Download Induction of Chromosomal Damage in Chinese

[CANCER RESEARCH
45, 2320-2325,
May 1985]
Induction of Chromosomal Damage in Chinese Hamster Ovary Cells by Soluble
and Particulate Nickel Compounds: Preferential Fragmentation of the
Heterochromatic Long Arm of the X-Chromosome by Carcinogenic
Crystalline NiS Particles1
Pramila Sen and Max Costa2
Department of Pharmacology, University of Texas Medical School at Houston, Houston, Texas 77025
ABSTRACT
Treatment of intact Chinese hamster ovary cells with crystal
line NiS and NiCI2 resulted in the induction of chromosomal
aberrations which included gaps, breaks, and exchanges. The
incidence of these aberrations increased in a time- and concen
tration-dependent fashion. NiCI2 was more potent in inducing
chromosomal aberrations in cells that were maintained with a
salts/glucose medium during metal treatment than when cells
were treated in culture growth medium. Chromosomal aberra
tions induced by NiCI2 occurred randomly among the autosomal
arms; however, the heterochromatic centromeric regions of the
chromosomes were preferentially damaged. In addition to induc
ing the same type of aberrations found with NiCI2, crystalline NiS
particles also caused the selective fragmentation of the hetero
chromatic long arms of the X-chromosomes. This fragmentation
was attributed to the difference in the mechanism of delivery of
nickel ions from phagocytized crystalline NiS particles which
aggregate around the nuclear membrane and release large
amounts of nickel ions from a dissolving phagocytized particle.
Previous studies have demonstrated that treatment of intact
cells with crystalline NiS particles produces a considerably higher
level of nickel in the nucleus compared with similar exposure to
water-soluble NiCI2. Since heterochromatin is known to form the
inside lining of the interface nucleus, nickel ions, as they are
solubilized from a phagocytized particle and enter the nucleus,
are likely to encounter heterochromatin before they interact with
euchromatin. In contrast, nickel ions derived from NiCI2 do not
preferentially accumulate in the cell, and those ions that enter
the cell are taken up by a nonphagocytic mechanism. It is
proposed that when cells are treated with high levels of NiCI2 in
an attempt to achieve the cellular levels of nickel produced by
NiS phagocytosis, this overloading results in cytotoxic responses
rather than the preferential fragmentation of heterochromatin
observed with particles. Since liposome-mediated delivery of
NiCI2 also results in fragmentation of the long arm of the Xchromosome, the selective breakage of heterochromatin by NiS
particles may be due solely to the mechanism of Ni2+ delivery in
cells.
INTRODUCTION
Nickel compounds represent well-established human carcino
gens based upon epidemiológica! studies (1-3). Certain nickel
1This work was supported by Grant CA 24581 from the National Cancer Institute
and by Contract DE AS05-81-ER 600 16 from the U.S. Department of Energy.
2 To whom requests for reprints should be addressed, at Department of Phar
macology, University of Texas Medical School at Houston, P. 0. Box 20708,
Houston, TX 77025.
Received 11/6/84; revised 1/29/85; accepted 2/1/85.
CANCER RESEARCH
compounds such as crystalline nickel sulfide and subsulfide are
extremely potent carcinogens in experimental animals (4-6),
while others such as amorphous NiS exhibit less potency (5). In
contrast to these particulate nickel compounds, water-soluble
nickel compounds are not considered carcinogenic in experimen
tal animals, even following multiple injections (7), and they are
less potent in transforming cells compared with the crystalline
NiS particles (8,9).
Based upon studies in tissue culture, it has been proposed
that the transforming and carcinogenic activity of crystalline
Ni3S2, crystalline NiS, and amorphous NiS was proportional to
their cellular uptake by potential cancer target cells that exhibited
facultative phagocytic properties (not macrophages) (10, 11).
The active phagocytosis of the potently carcinogenic crystalline
Ni3S2 and NiS represents a highly efficient mechanism for deliv
ering large quantities of nickel ions into the cells (12). Mounting
experimental evidence attests to the importance of such a phag
ocytic mechanism in cancer target cells as an initial step in the
carcinogenicity of crystalline nickel sulfide particles in vivo (13,
14), although the cause-and-effect relationship of this first step
has not been unequivocally proven (14).
Following phagocytosis, the particles are solubilized in the cell,
possibly as a result of the lysosomal acidification of the vacuole
containing the endocytized particle (15). In fact, recent studies
have shown a good correlation of cytotoxicity with the dissolution
of phagocytized particulate nickel compounds at acidic pH (16).
This intracellular solubilization is important for the nuclear uptake
of ionic nickel, since particles of NiS cannot cross the nuclear
membrane (8). Video intensification microscopy studies indicate
that phagocytized crystalline nickel sulfide particles which are
contained in highly acidified vacuoles aggregate around the
nucleus (12). It has been shown that solubilized nickel is gener
ated from these particles and that it enters the nucleus where it
interacts with DNA. The nuclear levels of nickel were shown to
be high in cells treated with crystalline NiS particles compared
to cells treated with equivalent concentrations of NiCI2 (13, 14).
Additionally, cells treated with crystalline NiS particles exhibited
a greater proportion of nickel bound to DNA relative to that
bound to protein in comparison with cells similarly treated with
NiCI2 (13, 14). In addition to the binding of nickel to the DNA
bases, lesions such as DNA-protein cross-links and single strand
breaks are induced in the DNA by this metal ion (17, 18). The
ability of Ni2+ to coordinate with protein and DNA in a highly
stable ternary complex may indicate a preference for Ni2+ to
remain in the nucleus (13,14,17,18).
Since the formation of this
Ni-DNA-protein complex is dependent upon the nuclear Ni2+
concentration and also upon the ability of cells to replicate their
VOL. 45 MAY 1985
2320
SELECTIVE
DAMAGE TO HETEROCHROMATIN
DMA (13, 14), the nuclear formation of this complex is favored
with NiS particles more than NiCI2 (see above).
In the present study, we have examined and compared the
effect of NiCI2 and crystalline NiS particles on the chromosomes
of Chinese hamster ovary cells. Our results show that both
compounds produce a number of chromosomal lesions, but in
general, heterochromatic regions appear to be a prevalent site
where chromosomes are damaged by ionic nickel. The phagocytized crystalline NiS particles appear to produce a more striking
effect on heterochromatin compared with NiCI2, since the long
arm of the X-chromosome is selectively fragmented by crystalline
NiS treatment under conditions where there are no observable
effects on other chromosomes. Water-soluble NiCI2 did not pro
duce any similar fragmentation of the long arms of the X-
BY NICKEL COMPOUNDS
plating. Mitotic cells were plated into 100-mm plastic Retri dishes and
were treated with either 100 or 500 MM NiCI2 for 2 hr in SGM, 1, 3, 5,
and 11 h after the initial plating, for the analysis of aberrations induced
by NiCI2 during early G,, late G,. early S, and late S phase, respectively.
After each treatment, a 24-h recovery time was given. Mitotic cells were
selected by Colcemid treatment (see above) and dislodged by gently
pipetting the overlaying medium. The cells were collected by centrifugation at 1200 rpm, treated with a hypotonie solution (0.56% KCI) for 5
min at room temperature, and fixed in 3:1 methanol/glacial acetic acid
fixative for 30 min with two changes of fixative. The cell suspension was
finally dropped onto clean wet slides and air dried. Slides were stained
with Giemsa and then mounted and subsequently scanned for qualitative
and quantitative analysis of the chromosomal aberrations. Cell cycle
position was monitored by mitotic index or [3H]thymidine labeling index.
chromosome, but the centromeric regions of different chromo
somes were often found to be involved in chromosomal aberra
tions with either compound. It is proposed that the long arm of
the X-chromosome is selectively damaged by crystalline NiS
particles because the heterochromatin of this chromosome forms
the bulk of an inside lining in the interface nucleus (15) and
represents the first chromatin site nickel ions encounter as they
enter the nucleus (12). The greater selectivity of NiS over NiCI2
to produce this fragmentation is thought to relate only to the
concentration of nickel that can be delivered into the nucleus
from localized particles surrounding the nuclear membrane.
RESULTS
Induction of Chromosomal Aberrations by NiCI2. Table 1
demonstrates the induction of chromosomal aberrations by NiCI2
in CHO cells maintained in a SGM or complete a-MEM medium.
Treatment of cells for 2 h with NiCI2 followed by a 24-h recovery
time resulted in a concentration-dependent induction of aberra
tions. The effect occurred at a lower concentration when cells
were treated with NiCI2 in a SGM compared with complete
growth medium. This was due to the higher uptake of nickel into
cells maintained in SGM. At least 100 metaphase cells were
evaluated for each treatment time shown in each table. Control
cells were routinely scored for each experiment; typical control
aberrations are given in Table 1. Table 1 also shows aberrations
in cells treated for 6 h with NiCI2; however, higher concentrations
for this time interval or similar concentrations for longer treatment
times resulted in no metaphases. As noted in the table, the
majority of chromosomal aberrations observed consisted of
gaps, breaks, and exchanges with rare occurrence of dicentrics
and fragments. At 1 mwi NiCI2, 14.4% of all aberrations were
found to be located in centromeric regions of the chromosomes.
This is in contrast to a random expected value of 1 to 2% for
MATERIALS AND METHODS
CHO3 cells were grown as monolayer cultures in plastic Retri dishes
with a-MEM (Grand Island Biological Co., Grand Island, NY). The medium
was fortified with 10% fetal bovine serum (Armour Pharmaceutical Co.,
Kankakee, IL) and with penicillin (100 U/ml), fungizone (0.25 ¿jg/ml),and
of streptomycin (100 nQ/m\). NiCI2 and crystalline NiS were purchased
from Alfa Inorganics (Danvers, MA). Crystalline NiS used was the low
temperature form (Millerite), which has a rhombohedral crystalline struc
ture (8). Paniculate NiS was ground with an impact mill, and particles
averaging 2 to 3 ^m in diameter were prepared by nucleopore filtration
as described previously (8).
Physiological concentrations of metal binding amino acids such as
cysteine and histidine have been found to exert strong inhibitory effects
on the toxicity and uptake of metal ions such as Ni2+ (19). They also
Tabtel
Concentrationdependenceand comparison of chromosomalaberrations induced
by NiCli in Chinesehamster ovary cells
Intact CHO cells were treated with NiCI2under the conditions shown. The metal
was removed, and the cells were allowed to recover for 24 h prior to collection of
mitotic cells, as described in "Materials and Methods." Previous studies have
appear to account for the majority of inhibitory activity of whole serum
toward uptake (19); therefore, in selected instances, cells were treated
with NiCI2 while maintained in a simple SGM. Synchronized or log-phase
shown that incubation of CHO cells in a SGM for at least 12 h does not affect
plating efficiency and trypan blue exclusion (19, 27).
cultures were placed in the SGM for relatively short time intervals (1 to
3 hr) only during the time of metal exposure. Previous studies have
demonstrated that incubation of CHO cells in this SGM did not decrease
cell viability or change other growth parameters (19). NiCI2 was dissolved
in SGM just prior to use and was filter-sterilized by passage through a
0.45-Mtn millipore filter. Stock suspensions of crystalline NiS particles
NiCI2con
with aberrationsG"868141813281547111315612B971291722573
ot
with
centration
damage
multiple
(%)9.1611.4"15.7"25.7"34.2*32.0"58.7"42.5"5.8C8.3C17.8C22.8C19.2e
GIM)001101001005001000001005001000250500Treatment
time
(h)2222222224822266Cells
damage000000150000000
were prepared by sonicating 2 to 3 ^m particles in ethanol, collecting
and drying the sterilized particles, and resuspending them in a stock of
sterile 0.9% NaCI solution. Chromosomes were prepared from Colcemidarrested (0.02 ^g/ml for 2 h) mitotic cells that had been treated with the
nickel compounds. In order to study the cell cycle specificity of NiClr
induced aberrations, cells were synchronized by the selective detach
ment of mitotic cells. Cells were plated in flasks 12 h prior to mitotic
selection. Four hr before selection of mitotic cells, Colcemid (0.02 ¿¿g/ml)
was added, and the cells were washed two times with medium before
* G. gaps; B, breaks; E, exchanges; D, dicentrics; F, fragments.
6 Treated in SGM.
c Treated in «-MEMwith fetal bovine serum.
3The abbreviationsused are: CHO, Chinesehamster ovary; SGM, salts/glucose
medium [50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid buffer, pH
7.2:100 mMNaCI:5 mw KCI:5 mw CaCI2];a-MEM, «-minimal
essential medium.
CANCER RESEARCH
VOL. 45 MAY 1985
2321
SELECTIVE
DAMAGE
TO HETEROCHROMATIN
centramene damages. Figure 1 illustrates the type of aberrations
frequently seen with NiCI2. The broken arrow in Figure 1a dem
onstrates an exchange induced by NiCI2, while the solid arrow
illustrates a nickel-induced gap. The broken arrow in Figure 10
shows a centromeric exchange induced by NiCI2, while the other
arrow illustrates a centromeric break.
Cell Cycle-specific Induction of Chromosomal Aberrations
by NiCI2. Table 2 shows the chromosomal aberrations induced
by NiCI2 in CHO cells at different stages of the cell cycle. There
was very little aberration above background level exhibited dur
ing the Gìphase (Table 2). During S phase and particularly late
S phase, there was a striking increase in the frequency of
chromosomal aberrations. These findings suggested that the
DNA replication phase is more susceptible to the induction of
chromosomal aberrations by NiCI2 and the aberration frequency
observed in a log-phase population of cells may be due to the S
phase population of that culture. It was also interesting to note
that most of the aberrations were caused during late S phase,
the cell cycle period when heterochromatic DNA replicates.
Effect of Crystalline NiS Particles on the Induction of Chro
mosomal Aberrations. Table 3 demonstrates that treatment of
intact CHO cells with crystalline NiS results in a time- and
concentration-dependent induction of chromosomal aberrations.
The nature of the aberrations observed with crystalline NiS were
very similar to those found with NiCI2 except that, with crystalline
NiS particles, there was also a selective fragmentation of long
arms of the X-chromosomes. Thus most of the NiS-induced
fragmentation shown in Table 3 represents the heterochromatic
long arms of the X-chromosomes. The induction of chromosomal
aberrations by crystalline NiS particles was dependent upon time
since, even at high concentrations, there were few aberrations
seen at 6 h, but these progressively increased after 24 or 48 h.
Cell cycle-specific
BonEarly
phase(1-3h)cLate
d
BY NICKEL COMPOUNDS
Figure 2 illustrates the fragmentation of the long arms of the Xchromosome caused by crystalline NiS particles. Progressing
from a to f in Figure 2, there was more striking fragmentation of
the long arm of this chromosome compared to other chromo
somes that are not heterochromatic. Even the short arm of the
same X-chromosome did not exhibit fragmentation. At higher
concentrations of NiS for longer exposure time intervals (I.e., 48
h), it was difficult to identify the chromosomes involved in the
fragmentation because fragmentation was extensive. However,
analysis of the intact chromosome morphology as well as the
morphology of the partially fragmented chromosome indicated
that the heterochromatic long arm of the X-chromosome was
often involved in this fragmentation. As many as 19.5% of the
total damaged cells had fragmentation after 48 h and, in most
instances, these involved breakage of the long arm of the Xchromosome (Table 3). Fewer cells with fragmentations were
found after 6- and 24-h treatments at all concentrations of
crystalline NiS.
The occurrence of selective fragmentation by NiS but not by
NiCI2 treatment suggested that this might be due to a difference
in the mechanism of delivery of nickel ions into the nucleus (13).
To test this hypothesis, CHO cells were treated with nickel ionsaturated albumen which was encapsulated into the liposomes.
This allowed the nickel to enter cells by a mechanism that may
model the delivery of nickel particles into the cells. To prepare
liposomes containing nickel-saturated protein, albumin was
treated with NiCI2, and unbound nickel was removed by Sephadex G-10 chromatography.4 CHO cells in logarithmic growth
phases were treated with various concentrations of this complex
in a SGM and in complete a-MEM medium for time intervals
ranging from 4 to 24 h. A minimum of 100 metaphase cells were
analyzed for the presence or absence of chromosomal fragmen
tation. Treatment of cells for 4 h with liposomes containing the
Table 2
Ni-albumen complex (NiCI2 concentration of 400 UM) resulted in
induction of chromosomal abbreviations by NiCI¡in CHO cells
a maximum of 4.2% cells with fragmentation, which, in many
Types of aberrations
cases, could be attributed to fragmentation of the X-chromo
NiClz
(JIM)"100500100500100500100500damage
(%)13.216.619.718.531.439.444.956.0G"21691516431519B131111630452427E2363351020D02326202F00001000
somes. Unencapsulated NiCI? alone, when added at similar or
higher concentrations, did not produce any chromosomal frag
mentation.4 Similarly, liposomes alone, liposomes with albumen,
phase(3-5
G,
h)Early
phase(5-7
S
h)Late
phase(11-13h)Of
S
or albumen with NiCI2 alone, when added at similar or higher
levels, did not cause any fragmentation of the long arm of the Xchromosome. Additional experiments were also conducted to
examine whether the irritant effect of an internalized particle may
produce fragmentation of the X-chromosome or other chromo-
* Cells were treated in a SGM for 2 h.
" G, gaps; B, breaks; E, exchanges; D, dicentrics; F, fragments.
: Time interval following release from mitosis during which NiClz was added.
4 P. Sen et al., unpublished observations.
Table 3
Induction of chromosomal aberrations by crystalline NiS in intact Chinese hamster ovary cells
Crystalline NiS
Types of aberrations
Cells with
mei na
(h)666242424484848OBIIä
(%)22.821.621.520.016.140.334.036.661.317.0G"1318141116221916388B2619242116313143497E36542681131D202313961
tion(%)0026377612
[ecumenitime Will]damage
uunuciiif
aiiuii(xQ/ml)510205102051020Untreatedi
a G, gaps; B. breaks; E, exchanges; D, dicentrics.
CANCER RESEARCH
VOL. 45 MAY 1985
2322
Cells with
SELECTIVE
DAMAGE
TO HETEROCHROMATIN
somes. Cells were treated with activated charcoal particles at
concentrations and for time intervals similar to those utilized for
analysis of chromosomal fragmentation by crystalline NiS parti
cles (see Table 3). Since activated charcoal particles were con
siderably less dense than crystalline NiS particles, there was a
greater particle exposure for a given mass of these particles
compared with crystalline NiS particles. There were no fragmen
tations of chromosomes induced by exposure of cells to acti
vated charcoal, despite the active phagocytosis of these particles
(data not shown).
DISCUSSION
The extent and nature of chromosomal damage induced by
nickel compounds was dependent upon the method of delivery
of nickel ions and upon the cell cycle position. NiCI2 was not
readily taken up by cells in tissue culture media, due to the
inhibitory effects of nickel binding amino acids such as cysteine
and histidine present in tissue culture growth media (19). Thus,
the potency of NiCI2 in inducing chromosomal aberrations was
considerably increased when cultured cells were exposed to this
metal ion in a minimal salts/glucose maintenance medium. Ex
posure of cells to the minimal salts/glucose medium per se did
not result in any measurable effects on a number of cellular
functions (19).
The mechanism of metal delivery also influenced the nature of
the chromosomal lesion observed. Although soluble NiCI2 pro
duced a high level of centromere-associated aberrations, it did
not, under any conditions, cause fragmentation of the long arm
of the X-chromosome as was observed following treatment with
crystalline NiS particles. However, introduction of nickel-albumen
complexes into cells by means of liposomes did produce heterochromatic fragmentation of the X-chromosome.4 In interphase
cells, condensed heterochromatin forms the inner lining of the
nuclear membrane (15). Thus, it is likely that the nickel ions
liberated after the solubilization of NiS particles in perinuclear
regions interact with heterochromatic DNA before interacting
with euchromatic DNA. Since the long arm of the X-chromosome
represents most of the heterochromatic DNA, it is likely to be
the most extensively damaged by NiS particles. In contrast, ionic
nickel from the NiCI2, which enters the cell less readily than the
particles, is distributed throughout the cell, interacting with nu
merous ligands in addition to DNA. Overloading of cells with
NiCI2 cannot produce an incidence of transformation equivalent
to the maximum possible transformation frequency found with
crystalline NiS (8, 13,14). The incidence of transformation and
the nuclear levels of Ni2+ are considerably greater with NiS
particles compared to NiCI2 (8,13,14). Nickel ions are known to
bind to the phosphate groups of DNA bases, but the ion also
has affinity for purine bases (20). The presence of GC-rich,
repetitive DNA in heterochromatin may cause more nickel ions
to interact with DNA per unit area in such regions as compared
to dispersed chromatin having unique or even moderately repe
titious DNA sequences. Thus, the number of initial lesions per
unit area in heterochromatin is expected to be many-fold greater
than euchromatin, resulting in the higher number of aberrations
in the heterochromatic regions. Another important factor affect
ing the incidence of chromosomal aberrations was the fraction
of cells in the late S phase of the cell cycle. According to the
studies illustrated in Table 2, those cells in the late S phase of
BY NICKEL COMPOUNDS
the cell cycle represent the most susceptible cell cycle population
for the induction of chromosomal lesion. Interestingly, NiCI2 and
crystalline NiS have been shown to cause an S-phase cell cycle
block (21), and this accumulation of cells in S phase may be one
of the reasons why the chromosomal aberrations induced by
crystalline NiS particles appeared to require longer exposure
times (i.e., 24 h; Table 3). During S phase, cells are probably
more susceptible to the DNA-damaging effects of nickel because
DNA is unfolded and more accessible to nickel ion binding during
its replication.
A major lesion induced by nickel in the DNA is the DNA-protein
cross-link (18). The formation of this lesion may involve the
chemical reactivity of ionic nickel in forming a ternary complex
with protein and DNA (22). The equilibrium binding constant of
nickel for DNA indicates very weak affinity of the metal for DNA
(22). The binding constant of nickel for protein indicates greater
affinity of the metal for protein than for DNA. However, the
formation of a ternary DNA-nickel-protein complex represents a
way of maintaining nickel bound to DNA in a very stable complex
(22). Our own studies have demonstrated that this ternary com
plex will form in vitro only if nickel is added to DNA prior to the
addition of nuclear protein (23). The formation of this ternary
complex may cause preferential retention of nickel in the nucleus,
making its entry into the nucleus irreversible. Additionally, the
proteins cross-linked to DNA by nickel either in vitro or in the
intact cells are the same proteins that are very tightly bound to
the DNA (23). Therefore, nickel appears to cross-link proteins
which have an inherently high binding affinity for DNA. Recent
studies5 have demonstrated that the nickel-induced DNA-protein
cross-link forms preferentially during the late S phase, and pro
teins present in the heterochromatin fraction are extensively
cross-linked to the DNA by nickel treatment of intact cells. The
observed fragmentations of the long arm of the X-chromosome
may result from incomplete replication of DNA in these crosslinked regions.
Earlier studies have shown that the aberrations induced by
carcinogenic and mutagenic agents are preferentially located in
heterochromatic regions (24, 25). These studies have suggested
that (a) concentration of repetitive DNA in these regions and (b)
condensed state of heterochromatin to form chromocenters
during interphase might be responsible for the increased aber
rations in the heterochromatic regions of the chromosomes. Our
results add support to these previous findings by demonstrating
that the highly carcinogenic NiS particles produce extensive
fragmentation of the heterochromatic long arm of the X-chro
mosome. The effect appeared to be due to the mechanism of
nickel delivery into cells. The significance of the heterochromatin
fragmentation in relationship to the carcinogenesis of crystalline
NiS is not known. It is, however, interesting to note the lack of
mutagenicity exhibited by potently carcinogenic nickel com
pounds in mammalian systems (26) which is consistent with a
site of action in heterochromatin. Heterochromatic DNA has few
actively transcribed genes and contains repetitive DNA. Little is
known about the function of this type of DNA, and therefore it is
difficult to ascertain the significance of the observed selective
interaction of nickel with heterochromatin.
5 Patierno et al., unpublished observations.
CANCER RESEARCH VOL. 45 MAY 1985
2323
SELECTIVE
DAMAGE
TO HETEROCHROMATIN
ACKNOWLEDGMENTS
The authors thank Faye Howard for secretarial assistance and S. R. Patierno
for his criticism of this manuscript. The authors thank Dr. R. L. Juliano for his
assistance in preparing the liposomes utilized in this study. The authors also thank
Dr. S. Pathak and Dr. T. C. Hsu for helpful discussion.
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**
I
/
\
b
Fig. 1. Photograph illustrating NiCI2-induced chromosomal damage in CHO cells. Cells in both a and b were treated for 2 h with 500 MM NiCI2 in a salts/glucose
medium. The cells were transferred from complete growth medium to the salts/glucose medium prior to the addition of NiCI2 (see "Materials and Methods"). Following
treatment, the metal compound was washed from the cellular monolayer, and the cultures were placed in complete culture medium for 24 h. At the end of this time
interval, mitotic cells were collected, and chromosomes were prepared as described in "Materials and Methods." The broken arrow in a illustrates a chromatid exchange,
while the solid arrow shows a chromatid gap. x 1600. The broken arrow in b shows a centromere exchange, while the solid arrow depicts a centramene break, x 1600.
CANCER
RESEARCH
VOL. 45 MAY 1985
2324
V^i X
Fig. 2. Selective fragmentation of the long arm of the X-chromosome of CHO cells by crystalline NiS particles. CHO cells were treated with crystalline NiS particles
(20 ¿ig/ml)for 48 h in complete culture medium. Following this treatment, the cultures treated with the particles were washed, and mitotic cells were collected by a 2-h
incubation with Colcemid (see "Materials and Methods"). The arrows in a to f show the fragmentation of the long arm of the X-chromosome. x 1600.