are statistically independent events
having the same probability of occurring, theni the probability
that two breaks will coincide (in the same cell or in the
same chromosome) is the square of the probability of
getting one break, and the probability that three will
coincide is the cube of the protability of a single break.
In fact, such non-linear kinetics were noted very early
in the study of radiation-induced chromosomal aberrations,
and formed the basis for much of the theory of chromosome
aberration production, Of course, the kinetics usually
observed are actually not precisely those expected from
such a simple analysis. In reality there are many
complications, such as the possibility that there is a
limit to the number of places in the chromosomas at which
breaks can participace in recombinations, and the possibility
that one piroton might actually induce more than one break,
to name just a few. Nevertheless, for practical purposes
the yields of deletions induced by acute X or y irradiation
are approximately a linear function of dose, and the
yields of rings, dicentrics, exchanges, and Inversions
increase approximately as the square of the dose.
As might be anticipated, there are situations where
the approximately second order kinetics expected ĉor two-
break aberrations (for example) do not occur. Irradiation
with densely ionizing particles, such as fission neutrons
or a particles, is characterized by approximately linear
dose-effect curves for multiple-break aberrations. This
is because the breaks induced by such radiations are not,
in fact, statistically independent. Iť a particle
dissipates enough energy while traversing a cell, the
probability that more than one break will be produced
II
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becomes virtually unity, and the observed linear dose-
effect kinetics result.
lleither does chronic irradiation with sparsely
ionizing X or y rays give the same dose-effect kinetics
as acute irradiation, Chromosome breaks have a finite
lifetime during which they remain available for recombination.
Although this does not affect the number of single-break
aberrations produced (provided the cell does not divide
during the irradiation), one would expect that the number
of multiple-break aberrations would be reduced, and that
the dose-effect kinetics would become more nearly linear
as the dose rate is decreased, which is just what is
actually observed. Ultimately, if the dose were given
over a long enough time, only those few multiple-break
aberrations which resulted from recombination of breaks
induced by the same lonizing event could occur at all,
and the dose-effect curve would be completely linear.
Fates
We have spoken, up to now, of aberrations as they
appear in the first metapahse following their induction.
But what happens to these aberrations when the cell
containing them actually divides? What is the fate of
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13
the daughter cells? How does the induction of chromosomal
aberrations in its cells affect the organism? We can
give some very general answers, even though they will,
of necessity, be somewhat oversimplified,
As already noted, many aberration types involve
acentric fragments.
Such fragments, lacking a centromere,
are not distributed to the spindle poles at anaphase like
the rest of the chromosomal material. They are only
included in one (or both) of the daughter nuclei by
accident. Generally, they will remain in the cytoplasm
of one of the daughter cells for a time, forming micronuclei,
but eventually they will be lost.
With the exception of isochromatid deletions with
sister union between the centric chromatids, deleted
chromosomes segregate normally at anaphase. One or botil
of the daughter nuclei, depending on the deletion type,
receives a daughter chromosome which is shortened by the
amount of material lost in the acentric fragment. A
diploid cell is thus made haploid for a portion of one
of the chromosomes of a pair, Where there is sister
union between the centric cliromatids in an isochromatid
deletion, however, normal segregation of the centromeres
is interfered with because the two centromeres, which
must try to move to opposite poles, are physically joined
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14
by a common chromatid strand. An an aphase bridge results.
The fate of such a bridge, and of the dividing cell
containing it, is not at all clear. One hears the famous
OUS
breakage-fusion-bridge cycle discovered by McClintock
(1938) mentioned frequently, but this process was discovered
in the triploid endosperm of maize, and has not been reported
11 other tissues or organisms. It actually seems quite
doubtful that the phenomenon is general. It is possible
that some or all bridges eventually break, permitting the
division to be completed, and leaving the daughter cells
with deleted chromosomes (frequently a duplication-deletion
in one of the daughters). If the bridge does not break,
the division cannot be completed, and the cell presumably
either dies or becomes a polyploid.
Symmetrical multiple-break aberrations can be
expected to segregate without any difficulty at the
anaphase. One or both of the daughter cells simply receives
one or more rearranged chromosomes.
In the case of a
symmetrical translocation, because there are two independently
assorting pairs of centromeres involved, the two translocated
chromosomes may go to the opposite poles at anaphase, and
duplication-deficiencies will result. No such problem
exists with Inversions,
15
Asymmetrical multiple-break aberrations, on the
other hand, involve acentric fragments. The same
aite
expectations apply to these fragments as to the fragments
from deletions. Ring chromosomes will segregate normally
..
at anaphase (provided two chromatid rings are not inter-
locked" by a twist or an odd number of exchanges between
chromatids). Dicentric chromosomes or chromatids, having,
in effect, two centromeres on one chromatid, are obvious
candidates for an aphase bridge formation. It should be
noted, however, that bridge formation is not necessary,
but only possible.
If both of the centromeres on one
chromatid attempt to go to one pole, only a twist which
looped one chromatid through the other could cause any
mechanical trouble. It is not uncommon that random
is te
IRO
segregation of the four centromeres of a dicentric
chromosome is assumed. If this were the case one would
expect bridge formation one-half of the time.
It seems
more itkely, though, that the probability of the "opposite
assortment" which would lead to bridge formation is a
SS
function of the distance between the centromeres on the
chromatids. Where the centromeres were very close together
one might expect virtually no bridge formation, but where
they were very far apart the probability of bridge formation
-
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-
inight approach 50 percent. As with the bridges formed by
16
some isochromatid deletions, little is known of how often,
if at all, they break, allowing the division to be completed.
But if they do break, the daughter cells must receive
what amount to deleted chroinosomes.
It can be seen that if cells containing most types
of aberrations do successfully divide, one or both daughter
cells will contain a chromosome that is deficient for
some of its genetic material. In a diploid cell, however,
the homologous chromosome is usually still intact, and
the cell is thus not lacking part of its geneta message,
but only haploid for it. Many of us are accustomed to
think of deficiencies of appreciable size as being lethal.
Any recessive lethals in the segment of the homologous
chronosome "uncovered" by a deletion can be expressed just
as though they were dominant. We must remember, however,
that this idea derives from genetic studies, and that the
lethals we have in mind are those that kill zygotes or
cmbryos, ivhat about deficiencies in somatic cells? Most
genetic lethals act by interfering with the production
of some critical compound in the cell. But to be a somatic
cell lethal a deletion would have to prevent the production
of a compound whose deficiency could not be made up by
supply from normal cells elsewhere in the organism, for
the deficient cell would otherwise be "saved." There
,
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17
is little information on the frequency with which deletions
are in fact lethal in diploid somatic cells, but it appears
from what little evidence there is that many, perhaps
most, are not lethal, nor even detrimental,
Several of the aberratior types, if they do get to
one or both of the daughter cells, have the same expectation
of causing mechanical trouble at each succeeding division
that they did in the first. Thus a dicentric which
segregates without difficulty at the first division still
has the same probability of forming a bridge at the next,
and so forth.
In a cell population, then, one would expect
a gradual. loss of dicentrics, leading ultimately to their
complete elimination.
In order to consider the effect of chromosome aberrations
on the organism in which they are induced, we must make
a distinction between somatic cells and gern line cells.
Aberrations certainly kill some somatic cells either outright
(at a division for example), or by removing třem from the
pool of cells capable of renewing the population. This
killing can be of more or less consequence to the organism,
depending on the particular tissue involved. Cell killing
in bone marrow, for example, has more dramatic effects than
cell killing in a tissue with less rapid cell turnover,
00S
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18
such as muscle.
Chromosomal aberrations are known to be
ass
associated with neoplasia. It is tempting, therefore,
to think that aberration induction might lead to neoplastic
diseases. I know, of no case, however, where this has
been demonstrated, although the case of the Phé chromosome
in chronic myeloid leukaemia (Nowell and Hungerford, 1960)
is certainly suggestive.
Chromosomal aberrations induced in germ : 1ine cells
may have genetic consequences, as distinct from the possible
clinical effects of aberrations in somatic cells, Germ
line aberrations can, of course, simply act as genetic
or zygotic lethals. On the other hand, they may be
transmitted to the next generation. Some will certainly
produce abnormal individuals, since the same chromosomal
abnormality is present in every cell of the offspring's
body. Some offspring may be balanced translocation
I
heterozygotes. Such individuals appear normal, but they
are semi-sterile and may even produce grossly abnormal
cffspring themselves.. Aberrations such as deletions
may spread in the population from generation to generation
acting as recessives. As with recessive gene mutations,
though, most may be expected to be detrimental when their
frequency in the population became high enough for homozygotes
to begin to appear.
19
Studies or Human Chromosomes
Since much is already known about radiation-induced
chromosomal aberrations, and since non-human material
has supplied most of the information we already have,
it is perhaps not unreasonable to ask for what reason
we bother to study human material at all. Studies on
man
human chromosomes have, perhaps, more glamour than studies
on toad or tomato chromosomes, but can human chromosomes
really give us more basic information on normal chromosome
structure and function than can the chromosomes of other
species? Failing that, are there actually sound practical
reasons for choosing human material over a multitude
of other possibilities?
In point of fact, in spite of their relatively high
chromosome number, human cells really have proved to
be superior mater! 11 for certain types of basic studies,
either because of the existence of "permanent" hutan
tissue culture cell lines or because of the large number
of chromosome abnormalities in man which have known
clinical consequences. Occasionally there are other
special technical reasons as well. This by no means,
however, is sufficient to explain the volume of work
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20
that has been done. Much human chromosome work has as
its objective the acquisition of practical information
of particular utility to humans themselves, information
examining human material. Several examples come readily
to mind.
in studies of radiation-induced chromosomal aberrations
in man is to evaluate liuman radiation hazards. Although
it is tempting to simply extrapolate from data on
non-human material, this is clearly a risky procedure
at best. And even if we could be confident of the
accuracy of such extrapolations, the practical importance
of the problein would still lead us to make checks with
liuman material anynyay, just to be sure. Several types
of study are indicated, but the answers sought are of
necessity all quantitative, rather than qualitative.
First, perhaps, one would like to know the radiosensitivity
of human chromosomes as compared with those of the more
thoroughiy studied "classical" materials. But we also
need evaluations of the specific peculiarities of human
material, of tissue-to-tissue differences in radiation
sensitivity, of the population dynamics of the cells in
the tissues of Interest, and so forth, in order to
U
21
meaningfully assess human radiation hazards.
A second major practical reason for studying human
material is the obvious possibility of using human chromosome
aberrations as a "biological dosimeter." If it is possible
to describe the yield of chromosomal aberrations as a
function of radiation dose by simple a'igebraic expressions,
and to accurately predict the yield from any dose, then
It is obviously also possible to calculate the dose that
must have been received in order to produce a given yield.
This possibility has been noted by several authors (Bender
and Gooch, 1962; Norman, et al., 1962; Shapiro, et al., 1963;
Kelly and Brown, 1965). Again, the prime requirement
is quantitative information on human chromosome radiosensitivity
and on cell and tissue dynamics, Practical confirmations
of the ability of this "dosłmeter" to measure radiation
exposures reasonably accurately are also in order, of course.
One can also think of other specific practical reasons
for the study of chromosomal aberrations induced in human
cells, but most are directed at much more limited and
specific objectives, such as manned spaceflight or
radiotherapy of specific types of tumors, for example,
They are consequently of more limited interest, and in
any case they must rest on the same quantitative foundation
as the radiation hazards and the biological dosime try
questions.
"Cellular" aberration rates
Germ line cells. From the point of view of the
human radiation hazard problem, one would really like
to have direct measurements of aberration yields in
.
human germ line cells.
To my knowledge, however, none
has as yet been made. All the information available is
on somatic cells.
It is unfortunately not at all clear
to what extent it is valid to extrapolate fror somatic
ce11 aberration rates to germ line aberration rates.
Somatic cells in vitro. The earliest studies of
radiation-induced human chromosomal aberration rates
were made on diploid tissue culture cells which had
were
ue C
been in culture only a short time and should thus, it
was hoped, have about the same radiosensitivity as cells
in vivo (Bender, 1957, 1960; Puck, 1958; Lindsten, 1959;
Fraccaro, 1960; Dubluin, et al., 1960; Chu, et al., 1961).
The cells were irradiated with X rays in vitro and
examined for (mainly) chromatid-type aberrations. A
YO
were
similar chromatid aberration experiment has been done
with leukocytes in short-term culture (Bender and Gooch,
1963a), Such studies presenced many difficulties of
various kinds (Bender, 1963). While it is not possible
to 30 into these extensively here, two of them deserve
er ve
SORE
some comment. First, there is the question of how well
actually be expected to agree with ratas induced in cells
PUU
In vivo. Second, chromatid aberration yields induced
by a given dose are known to vary markedly as the cells
move through the cell cycle. This, together with
differences in the scoring methods for chromatid aberrations
mov
employed by various investigators makes it difficult to
precisely reconcile the various reports. Nevertheless,
it is certainly possible to conclude that the radiosensitivity
froin that of the chromosomes of other species.
The peripheral leukocyte technique, when it became
available, offered a superior and much more convenient
means of detennining the radiosensitivity of human
chromosomes. Blood samples can be obtained quite easily,
and may be irradiated immediately after they are drawn,
thus approximating an 111 vivo irradiation as closely as
seems possible withouo actually irradiating people,
Furthermore, when one irradiates before placing the
cells in the short-term cultures necessary to obtain
cm
mitoses for examination, or even if the cells are
only chromosome-type aberrations are induced, and the
problems associated with the variation of chromatid
aberration induction rates with position in the cell
cycle is avoided. Many determinations of aberration
yields in the leukocyte system have been reported for
acute exposures to X and y brradiation (Olinuki, et al.,
1961; Bender and Gooch, 1962 a; Bell and Baker, 1962;
Sasaki, et al., 1963; de Grouchy, et al., 1963; Norman,
et 31., 1964; Kelly and Brown, 1965; Schmickel, 1967),
fast neutron irradiation (Gooch, et al., 1964; Bender
and Gooch, 1966), and even for 32p B particles (Bender,
et al., 1967). Although the leukocyte system appears
to be a relatively simply one, it was probably inevitable
that confusion would arise over such experiments. The
VE
IS
Over S
yields of the various aberration classes per unit dose)
obtained by various workers have not always agreed very
well. Sometimes the reason is simply that extremely
high radiation doses, for which the simply kinetic models
are not applicable, were used; in other cases the
disagreement is actually more apparent than statistical,
since the authors scored only small samples of cells.
There is a third source of confusion, however, which
deserves closer examination,
i
25
25
een
measure
Although it seems quite obvious that to measure
the primary aberration frequencies in irradiated cells
one must observe the cells in their first post-irradiation
division, this has not always been done in experiments
with irradiated human leukocytes. In one case, when the
investigators finally became aware that they were not
In fact scorin: only first in vitro divisions, they
actually reported this requirement as a novel discovery
(Buckton, and Pike, 1964a, 1964b). Unfortunately, the
rather naive assumption that if second post-irradiation
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.. ...
divisions could be found in any leukocyte culture fixed
.
.
..
.
at a particular incubation time, they must then be
.
..
... ...
..
present in all leukocyte cultures fixed at that time
(Buckton and Pike, 1964a, 1964b; Sasaki and Norman, 1966)
..
---
.
.
.
has led to fairly general doubts about the validity of
much of the data accumulated from in vitro irradiation
of blood samples. Consequently, even though we had
naturally made tests before hand to be sure that we
were in fact going to be dealing virtually exclusively
PA
with first post-irradiation division cells, and in spite
of the fact that the complete data from such experiments
actually provides an internal check on this point (1.e.,
the frequency of cells which should have acentric fragments
but do not), our group felt compelled to look into tlie
2:26
"time of fixation" problem in detail (Bender and Brewen,
1967).
I use the word "compelled" deliberately because
Use
we were reluctant, to say the least, to spend our time
and efforts on cedious experiments designed to prove
RES
that which was obvious (to us at least) in the first
place. And we did, in fact, find that various culture
parameters, particularly incubation temperature,
markedly affected the timing of divisions in leukocyte
cultures, jusť as any biologist would predict. But we
also discovered some thing which I, for one, did not
expect. Most workers, myself included, had assumed that
the leukocytes which divide in our short-term cultures
must represent a single population of cells with uniform
radiosensitivity. It is just this tacit assumption
which leads to the expectations that aberration yields
will drop as one begins to include significant numbers
of second post-irradiation divisions in one's sample,
SO
*S
and that aberration yield will be independent of culture
time as long as second divisions are excluded. Our
experiments (which will be reported in detail elsewhere)
showed my assumption to be completely wrong. We did
observe changes in aberration yields as a function of
culture time all right, but we observed them before we
-
-
-
-
.
-
-
-
27
...
began to get significant numbers of second post-irradiation
....
divisions in our samples. Furthermore, we did not observe
........
any marked cliange in aberration yields when the second
.
post-irradiation divisions finally did begin to appear.
..........
.
This latter result has also been observed in another
laboratory as well. Sugahara, et al (1967) compared
aberration yields at various culture intervals before
and after the appearance of second divisions, and actually
found aberration yields slightly higher in cultures they
harvested later. It is impossible to reconcile such a
result with the assumption of a single population.
The simplest explanation is that there are actually at
least two populations of leukocytes which divide in our
cultures, one which is more radiosensitive and has a
shorter cell cycle and another which is less radiosensitive
and has a longer cell cycle.
It appears, then, that the leukocyte system cannot
be expected to provide a single simple answer to the
question of human chromosome radiosensitivity after all.
Nevertheless, it is by far the best system we have. The
results obtained if the experiments are controlled carefully
are ren
are remarkably reproducible (Bender and Gooch, 1962a;
Gooch, et al., 1964), and the difference in primary
aberration coefficients appears to vary a maximum of
-
-
-
-
--
--
-
-
-
--
--
28
28
only a factor or two from the beginning to the end of
the first division interval, and frequently much less.
(Bender and Brewen, 1967). The experiments of
de Grouchy (1963) suggest coefficients in this range
are a fair measure of the radiosensitivity of human
-
-
chromosomes in vivo. X-ray-induced aberration yields
were compared in leukocytes and in bone marrow cells
under comparable conditions. Although the yields from
-
the bone marrow cells were slightly higher than those
from the leukocytes in de Grouchy's experiment, they
fall well within the range we have observed for
leukocytes. Additional evidence on the validity of
in vitro leukocyte determinations can be obtained from
in vivo irradiations.
Somatic cells in vivo.
There have been a great
many reports of chromosome aberrations in peripheral
leukocytes following human radiation exposures. Unfortunately,
quantitative interpretation of the aberration yields
reported and the estimation of coefficients of aberration
production from them is difficult or inpossible in many
cases.because the people studied were exposed to chronic
or fractionated extemal doses, frequently to only part
of the body, or to radiation from internally-incorporated
radioisotopes (Boyd, et al., 1961; Conen, 1961; Stewart
29
and Sanderson, 1961; Buckton, et al., 1962; Lindgren
and Norryd, 1962; lacintyre and Dobyns, 1962; Conen,
et al., 1963; Moore, et al., 1963; Engle, et al., 1964;
Moore, et al., 1964; Nofal and Beierwaltes, 1964;
Visfeldt, 1964; Amaroge and Baxter, 1965; Court Brown,
et al., 1965; Doida, et al., 1965; Norman, et al., 1965;
Warren and Meisner, 1965; Bauchinger and llug, 1966).
Furthermore, the value of some of these reports has been
further decreased by failure of the authors to familiarize
de.
Om
el
themselves with (or perhaps to recognize) the previous
work in radiation cytology.
Chromatid aberrations are
confused with chromosome types, new aberration categories
are confused with deletions, and so forth. The authors
of some of these papers have apparently thought they
were making useful quantitative measures of human
radiosensitivity, but frequently all that they managed
to establish was that radiation really does break
chromosomes. Unfortunately, many of the reports of
chromosome aberration levels in people who received
acute whole-body exposures from external sources are not
OUTCAS are
of any value for determining.coefficients of aberration
production either because cells could be obtained only
Use
-
long after the exposure (Bender and Gooch, 1962b, 1963b;
..
-
-
..
.
-.
.
.
.
.
.
.
-
.
.
.
.
.
.
30
Sasaki, et al., 1963; Papiernik-Berkkaver, et al., 1963;
Doida, et al., 1965; Bloom, et al., 1966; Goh, 1966;
Lisco and Conard, 1967). In a few cases, however, it has
been possible to obtain leukocyte samples within a matter
Jever
of hours or at most a few days after acute human whole-
body exposures, received either in radiation accidents
(Gooch, et al., 1964; Bender, 1964; Bender and Gooch,
1966) or in the course of therapy (Norman, et al., 1962;
Sasaki, et al., 1963). Although only a few cases have
been studied in this way, the agreement between the
radiation doses estimated from aberration yields and the
doses neasured or estimated from physical evidence has
been reasonably good. More studies of this type are
needed before a final conclusion is drawn, of course,
and it is to be hoped that it will be acquired and
reported in a more conventional manner than has sometimes
been the case in the past. It nevertheless appears that
the leukocyte system Irradiated in vitro does actually
provide a fair measure of in vivo human chromosome
radiosensitivity,
Aberration rates in populations
It seems obvious that although most of the efforts
in human radiation cytology to date have been directed
.....
31
res
toward cases of individual. exposures, studies of exposed
human populations are of much greater importance from
the point of view of radiation hazards. Because of the
very large volume of material that must be examined,
such population studies are extremely time consuming.
Nevertheless, a beginning has been made. Several groups
have compared the frequencies of chromosomal aberrations
in leukocytes of survivors of the Hiroshima and Nagasaki
atomic bomb explosions with those in unexposed controls
(Doida, et al., 1965; Bloom, et al., 1966). Other studies
have been made of radiation workers who accumulated above-
background exposure totals in the course of their normal
occupations (Sasaki, et al., 1963; Court Brown, et al.,
1965; Doida, et al., 1965). Of particular interest and
importance is the study of chromosome aberration levels
in the leukocytes of a population living in a monazite
sand natural high radiation area in Brasil (Penna-Franca,
et al., 1965). All of these studies may be considered
to be in their early stages, since the population samples
are still quite small. Nevertheless, it already appears
that some increase in the frequency of chromosomal
abnormalities is detectable even in groups where the
radiation exposure is chronic and the dose' rate extremely
-
-
-
-
.
low. of particular interest and importance is the
possibility of determining the frequency of viable
chromosome alterations by examining the children of
Ome
people in these exposed populations for altered chromosome
constitutions.
Far more information will be nuaded beforo we can
confidently predict the effects of radiation exposures
on aberration levels in human populations.
To accumulate
the vast amount of data required will certainly necessitate
(and certainly merits) higher levels of support by funding
agencies. Even with increased support, however, the
accumulation of tire vast amount of data required will
be very slow unless sone means of automating some or all
of the process of chromosome scoring is developed.
Fortunately, quite a lot of effort is currently being
expended on the development of such aids, and we may be
hopeful that some practical device will be forthcoming
in the naar future.
Biological dosimetry
Quite a lot has already been said about biological
dosimetry by means of chroniosome aberration analyses
on leukocyte samples.
The work on in vitro and in vivo
33
irradiations already mentioned provides both confidence
that such dosimetry is practical and the coefficients of
aberration production required. Our group has, in fact, .
already made useful dose estimates in cases of accidental
human exposures (sce Bender, 1965, 1966; Bender and Gooch,
1966). But it is easy to expect too much quantitative
infornation from this technique. It is applicable in: a
quantitative way only under certain special circumstances.
The exposures must have been high enough, but not too
high; the exposures must have been acute. Sampling
must be done before aberrations are lost through cell
S
2
division, or at least at post-irradiation times for
which we know the aberration 1098 to be expected
(Sasaki and Norman, 1967). In cases where the whole
body was not exposed or where there was considerable
variation in dose to different parts of the body, the
technique can only give an estimate of what we might
call the "average biologically effective dose." And
even where the circumstances are not such as to allow
an estimation of dose to be made confidently, the
qualitative results of an aberration analysis can
sone times be of value. We have used them, for.example,
to check a case where a film badge indicated a radiation
exposure which it seemed un likely that the wearer actually
weas
34
34
Conclusions
In summary, work on human chromosome aberrations is,
in general, nothing more than the extension of an existing
body of Information to include another organism. Very
little new Information of basic significance has come
out of such studies.
The human chromosome aberration
work is nevertheless of extreme practical inportance,
simply because we are the organism being studied. Much
of the Informacion gained is of direct use to us. To
get the most out of it, however, will require both
larger scale population studies and the sensible application
of the pertinent scientific data we already have.
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43
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I
44
44
FIGURE LEGENDS
Fig. 1. Chromosome spreads from short-term cultures
080ne
res
of human peripheral leukocytes. a - normal
male complement. b - complement with chromosome
deletion, c.- complement with dicentric chromosome
and acentric fragment, d - complement with ring
chromosome but lacking the accompanying acentric
fragments which would normally have been present
in a first post-irradiation division.
Fig. 2. Karyotype prepared from the chromosome spread
shown in Fig. la.
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