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MICROCOPY RESOLUTION TEST CHART
NATIONAL BUREAU OF STANDARDS - 1963
ORNnP.HH
2775
Conf-660412-2
Symposium on: Accidental
Irradiation at Place of Work
Nice, France, April 26-29, 1966
mento
JUN 27 the
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H.C. $ 4.00; MN 30
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RELEASED FOR ATROVICRIT
IN NUCLEAR SCIENCE ABSTRACTS
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Cytogenetic Methods: Somatic Chromosome Aberrations
in Occupationally Irradiated Humans
M. A Bender and P. C. Gooch
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Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, U.S.A.
CA
Research sponsored by the U. S. Atomic Energy Commission under contract
with the Union Carbide Corporation.
Running head:
Chromosome aberrations
Send proof to: M. A Bender
Biology Division
Oak Ridge National Laboratory
P. 0. Box Y
Oak Ridge, Tennessee 37830
U.SA.
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Chromosomal aberrations have been known to be induced
by ionizing radiations for more than thirty years. Actually,
they now constitute one of the most thoroughly studied
phenomena in radiobiology, as may be seen from reviews such
as the recent one of Evans (1962). Because breaks and
rearrangements of chromosomes are actual changes in the
cell's genetic mechanism, they seem a particularly
suitable measure of radiation damage. Furthermore,
chromosomal aberration frequencies can be determined
quantitatively, and their dose-effect relationships are
reasonably well known. They thus offer the possibility
of estimating an unknown dose from the yield. We have,
in fact, already used aberration yields for biological
dosimetry.
It is only recently that induced chromosomal
aberrations have been studied in humans. The reason is
that previously no techniques were available which
yielded adequate human chromosome preparations. In fact,
the human diploid chromosome number of 46 was finally
determined just ten years ago by Tjio and Levan (1956);
study of radiation-induced aberrations in human
chromosomes came the next year (Bender, 1957).
Aberrations may be induced in either somatic or .
germinal cells, but unfortunately very little work has
yet been done on human meiotic cells. In any case, the
effects of most interest from the point of view of the
accidentally exposed individual, and of the physician
treating him, are the somatic effects. While it is
feasible to study somatic chromosome aberrations in a
number of different tissues, only the peripheral leukocytes
and their precursors have received substantial attention.
A method for inducing mitosis in short-term cultures of
leukocytes from peripheral blood samples, described by
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Moorhead et al. (1960), offers many advantages over other
techniques. Peripheral blood samples are relatively easily
obtained, even when repeated sampling is necessary.
Furthermore, all of the pe 'ipheral leukocytes which later
divide in culture are in the pre-DNA-synthetic stage of
the cell cycle while in the circulation, and the aberrations
induced in them are chromosome types, rather than chromatid
types. Thus the problem of differential stage sensitivity
is avoided. The only other suitable tissue is the bone
marrow, which offers neither advantage, and has consequently
been used to study chromosomal aberrations on only one
occasion.
It was only to be expected that chromosomal aberrations
would be found in somatic cells of irradiated people once
they were looked for. Fleidner et al. (1959) observed
anaphase bridges and fragments in bone marrow samples from
men irradiated in the Y-12 criticality accident in 1958.
We observed aberrations at metaphase in leukocyte preparations
from peripheral blood samples obtained from the same men
several years later (Bender and Gooch, 1962a; 1963).
Neither observation was of particular value, however, in
providing a quantitative measure of radiation damage.
Since then, chromosome aberration analyses have been made
on many persons receiving various types of occupational
radiation exposure. Before these observations are discussed,
though, we will first consider what our large previous
experience with other species would predict for the Leukocyte
system in exposed humans.
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THEORETICAL EXPECTATIONS
Aberration tyres.
As mentioned, chromosome type aberrations are expected
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from irradiation of peripheral leukocytes. All aberrations
involve a break in the continuity of the chromosome. Induced
breaks are (with radiations of low LET) distributed at
random among cells. Any break may close again (in which
case nothing is observed at the following metaphase).
If it does not close, it may either simply remain open,
or the broken ends may recombine with other broken
chromosome ends. The simple open break is called a deletion.
It results in a shortened chromosome plus & separate piece
without any centromere (an acentric fragment). There are
many types of aberrations involving recombination of
broken ends. Because those involving three or more breaks
are rare and because all multiple-break aberrations fall
into a few general classes, only the two-break aberrations
will be discussed here.
Two chromosome breaks may be in either the same or
different chromosomes. If the latter, two types of
recombination, symmetrical and asymmetrical, are possible.
Symmetrical exchange results in transposition of the
acentric fragments, i.e., a translocation. An asymmetrical
exchange joins the two centric chromosome portions,
resulting in a dicentric chromosome plus one or two
acentric fragments. If the breaks are in the same
chromosome analogous aberrations result. The symmetrical
form is an inversion. The asymmetrical recombination
produces a ring chromosome plus an acentric fragment.
Detection of the symmetrical recombination aberrations ia
uncertain and subjective, and it is common therefore to
use only the asymmetrical types as a quantitative measure
of radiation damage. Thus for practical purposes only
three aberration types are scored: deletions, dicentrics,
and rings..
Spontaneous aberration rate.
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A few chromosome breaks occur spontaneously, without
any increase in exposure above background levels. The
spontaneous frequency of chromosome deletions (plus a chromatid
deletion type which is indistinguishable from it) observed
in our laboratory (s only about 0.005 per cell. The
actual frequency of deletions in vivo may be substantially
lower. Spontaneous multiple-break aberrations are, as
might be expected, very much rarer. As a consequence of
these low background levels, the level at which radiation
effect can be detected is quite low.
It should be mentioned here that factors other than
radiation exposure can induce chromosomal aberrations.
Virus infections produce mainly (perhaps entirely) chromatid
type aberrations and thus cannot significantly confuse
measurement of radiation effect. Various chemical agents
used in therapy, however, do cause chromosome type
aberrations. In interpreting, the results of chromosome
aberration analyses It is therefore necessary to have
information on any prior chemotherapy, as well as prior
radiation exposure.
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Dose-effect kinetics.
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Chromosome breaks increase as a linear function of
radiatiou dose. Thus deletions show linear dose-effect
kinetics of the form
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Y = a + bD
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where Y is the yield, & the spontaneous frequency, D the
dose and b the coefficient of deletion production. With
low Le radiation the breaks involved in multiple-break
aberrations are independent events. Thus ring and dicentric
34
in
chromosomes, for example, are expected to increase as
the square of the dose. For practical purposes this
may be expressed
where c is the coefficient of ring and dicentric production.
(aberrations involving more than two breaks are, of course,
expected to display higher order kinetics).
High LET radiations.
Radiations of high LET, such as alpha particles or
fission neutrons, produce multiple-break aberrations with
first order kinetics. This is because more than me break
is likely to be produced in any cell that is "hit," and
the breaks involved are thus no longer independent.
Dose rate
What has been said so far has been based on acute
radiation exposures. Protraction or fractionation of
the exposure is expected to influence aberration yields.
If enough time elapses between the induction of various
breaks in a cell, the first break may have become
vnavailable for recombination by the time the second
occurs. Thus as the duration of the exposure (or the
time between dose fractions) increases, the yield per
rad will decrease, and the dose-effect kinetics will
approach linearity. The "half life" of breaks is of the
order of a few hours.
Cell division.
Cell division changes observed aberration yield
through elimination or alteration of aberrations. Acentric
chromosome fragments, by means of which most deletions are
detected, are generally lost at mitosis because they fail
to move to the poles of the spindle. The centromeres of
dicentric chromosomes sometimes segregate in such a way
as to cause the production of an anaphase bridge. Bridge
formation apparently leads to cell death and loss of the
averration. On the other hand, if a cell containing a
dicentric or a ring divides successfully a number of
times a clone of cells with the same aberration (but
lacking the acentric fragments) may become established.
A cell division, whether in vivo or in vitro, will
lead to a decrease in apparent aberration yield. Obviously,
then, leukocyte cultures must be harvested before any
second in vitro divisions occur.
Although the peripheral leukocytes which are made
to divide in culture do not normally divide while in the
peripheral circulation, they must ultimately be replaced
either directly or indirectly by cell division. One
might expect this replacement to be gradual, and expect
to find a decrease in the apperent frequency of deletions
and an increase in the proportion of multiple-break
aberrations without fragments with increasing time between
irradiation and sampling.
Partial-body exposure
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The leukocyte system is unique in that a partial-body
or seriously inhomogeneous exposure would be integrated
over the entira body by the rapid circulation and mixing
of the cells in the peripheral blood. For aberrations
with one-hit dose-effect kinetics such as deletions, or
as rings and dicentrics induced by f1ssion spectrum
neutrons, the effect of irradiating 10% of the body with
1,000 rad is expected to be the same as that of irradiation
of the whole body with 100 rad. For aberrations induced
with second order kinetics, however, this is not true; ten
times the yield would be expected in the case where only
10% of the body was exposed. Thus for acute, low LET
irradiation the ratio of two-hit to one-hit yields becomes
larger with increasing inhomogeneity of dose over the body.
Biological dosimetry.
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Obviously, if it is possible to describe the yield
of chromosome aberrations per unit dose with reasonable
accuracy, then it is also possible to calculate the dose
from the observed aberration yield. All that is required
is an accurately-known coefficient of aberration production.
It would be a mistake, however, to expect too much from
such dosimetry. Accurate dosimetry should indeed be
possible by measuring aberration yield immediately after
& person received an acute, homogeneous, whole-body
exposure to radiation of known quality. Unfortunately,
actual occupational exposures rarely, if ever, meet these
criteria. Furthermore, determination of the coefficients
of aberration production (the calibration of the dosimeter)
presents some problems. On the other hand, there is
usually some information available about the nature of the
exposure and in many cases the very complexity of the
system should allow useful conclusions to be drawn from
aberration analyses. For example, if it were known that
an acute, homogeneous, whole-body exposure had occurred,
the quality of the radiation might be deduced from the
ratio of one- to two-break aberrations. Similarly, in a
case where the quality of the radiation was known, the
one- to two-break aberration ratio might be used as a
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measure of inhomogeneity of exposure over the body.
RESUITS FROM OCCUPATIONALLY-EX.POSED PEOPLE

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The first cytogenetic studies of occupationally-exposed
people were those mentioned previously of eight men involved
in the Y-12 criticality accident (Fleidner, et al., 1959;
Bender and Gooch, 1962a, 1963). More recently, we have
had the opportunity to thoroughly study three men involved
in a plutonium criticality accident (Gooch, et al., 1964;
Bender, 1964; Bender and Gooch, 1966). Sasaki et al. (1963)
found increased aberration frequencies in "distinguished
radiologists" and in "radiation workers." Doida et al.
(1965) described aberrations in two "radiation workers"
who had received single moderate doses of X or gamma rays
in accidents nine rnths and one year before sampling,
but failed to detect increased aberration frequencies in
others receiving "negligible" occupational exposures over
& ten year period.
Increased aberration frequencies have been detected
in persons formerly employed in a thorium separation plant
(Dr. J. C. Cabral de Almeida, personal communication) and
also in former radium dial painters with high radium body
burdens (Dr. H. Lisco, personal comarunication). In addition,
we have results from cytogenetic studies of a number of
other accidentally exposed persons (Bender and Gooch,
unpublished). Included are four persons who received doses
in a gamma ray accident estimated to range from 10 to 100
rad, and two men who received low doses in connection with
a uranium criticality accident, as well as a number of
persons who received (or were thought to have received)
moderate partial body exposures to X or gamma rays.
While our experience with occupational y-exposed
people 18 still very limited, the results of all of these
Die utilitatii interidrive to
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studies are completely consistent with the predictions from
work with non-human material. All of the expected aberrati.on
types are found in cells from irradiated people. Immediately
after an acute radiation exposure acentric fragments are
present, but after a few weeks cells without the expected
scentric fragments begin to appear in appreciable numbers.
At the same time the aberration frequency also declines,
particularly the deletions. Even after many years, however,
an occasional call with acentric fragments is still found,
suggesting that a few cells may persist for years before
division. All of the expected aberration types, even
dicentrics, persist for long periods, and have been
observed in peripheral leukocytes many years after
irradiation. Presumptive clones of cells with apparently
identical aberrations have been observed. It appears that,
many of the aberrations, at least, confer no selective
disadvantage on the cells containing them.
We feel that the studies made to date have already
clearly demonstrated the utility of aberration analyses
in at least some classes of occupational radiation exposure.
In several cases of possible large overexposures, cytogenetic
analysis has enabled us to conclude positively that an
exposure of the magnitude suggested had not in fact
occurred. In another case a large disparity between the
yields of one- and two-break aberrations after an
accidental X ray exposure made us suspect that some part
of the body had received a much larger dose than indicated
by the worker's film badge. Further questioning and
reconstruction of the accident showed that his lower
body must have been in the beam for some time while the
film badge, worn higher, had not. Similarly, a high
two-break aberratio yield in the leukocytes of two men
who had "cleaned up" after a criticality accident led us
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to suspect that a low-level neutron pulse had occurred after
the original criticality, though the early evidence did not
suggest this possibility. Additional physical data confirming
the men's neutron exposure was later obtained.
Probably the most valuable application of cytogenetics
to occupational radiation exposure will ultimately be
biological dosimetry (Bender and Gooch, 1962b; Kelly and
Brown, 1965; Sugahara, et al., 1965). To this end, we
have experimentally determined the required coefficients of
aberration production by irradiating fresh whole human
blood samples (Bender and Gooch, 1962b; Gooch, et al., 1964;
and unpublished data). We have used the coefficients for
X and gamma rays and for f1ssion spectrum neutrons to
estimate doses in a number os the accidental exposures
we have studied. Although the observed prompt aberration
yields in all the cases were low, and sampl.ing errors
consequently high, the doses estimated agreed fairly
well with what physical estimates were available. While
the argument is admittedly somewhat circular, this agreement
has given us confidence ti at the coefficients derived from
in vitro irradiations may be applied to the case of
irradiation in vivo as well.
It is obvious that a good deal more experience will
be required before we can completely evaluate the potential
of chromosome analysis as a tool for detecting and measuring
occupational radiation exposures. Chromosome aberration
monitoring will certainly never replace physical dosimeters
for routine personnel monitoring. It is already clear,
however, that chromosome analyses can provide valuable
information, both qualitative and quantitative, in certain
cases.

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References
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Bender, M. A, X ray Induced chromosome aberrations in normal
diploid human tissue cultures. Science 126, pp. 974-975
(1957).
Render, M. A, Chromosome aberrations in irradiated human subjects.
Ann. N. Y. Acad. Sci. 214, pp. 249-251 (1964).
Bender, M. A, and P. C. Gooch, Persistent chromosome aberrations
in Irradiated human subjects. Radiation Res. 16, pp. 44-56
(1962a).
Bender, M. A, and P. C. Gooch, Types and rates of X ray induced
chromosome aberrations in human blood irradiated in vitro.
Proc. Natl. Acad. Sci. U.S. 48, pp. 522-532 (1962b).
Bender, M. A, and P. C. Gooch, Persistent chromosome aberrations
in irradiated human subjects II. Three aná one-half year
Investigation. Radiation Res. 18, pp. 389-396 (1963).
Bender, M. A, and P. C. Gooch, Somatic chromosome aberrations
Induced by human whole-body irradiations I. The "Recuplex"
criticality accident. Radiation Res. In press (1966).
Doida, Y., T. Sugahara, and M. Horikawa, Studies on some radiation-
induced chromosome aberrations in man. Radiation Res. 26,
pp. 69-83 (1965).
· Evans, H. J., Chromosome aberrations induced by ionizing radiation.
Intern. Rev. Cytol. 12, pp. 221-321 (1962).
Fleidner, T. M., E. P. Cronkite, V. P. Bond, J. R. Rubini, and
G. Andrews, The mitotic index of human bone marrow in healthy
individuals and irradiated human beings. Acta haemat. 22(2),
pp. 66-78 (1959).
Gooch, P. C., M. A Bender, and M. L. Randoluh, Chromosome aberrations
induced in human somatic cells by neutrons. Biological Effects
of Neutron and Proton Irradiation I, pp. .325-342, International
Atomic Energy Agency, Vienna, 1964.
Kelly, S., and C. D. Brown, Chromosome aberrations as a biological
dosimeter. Am. J. Public ilealth 5519), pp. 1419-1429 (1965).
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Moorhead, P.Si., P. C. Nowell, W. K. Mellman, D. M. Battips, and
D. A. Hungerford, Chromosome preparations of leukocytes
cultured from human peripheral blood. Exptl. Cell Res. 20,
pp. 613-616 (1960).
Sasaki, M., R. E. Ottoman, and A. Norman, Radiation-induced :
chromosome aberrations in man. Radiology.87, pp. 652-656
(1963).
Sugahara, T., Y. Doida, Y. Veno, and T. Hashimoto, Biological
dose estimation by means of radiation-induced chromosome
aberrations in human blood. Nippon Acta Radiol. 25,
pp. 816-823 (1965).
Tiia, J. A., and A. Levan, The chromosome number of man.
Hereditas 42, pp. 1-6 (1956).
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