Mutagenesis vol.12 no.4 pp.195-200, 1997
Detection of chromosomal alterations affecting the Icen-lql2
region in irradiated granulocytes and lymphocytes by multicolour
FISH with tandem DNA probes
Doppalapudi S.Rupa, Leslie S.Hasegawa and
David A.Eastmond1
Environmental Toxicology Graduate Program, Department of Entomology,
University of California, Riverside, CA 92521, USA
'To whom correspondence should be addressed
A multicolour tandem labelling fluorescence in situ hybridization (FISH) procedure was used to compare the frequencies of radiation-induced chromosome breakage and
hyperdiploidy of chromosome 1 occurring in non-cultured
granulocytes and Go lymphocytes with those observed in
cultured metaphase and interphase lymphocytes. Whole
blood, obtained from healthy male donors, was exposed
in vitro to 0,100,200,300 and 400 cGy of ionizing radiation
from a 137Cs source. Aliquots containing granulocytes and
Go lymphocytes from each dose were treated immediately
with hypotonic KC1 on ice and harvested. Cells were
hybridized with a- and classical satellite probes to the
Icen-ql2 region of chromosome 1 and the frequencies of
hyperdiploidy and breakage affecting this region were
determined. Elevated dose-related frequencies of breakage
were detectable hi both lymphocytes and granulocytes
immediately following radiation and decreased rapidly over
the first 0.25-2 h. In a second series of experiments, the
frequencies of hyperdiploidy and breakage for uncultured
granulocytes and Go lymphocytes were compared with
interphase and metaphase cells following 48-51 h of culture.
Similar and significant dose-related increases in breakage
were seen for the granulocytes, Go lymphocytes, 48 h
cultured interphase and metaphase lymphocytes. A minor
increase in hyperdiploidy was seen in the irradiated
cultured cells, whereas no hyperdiploid cells were detected
in the non-cultured cells. These results indicate that, in
general, granulocytes and lymphocytes show similar sensitivity to radiation-induced damage and that cell culture is
not required for chromosome breakage to be observed
microscopically using this FISH procedure.
Introduction
Human peripheral blood has been widely used in genetic
toxicology as a test system for in vivo and in vitro studies. It is
composed primarily of two distinct cell populations: nucleated
leukocytes and enucleated erythrocytes. Leukocytes include
granulocytes, lymphocytes and monocytes, all of which differ
in function, size and half-life. For many years structural and
numerical chromosomal aberrations have been evaluated in
human biomonitoring and in vitro studies by using conventional
cytogenetic analyses of metaphase preparations of cultured
lymphocytes. However, a major drawback of this approach is
that metaphases can only be obtained from actively dividing
cells, restricting the tissues and cell types which can be
evaluated. This type of analysis cannot be performed on
granulocytes because these cells have terminably differentiated
© UK Environmental Mutagen Society/Oxford University Press 1997
prior to their release into the peripheral blood and are no
longer capable of mitosis. Since granulocytes comprise 5070% of the peripheral blood leukocyte population and are the
lineage most affected by leukaemia-inducing agents, detection
of DNA damage in these cells may be important for human
biomonitoring and in understanding leukemogenesis.
Fluorescence in situ hybridization (FISH) widi chromosomespecific DNA probes has been used to detect aneuploidy in
interphase cells (Pinkel et al, 1986; Eastmond and Pinkel,
1990), significantly increasing the number and types of tissues
available for analysis. In earlier banding studies it has been
shown that the pericentric heterochromatic regions frequently
exhibit breakage following treatment by ionizing radiation and
a variety of chemical agents (Brogger, 1977; Yunis et al,
1987; Sabatder et al, 1989). In view of these studies, we have
recently developed a new multicolour FISH tandem labelling
approach to differentiate between hyperdiploidy and breakage
affecting the centric/pericentric regions of chromosomes 1 and
9 in interphase human lymphocytes. Previous studies in our
laboratory have shown that this FISH approach can detect
breakage induced in vitro by hydroquinone and ionizing
radiation as well as in vivo in a pesticide-exposed population
(Eastmond et al, 1994; Rupa et al, 1995; Hasegawa et al,
1995). We have extended these studies to determine whether
this FISH approach could be used to detect chromosomal
breakage in non-dividing granulocytes and lymphocytes and
to compare the relative sensitivity of these cells to cultured
interphase and metaphase lymphocytes following treatment
with ionizing radiation.
Materials and methods
Irradiation and slide preparation
Peripheral blood was collected in heparinized vacutainers from healthy male
donors. Blood from at least two different donors was used for each experiment.
Aliquots of blood were exposed to 0, 100, 200, 300 or 400 cGy of ionizing
radiation from a 137Cs source at room temperature at a dose rate of 85.8 cGy/
min. For the initial repair studies, an aliquot from each exposed sample was
subjected to hypotonic treatment (0.075 M KC1) for 30 min on ice immediately
following radiation, fixed using Carnoy's fixative (methanol:acetic acid, 3:1)
and dropped on slides for the analysis of granulocytes and Go lymphocytes.
Additional aliquots were processed in the same fashion at 0.25 and 2 h
following irradiation.
In the second series of experiments, the irradiated blood was harvested
~3 h post-irradiation by treating with hypotonic KC1 treatment for 30 min at
room temperature and fixation as described above. The remaining blood was
cultured with phytohaemagglutinin (PHA) stimulation (2.36%; Gibco-BRL,
Grand Island, NY, USA) for 48-51 h and harvested foT interphase and
metaphase analyses. Colcemid (0.05 Ug/ml) (Sigma Chemical Co, St Louis,
MO, USA) was added to metaphase cultures for the final 3 h. Cells were
treated with hypotonic KC1 at room temperature, fixed with Camoy's fixative,
and dropped onto cleaned slides. All slides were stored at - 2 0 T under a
nitrogen atmosphere until used.
Fluorescence in sin: hybridization
Two chromosome 1-specific DNA probes were used for tandem labelling: an
a-satellits prcttt specific for the centrorrxric region (lcenj fwlllard and Waye,
1987) and a classical satellite probe, pUC1.77, (Cooke and Hindley, 1979)
specific for the pericentric heterochromatin region (Iql2). The classical
satellite prcbe was labelled v, ith digoxigenin-11 -dUTP (Bcehringer Mannheim,
195
D-S.Rupa, L.E.Hasegawa and D.A.Eastmond
Indianapolis, IN, USA) by nick translation in our laboratory according to the
protocol of the manufacturer (Bethesda Research Laboratories, Gaithersburg,
MD, USA) and the biotinylated a-satellite probe was purchased from Oncor
(Gaithersburg, MD, USA)
Slides were denatured in 70% formamide/0.3 M NaCl plus 0.03 sodium
citrate (2X SSC; pH 7.0) and dehydrated in an ethanol series (70, 85 and
100*). An aliquot of 10 ul of a hybridization mixture containing 1 |il of
digoxigenin-labelled classical satellite probe (5-20 ng), 1 \i\ biotin-labelled
a-satellite probe (5—20 ng), 1 u.1 sonicated herring sperm DNA (1 mg/ml)
and 7 ul of MM 2.1 hybridization mix (final concentration of 55% fonmamide/
IX SSC/10% dextran sulphate) was denatured at 70°C and applied to the
slides. Coverslips were sealed on the slides which were then hybridized
overnight at 37°C Slides were washed in 60% formamide/2X SSC, three
times for 5 min each and once in 2X SSC all at 45°C. The slides were then
rinsed twice in PN buffer (0.1 M phosphate buffer, pH 8.0, containing 0 5%
NP-40) at room temperature. The digoxigenin-labelled classical satellite probe
was detected using a mouse anti-digoxigenin antibody [3.2 ug/ml in PN buffer
with 5% non-fat dry milk supernatant (PNM); Boehnnger Mannheim] followed
by a Texas Red-labelled goat anti-mouse antibody (10 ug/ml in PNM;
Molecular Probes, Eugene, OR, USA). The biotinylated a-satellite probe
was detected using fluorescein-conjugated avidin (5 Ug/ml in PNM; Vector
Laboratories, Burhngame, CA, USA). The fluorescein signals were amplified
once using a biotinylated anti-avidin antibody (5 ng/ml in PNM; Vector
Laboratories) followed by another layer of fluorescein-conjugated avidin 4',6diamidino-2-phenyhndole (DAPI, 2.5 |ig/ml in diphenylenediamine anufade)
was used to counterstain the DNA
Scoring criteria
Slides were observed using a Nikon Optiphot II microscope equipped with
fluorescence attachment and a triple-band-pass (Chroma, Brattleboro. VT,
USA), FITC (Nikon B2A) and Texas Red (Chroma) filters. For each experiment
1000 cells per dose for each cell type were scored for aberrations involving
the Icen-ql2 region. The criteria used for scoring the coded slides were: (i)
polymorphonuclear granulocytes were identified by their irregular shape or
lobular structures distinguishing them from round mononuclear lymphocytes;
(li) hybridization regions containing both the a-satellite (green/yellow) and
classical satellite (red) signals were scored as one copy of chromosome 1
Doublets or diffused hybridization signals were scored as one hybridization
region. Whenever the FTTC-a-satellite signals were weak, a Nikon B2A filter,
which is specific for fluoroscein, was used to verify the signal; (in) two
hybridization regions containing both a- and classical satellite signals and
another hybridization region containing only the classical satellite region were
scored as two copies of chromosome 1 with a break in the classical satellite
region of one chromosome. A Texas Red filter was used to confirm the breaks;
(iv) a clear separation (generally more than the width of the probe region)
between the a- and classical satellite regions was scored as a break between
probes, (v) breakage events in metaphases were classified into two categories.
clear interphase breaks and possible interphase breaks. Clear interphase breaks
occurred within the classical satellite region and would most likely be seen
in interphase as well as metaphase cells. Possible interphase breaks were
classified as those which occurred within or distal to the classical satellite
region and where the acentric fragment was not present in the metaphase In
these cases, it is not certain whether or not these types of breaks would be
seen in interphase cells. Previous studies had indicated the frequency of 'clear
interphase breaks' in the metaphase cells corresponded more closely with the
breakage frequency observed in the interphase cells. As result the primary
focus of this paper will be on those metaphase cells containing 'clear
interphase breaks'.
Statistical analysis
The presence of a dose-related trend for the breakage and hyperdiploid>
induced by radiation was determined for each cell type and using the CochranArmitage binomial trend lest (Margolin and Risko, 1988). The CochranArmitage binomial trend test was also used to assess DNA repair during the
first 2 h for each dose and cell type. The frequency of hyperdiploidy and
breakage in each cell type was compared with its respective control using a
one-tailed Fisher's exact test A two-tailed Fisher's exact test was used to
compare the frequencies of breakage and hyperdiploidy between the granulocytes and the lymphocytes. Critical values were determined using a 0.05
probability of type 1 error.
Results
The tandem labelling approach was effective in distinguishing
normal cells from cells having aberrations in the Icen—q 12
region in the four cell types: gTanulocytes, Go lymphocytes,
interphase and metaphase cells. Examples of cells following
i96
hybridization with the tandem-labelled a- (green/yellow) and
classical satellite (red) regions of chromosome 1 are illustrated
in Figure 1. Figure la shows a granulocyte and Go lymphocyte
containing the signals for two normal copies of chromosome
1. A granulocyte exhibiting a break within the classical satellite
region is presented in Figure lb and another granulocyte with
a break between the a- and classical satellite regions is shown
in Figure lc.
To determine the influence of DNA repair occurring following irradiation, the frequencies of breakage and hyperdiploidy
were determined in cells harvested immediately and at 0.25
and 2 h following radiation treatment. Highly significant doserelated increases in breakage were detected in both granulocytes
and lymphocytes at each of three time points (Figures 2
and 3). Repair occurred rapidly with breakage frequencies
decreasing by ~30% over the first 0.25 h and by 55% over 2
h. No effect of radiation was seen in the frequencies of
hyperdiploid cells.
In the second series of experiments, the blood was irradiated
and held for ~3 h prior to harvest or culture, to allow initial
DNA repair to be completed. The frequencies of breakage and
hyperdiploidy affecting the Icen-ql2 region were determined
for the non-cultured Go lymphocytes and granulocytes and
compared with those seen in the interphase and metaphase
preparations following culture for 48-51 h. A significant doserelated trend in the induction of breaks was seen in the
irradiated granulocytes, Go lymphocytes and 48 h cultured
interphase and metaphase cells (Figure 4). The frequency of
breakage was significantly increased over the respective control
value for each cell type and at all irradiated dose levels except
at the lowest dose (100 cGy) tested in the cultured interphase
cells. In the control granulocytes, the frequency of breakage
was 7/2000 whereas the frequency of breakage in the irradiated
granulocytes ranged from 23 to 50 per 2000 cells over the
range of doses tested. The frequency of induced breaks in
the irradiated granulocytes was slightly higher than the Go
lymphocytes but the difference was not significant. A similar
dose response was seen for each of the lymphocyte cell types,
the Go lymphocytes, cultured interphase and metaphase cells
(Figure 4). The frequency of radiation-induced breaks in the
Go lymphocytes was 14-37 per 2000 cells for tested doses
and 5 per 2000 in controls. In the irradiated cultured interphase
cells, the frequencies ranged from 11 to 37 per 2000 cells in
the treated cultures and 5 per 2000 in controls. In the metaphase
cells, the frequencies of 'clear interphase breaks' ranged from
17 to 41 per 2000 cells in the treated cultures, and 2 per 2000
in the controls, whereas 'possible interphase breaks' were 516 per 2000 in the irradiated cells and 1 per 2000 in the controls.
Of the "clear interphase breaks" that could be evaluated,
61% represented chromosomal breaks and 39% represented
chromosomal exchanges.
The incidence of hyperdiploidy in the various cell types
following irradiation is given in Figure 5. A weakly significant
increase in hyperdiploidy was seen in both the cultured
interphase and metaphase cells but not in non-cultured cells.
None of the individual treatments differed significantly in the
interphase cells whereas significant differences in hyperdiploidy were seen in the 100 and 400 cGy treatments of the
metaphase cells.
Discussion
In this study, multicolour FISH with adjacent DNA-specific
probes for chromosome 1 was used to detect breakage affecting
Detection of chromosomal alterations by multicolour FISH with tandem DNA probes
Fig. 1. Granulocytes and a Go lymphocyte following fluorescence in situ hybridization with biotin-labelled a-satellite probe and a digoxigenin-labelled
classical satellite probe for chromosome 1. Using a triple-band-pass filter and DAPI as a nuclear counterstain, the a-satellite region will appear as green/
yellow and the adjacent classical satellite region will appear red. (a) Granulocyte and Go lymphocyte containing two hybridization regions indicating the
presence of two normal copies of chromosome 1 in each cell, (b) Granulocyte containing two copies of chromosome 1 and a break within die classical
satellite region, (c) Granulocyte exhibiting one intact copy of chromosome 1 and a second containing a break between the a- and classical satellite regions.
the Icen-lql2 region in non-cultured granulocytes, Go
lymphocytes and cultured interphase and metaphase lymphocytes from irradiated peripheral human blood. Each of these
cell types showed a significant increase in breakage following
exposure to radiation at doses between 100 and 400 cGy. High
frequencies of breakage affecting the centric/pericentric region
of chromosome 1 were observed immediately following irradiation and significant repair occurred over the initial 0.25-2 h
after treatment. After the initial repair period, similar frequencies of breakage were seen in the cultured and non-cultured
cells.
In general, these results are similar to those reported in
previous studies measuring genetic damage in granulocytes and
non-cultured lymphocytes following treatment with radiation or
chemical clastogens. For example, previous reports of irradiated
interphase lymphocytes combined with premature chromosome
condensation have shown that repair of DNA breaks occurs
rapidly following irradiation with >50% of breaks being
repaired within 2 h (Pantelias and Maillie, 1985; Greinert
et al, 1995). In these experiments, rapid repair was observed
in both the Go lymphocytes and the granulocytes. Using this
FISH technique, similar frequencies of breakage were also
detected in Go lymphocytes harvested ~3 h following irradiation and those detected in 51 h cultured metaphase preparations indicating diat relatively little repair occurred during
this period. A comparison between the breakage frequencies
observed in these studies and those previously reported can
be made after correcting for the proportion of the human
genome targeted during the FISH procedure. The heterochromatin of chromosome 1 comprises -0.7% of the human genome
based upon high resolution banding (Hamden and Klinger,
1985). In general, the breakage frequencies observed with this
FISH procedure range between 50 and 126% of those reported
using conventional metaphase analyses or studies of premature
19?
D^.Rupa, L.E.Hasegawa and D.A.Eastmond
D
•
E3
0
e
OcGy
100 cQy
200 c<3y
300 c<3y
400 cOy
3
CO
O
o
s
8.
a
a
JC
Qranulocytaa Qo lymphocyte* Intarphaaas
ID
CD
100
200
300
Cell Type
400
Dose (cGy)
Fig. 2. The frequencies of breakage in the lcen—ql2 region in irradiated
granulocytes immediately (0 h), 0.25 and 2 h following irradiation. Data
points represent the mean and SE of two or three separate experiments.
Fig. 4. The frequencies of breakage/exchanges in the Icen-ql2 region in
granulocytes, Go lymphocytes and cultured interphase and metaphase cells
following various doses of radiation to whole blood
30
(0
a
o
o
Uttaphasas
o
o
o
20-
B
10-
D OcQy
E3 100cGy
•
•
•
200 cOy
300 cGy
400 cGy
a
E
S
a
a.
Qranulocytaa Qo lymphocytaa
Intarphaaaa
Uataphaaea
Cell Type
Z
m
100
200
300
400
Fig. 5. The frequencies of hyperdiploidy detected in various cultured and
non-cultured cells following irradiation of whole blood
Dose (cGy)
Fig. 3. The frequencies of breakage in the Icen-ql2 region in irradiated
lymphocytes immediately (0 h), 0.25 and 2 h following irradiation. Data
points represent the mean and SE of two or three separate experiments.
condensed chromosomes of interphase lymphocytes (Caspersson et ai, 1972; Holmberg and Jonasson, 1973; Buckton,
1976; Dubos et ai, 1978; Pantelias and Maillie, 1985; Greinert
et ai, 1995). These estimates are in generally good agreement
considering the differences in experimental protocol, doses,
ionization source, scoring criteria and staining procedures
between the two types of studies. In concordance with the in
situ hybridization results reported here, other studies in which
DNA damage was measured using alkali-labile sites have
reported similar frequencies of damage in granulocytes and
lymphocytes following exposure of cells to ionizing radiation
or ethylating agents (Schutte et ai, 1988; Vijayalaxmi et ai,
1993).
In both the interphase and metaphase cultures, a weak but
significant increase in hyperdiploidy was seen with increasing
dose of radiation, agreeing with previous studies that have
reported that radiation is capable of inducing hyperdiploidy in
cultured lymphocytes (Barquinero et ai, 1993). The increases
in hyperdiploidy in this study were seen only in cultured
interphase and metaphase lymphocytes but not in the noncultured cells, a result consistent with a real radiation-induced
increase in hyperdiploidy which would require at least one
cell division following exposure. The relatively low magnitude
of the increase is probably related to the use of probes to a
198
single chromosome and 48-51 h cultures which would contain
both first and second division cells. Previous results from our
laboratory and others have indicated that a significant number of
second division metaphases are present in 48-51 h lymphocyte
cultures (D.Eastmond and D.Rupa, unpublished data; Galloway
et ai, 1986). However, the presence of significant numbers of
first division cells in the cultures would tend to reduce the
magnitude of the hyperdiploid response. As an additional
consideration, the higher frequencies and lack of a clear dose
response seen in our metaphase studies indicate that a portion
of the hyperdiploidy seen in the metaphase cells may be related
to a scoring artefact rather than a radiation effect. A fraction
of the metaphases containing three or four hybridization spots
are likely to have resulted from scoring two overlapping
diploid metaphases with similar condensation rather than true
hyperdiploid or polyploid cells.
The ability to detect chromosomal breakage using the tandem
label approach in non-cultured lymphocytes and granulocytes
indicates that cell culture is not required to detect radiationinduced breakage. This is somewhat surprising given the wide
separation of the chromosomal fragments that must occur for
the break to be visible through the microscope. Although
hypotonic treatment may contribute to the separation, we
believe that the movement of the fragments occurs primarily
as a result of physical forces exerted within the nucleus during
the drying of the cells on the microscope slides. In other
studies, we have detected breakage affecting the lq 12 region
in the nucleated cells of blood smears that had not previously
Detection of chromosomal alterations by multicolour FISH with tandem DNA probes
been subjected to hypotonic treatment (D.Rupa and
D.Eastmond, unpublished data). These results suggest that
with proper slide preparation it may be possible to use
the tandem label approach to detect chromosomal changes
occurring in other non-cultured cells or tissues.
Another advantage of using non-cultured cells such as Go
lymphocytes or granulocytes for cytogenetic analysis is that
the lack of a requirement for cell culture should facilitate the
processing of samples, particularly in remote field locations.
Moreover, the use of Go lymphocytes for cytogenetic studies
may eliminate the individual variation in the stimulation of
lymphocytes and exposure-related cell cycle delay as well as
variability in the rates of cell growth and death. This approach
will, however, have limited usefulness for detecting hyperdiploidy or breakage in cases where in vitro DNA synthesis or a
mitosis is required for the lesion to be expressed (e.g. S phasedependent clastogens).
The use of short-lived cells such as granulocytes may allow
a temporal relationship to be established between exposure to
an agent and the subsequent induction of chromosomal damage
in human biomonitoring studies. It should be noted that the
half-life of circulating granulocytes is quite short (~7-24 h)
for most normal subjects (Dancey et al, 1976; Athens, 1993;
Vijayalaxmi et al., 1993). As a result, it may be necessary to
process blood samples relatively quickly after collection to
obtain an adequate recovery of granulocytes. In some studies
from our laboratory, we have found that samples processed
~24 h after collection did not yield adequate numbers of
granulocytes for scoring, presumably due to the lysis of these
cells in the heparinized tubes during transport. As an additional
note, the hypotonic treatment used in these studies slightly
altered the lobular structure of the granulocytes into more
roundish structures. However this did not interfere significantly
with scoring.
There is increasing evidence that indicates that the heterochromatin regions of the human genome, particularly qh regions
of chromosomes 1,9, 16 and Y are important origins of stable
chromosome rearrangements which play a role in both the
early and late stages of tumour development (Atkin and BritoBabapulle, 1981; Larizza et al, 1988, 1989; Doneda et al,
1989). In a survey of chromosome 1 abnormalities in 217
neoplasms, it was observed that 49.9% of the breaks were in
the centromeric region (Brito-Babapulle and Atkin, 1981). In
addition, recent evidence indicates that breakage affecting the
centromeric and pericentric heterochromatin regions of human
chromosomes can lead to mutations, delayed chromosomal
rearrangements and genomic instability (Smith and Grosovsky,
1993; Grosovsky et al, 1996). In view of these associations,
the ability to detect chromosomal aberrations in the heterochromatin of a variety of human cell types promises to be a
potentially powerful tool for studies of carcinogenic agents
and cancer developing in a wide range of human tissues.
In summary, the present study demonstrates that cytogenetic
information on structural and numerical aberrations can be
obtained using FISH with tandem labelling in non-stimulated
Go lymphocytes and in terminally differentiated granulocytes.
In addition, similar frequencies of breakage were seen between
the non-cultured and the cultured interphase and metaphase
cells. Although this type of analysis provides only limited
information about specific types of chromosomal aberrations,
the flexibility of the method and its potential for use on any
nucleated cell or tissue suggest that it might be valuable for
studies of human malignancies and for human biomonitoring.
Acknowledgements
This research was supported in part by the US Environmental Protection
Agency grant (R820994-01-1).
References
AthensJ.W. (1993) Granulocytes-neutrophils. In Lee.R.G., Bithell.T.C,
FoersterJ., Athens J.W. and Lukensj.N. (eds), Wintrobe's Clinical
Hematology. Vol. 1. Lea and Febiger, Philadelphia, London, pp. 223-298.
Atkin,N.B. and Brito-Babapulle.V. (1981) Heterochromatin polymorphism and
human cancer. Cancer Genet. Cytogenet., 3, 261-272.
BarquineroJ.F., Barrios.L-. Caballing.R., Miro.R., Ribas.M., Subias,A. and
EgozcueJ. (1993) Cytogenetic analysis of lymphocytes from hospital
workers occupationally exposed to low levels of ionizing radiation. Mutat.
Res.. 286, 275-279.
Brito-Babapulle.V. and Atkin^N.B. (1981) Break points in chromosome #1
abnormalities of 218 human neoplasms. Cancer Genet. Cytogenet., 4,
215-225.
Brogger.A. (1977) Non-random localization of chromosome damage in human
cells and targets for clastogenic action. Chromosomes Today, 6, 297—306.
Buckton.ICE. (1976) Identification with G and R banding of the position of
breakage points induced in human chromosomes by in vitro X-irradiation.
Int. J. Radiat. Biol, 29, 475-488.
Caspersson.T, Haglund.U., Lindell.B. and Zech.L- (1972) Radiation-induced
non-random chromosome breakage. Exp. Cell. Res., 75, 541—543.
Cooke.HJ. and HindleyJ. (1979) Cloning of human satellite IE DNA:
different components are on different chromosomes. Nucleic Acids Res., 6,
3177-3197.
DanceyJ.T., Deubelbeiss.K.A., Harker.LA. and Finch.C.A. (1976)Neutrophil
kinetics in man. /. Clin. Invest., 58, 705-715.
DonedaX.. Ginelli.E., Agresti.A. and LarizzaX. (1989) In situ hybridization
analysis of interstitial C-heterochromatin in marker chromosomes of two
human melanomas. Cancer Res., 49, 433-438.
Dubos.C, Pequignot,E.V. and Dutrillaux.B. (1978) Localization of y-rays
induced chromatid breaks using a three consecutive staining technique.
Mutat. Res., 49, 127-131.
Eastmond,D.A. and Pinkel.D. (1990) Detection of aneuploidy and aneuploidyinducing agents in human lymphocytes using fluorescence in situ
hydridization with chromosome-specific DNA probes. Mutat. Res., 234,
303-318.
Eastmond.D.A., RupaX>S. and HasegawaX.S- (1994) Detection of
hyperdiploidy and chromosome breakage in interphase human lymphocytes
following exposure to the benzene metabolite hydroquinone using multicolor
fluorescence in situ hybridization with DNA probes. Mutat. Res., 322, 9-20.
Galloway.S.M., Berry.P.K., Nichols.W.W., Wolman.S.R., Soper.K.A.,
StolleyJ'.D. and Archer.P. (1986) Chromosome aberrations in individuals
occupationally exposed to ethylene oxide, and in a large control population.
Mutat. Res., 170, 55-74.
Greinert,R., Detzler.E., Volkmer.B and Harder.D. (1995) Kinetics of the
formation of chromosome aberrations in X-irradiated human lymphocytes:
analysis by premature chromosome condensation with delayed fusion.
Radiat. Res., 144, 190-197.
Grosovsky.A J., Parks.K.K., Giver.C.R. and Nelson.S.L. (1996) Clonal analysis
of delayed karyotypic abnormalites and gene mutations in radiation-induced
genetic instability. Mol. Cell. Biol., 16, 6252-6262.
Harnden.D.G. and Klinger.G.P. (eds) (1985) An International System for
Human Cytogenetic Nomenclature. Vol. 21. ISCN/Birth Defects Foundation,
March of Dimes, pp. 49-65.
HasegawaX.S., Rupa,D.S. and Eastmond.D.A. (1995) A method for the rapid
generation of alpha- and classical satellite probes for human chromosome
9 by polymerase chain reaction using genomic DNA and their application
to detect chromosomal alterations in interphase cells. Mutagaxesis, 10,
471-476.
Holmberg,M. and JonassonJ. (1973) Preferential location of X-ray induced
chromosome breakage in the R-bands of human chromosomes Hereditas,
74, 57-68.
LarizzaX., DonedaX., GinelliJE. and Fossati.G. (1988) C-heterochromatin
variation and transposition in tumor progression. Adv. Exp. Med. BioU, 233,
309-318.
LarizzaX., DonedaX., Rodolfo,M. and Fossati.G. (1989) High incidence of
chromosomal lesions involving C-heterochromatin in four human melanoma
lines. Clin. Exp. Metastasis, 7, 633-644.
MargoIin,B.H. and Risko.KJ. (1988) The statistical analysis of in vivo
genotoxicity data: case studies of the rat hepatocyte UDS and mouse bone
marrow micronucleus assays. In AshbyJ., de SerresJU., Shelby,M.D.,
Margolin.B.H., Ishidate.M. J r and Becking.G.C. (eds), Evaluation of Short-
199
D.S.Rupa, L.E.Hasegawa and D.A.Eastmond
Term Tests for Carcinogens Vol. 1. Cambridge University Press, Cambridge.
pp 129-142.
Pantehas.G.E. and Mailhe.D.H. (1985) Direct analysis of radiation-induced
chromosome fragments and rings in unstimulated human penpheral blood
lymphocytes by means of the premature chromosome condensation
technique. Mutat. Res., 149, 67-72.
Pinkel.D., GrayJ.W., Trask,B., van den Engh.G., FuscoeJ. and van Dekken,H.
(1986) Cytogeneuc analysis by in situ hybridization with fluorescently
labeled nucleic acid probes. Cold Spring Harbour Symp. Quant. Biol, 51
(Part 1), 151-157.
Rupa,D.S., Hasegawa,L. and Eastmond.D.A. (1995) Detection of chromosomal
breakage in the Icen-lql2 region of interphase human lymphocytes using
multicolor fluorescence in situ hybridization with tandem DNA probes.
Cancer Res., 55, 640-645.
Sabatier.L , Muleris.M, Prieur,M., Al Achkar.W., Hoffschir.F, Prod'hommeRicoul.M., Gerbault-Sercau.M., Viegas-Pequignot.E. and Dutrillaux,B.
(1989) Specific sites of chromosomal radiation-induced rearrangements. In
Jolles.G. and Cordier,A. (eds), New Trends in Genetic Risk Assessment
Academic Press, London, pp. 211-224.
Schutte.H.H., van der Schans.G.P. and Lohman.P.H (1988) Comparison of
induction and repajr of adducts and of alkali-labile sites in human
lymphocytes and granulocytes after exposure to ethylating agents. Mutat.
Res., 194, 23-37.
Smith.L.E. and Grosovsky,A J. (1993) Genetic instability on chromosome 16
in a human B lymphoblastoid cell line. Somatic Cell Mol. Genet, 19,
515-527.
Vijayalaxmi, Strauss,G H. and Tice.R.R. (1993) An analysis of gamma-rayinduced DNA damage in human blood leukocytes, lymphocytes and
granulocytes. Mutat. Res., 292, 123-128
Willard.H.F. and WayeJ.S (1987) Hierachial order in chromosome-specific
human alpha satellite DNA. Trends Genet., 3, 192-198.
YunisJ J., Soreng.A.L. and Bowe.A.E. (1987) Fragile sites are targets of
diverse mutagens and carcinogens Oncogene, 1, 59-69.
Received on July 10, 1996; accepted on March 6, 1997
2C0