Research Article
Chronic Exposure to Lead Chromate Causes Centrosome
Abnormalities and Aneuploidy in Human Lung Cells
1,2
1,2
1,2
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Amie L. Holmes, Sandra S. Wise, Sarah J. Sandwick, Wilma L. Lingle,
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Vivian C. Negron, W. Douglas Thompson, and John Pierce Wise, Sr.
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Wise Laboratory of Environmental and Genetic Toxicology, 2Maine Center for Toxicology and Environmental Health, and 3Department of
Applied Medical Science, University of Southern Maine, Portland, Maine and 4The Tumor Biology Program, Division of Experimental
Pathology, Mayo Clinic and Foundation, Rochester, Minnesota
Abstract
Hexavalent chromium [Cr(VI)] compounds are established
human lung carcinogens. The carcinogenicity of Cr(VI) is
related to its solubility, with the most potent carcinogens
being the insoluble particulate Cr(VI) compounds. However,
it remains unknown why particulate Cr(VI) is more carcinogenic than soluble Cr(VI). One possible explanation is that
particulates may provide more chronic exposures to chromate over time. We found that aneuploid cells increased in a
concentration- and time-dependent manner after chronic
exposure to lead chromate. Specifically, a 24-hour lead
chromate exposure induced no aneugenic effect, whereas a
120-hour exposure to 0.5 and 1 Mg/cm2 lead chromate
induced 55% and 60% aneuploid metaphases, respectively.
We also found that many of these aneuploid cells were able
to continue to grow and form colonies. Centrosome defects
are known to induce aneuploidy; therefore, we investigated
the effects of chronic lead chromate exposure on centrosomes. We found that centrosome amplification in interphase and mitotic cells increased in a concentration- and
time-dependent manner with 0.5 and 1 Mg/cm2 lead
chromate for 120 hours, inducing aberrant centrosomes in
18% and 21% of interphase cells and 32% and 69% of mitotic
cells, respectively; however, lead oxide did not induce centrosome amplification in interphase or mitotic cells. There was
also an increase in aberrant mitosis after chronic exposure
to lead chromate with the emergence of disorganized
anaphase and mitotic catastrophe. These data suggest that
one possible mechanism for lead chromate–induced carcinogenesis is through centrosome dysfunction, leading to the
induction of aneuploidy. (Cancer Res 2006; 66(8): 4041-8)
Introduction
Hexavalent chromium compounds [Cr(VI)] are well-known
human lung carcinogens (1–4). However, their carcinogenic
mechanisms remain poorly understood. Epidemiologic, whole
animal, and cell culture studies indicate that water solubility plays
a key role in the carcinogenicity of Cr(VI) with water-insoluble or
‘‘particulate’’ Cr(VI) compounds as the most potent form (1–4).
Human pathology studies show that Cr(VI) deposits and persists at
bronchial bifurcations where Cr-associated cancers occur (5, 6),
which is consistent with a particulate exposure.
Requests for reprints: John Pierce Wise, Sr., University of Southern Maine, 96
Falmouth Street, 178 Science Building, Portland, ME. Phone: 207-228-8050; Fax: 207228-8057; E-mail: [email protected].
I2006 American Association for Cancer Research.
doi:10.1158/0008-5472.CAN-05-3312
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It remains unclear why particulate Cr(VI) compounds have a
greater carcinogenic potential than soluble Cr(VI) compounds.
Studies using lead chromate as a model particulate Cr(VI)
compound show that the particles partially dissolve outside the
cell releasing Pb cations and chromate anions (7–9). The internalized Cr ions induce chromosome aberrations, DNA adducts, and
DNA double-strand breaks (10–15), whereas the internalized Pb
ions are generally nongenotoxic (8, 16). Two studies investigating
potential epigenetic effects of Pb in lead chromate–induced
carcinogenesis also show that Pb does not interfere with Crinduced cell death (17) or cause mitotic stimulation of Cr-damaged
cells (18). In addition, internalized particles seem to have no toxic
effect except for possibly a small contribution to cytotoxicity (17).
Thus, the differences between particulate and soluble exposures
remain unclear.
One possible explanation is that particulates may provide more
chronic exposures to chromate over time. The particulates may
have an effect at bronchial bifurcation sites, leading to more
chronic exposures, whereas soluble chromate is more rapidly
cleared. However, no studies have considered chronic exposures
to particulate chromate. All of the studies thus far focused on
24 hours or shorter time points (8, 10–16). Thus, in this study, we
investigated longer exposure times to better mimic the exposures
to particulate chromate in humans.
Lung cancers exhibit a high incidence of chromosomal
instability (19). Specifically, 70% to 80% of malignant lung
tumors exhibit a complex karyotype with severe aneuploidy, often
a triploid to tetraploid complement of chromosomes (19). One
possible mechanism of chromosomal instability is centrosome
amplification, which is also a common event in lung cancer (19).
Centrosome amplification is characterized by either an increase
in centrosome number (>2) or in size. These centrosome
abnormalities result in multipolar spindle formation and/or
an increased microtubule nucleating capacity, which in turn
causes chromosome missegregation and aneuploidy (as reviewed
in ref. 20).
Metals do induce chromosomal instability and abnormal
centrosomes. For example, one study found that vanadium
inhibited the separation of the centrosome after duplication
causing a monopolar spindle apparatus (21). Three studies on
dimethylarsinic all revealed that arsenic induces multiple centrosomes and multipolar spindle formation, which leads to the
induction of aneuploidy (22–24). One study on mercury revealed
that methylmercury but not inorganic mercury induced aberrant
centrosomes and multipolar spindles (25). Cell culture studies
show that particulate chromate induces structural chromosomal
instability in the form of chromosomal aberrations (8, 10–14).
However, the effects of particulate chromate on numerical chromosomal instability and centrosome amplification have not been
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investigated. Accordingly, this study investigates the hypothesis that
chronic exposures to lead chromate induce chromosomal instability
and centrosome dysfunction in human bronchial cells.
Materials and Methods
Chemicals and reagents. Lead chromate, lead oxide, colcemid,
potassium chloride, magnesium sulfate, EGTA, PIPES, anti-g-tubulin (clone
GTU-88), anti-a-tubulin-FITC conjugate antibody, and Triton X-100 were
purchased from Sigma-Aldrich (St. Louis, MO). Giemsa stain was purchased
from Biomedical Specialties, Inc. (Santa Monica, CA). Alexa Fluor 555 goat
anti-mouse IgG, TOPRO-3, and Prolong were purchased from Molecular
Probes (Eugene, OR). Trypsin/EDTA, sodium pyruvate, penicillin/streptomycin, L-glutamine, and Gurr’s buffer were purchased from Invitrogen Corp.
(Grand Island, NY). Methanol, acetone, and acetic acid were purchased
from J.T. Baker (Phillipsburg, NJ). DMEM and Ham’s F-12 (DMEM/F-12)
50:50 mixture was purchased from Mediatech, Inc. (Herndon, VA).
Cosmic calf serum (CCS) was purchased from Hyclone (Logan, UT). Tissue
culture dishes, flasks, and plasticware were purchased from Corning, Inc.
(Acton, MA).
Cells and cell culture. WTHBF-6 cells, a clonal cell line derived from
normal human bronchial fibroblasts that ectopically express human
telomerase, were used in all experiments. These cells have a similar
clastogenic and cytotoxic responses to metals compared with their parent
cells (13). Cells were maintained as subconfluent monolayers in DMEM/F12 supplemented with 15% CCS, 2 mmol/L L-glutamine, 100 units/mL
penicillin/100 Ag/mL streptomycin, and 0.1 mmol/L sodium pyruvate and
incubated in a 5% CO2 humidified environment at 37jC. They were fed
thrice a week and subcultured at least once a week using 0.25% trypsin/
1 mmol/L EDTA solution. All experiments were done on logarithmically
growing cells.
Preparation of chemicals. Lead chromate and lead oxide were
administered as a suspension in acetone, as previously described (11). We
treated with a concentration range of 0.1 to 1 Ag/cm2 lead chromate as it is
consistent with the published literature on lead chromate, and we and
others have shown this range to model both environmental and
occupational exposures (10, 11).
Aneuploid analysis. Aneuploidy was determined by counting the
number of chromosomes in solid stained metaphases. Preparation of
chromosomes was done as previously described (11). Each experiment was
repeated at least thrice. A minimum of 100 metaphases were analyzed per
concentration.
Clonogenic aneuploidy. Cells were seeded into 60-mm dishes, and
the monolayer of cells was treated with 0 or 0.5 Ag/cm2 lead chromate for
96 or 120 hours. The cells were then harvested and plated onto coverslips
at a very low density so they would form colonies derived from a single
surviving cell as opposed to forming a monolayer. Once the colonies were
at least 20 to 30 cells, the cells were incubated for 23 minutes in 0.8%
sodium citrate and fixed with 3:1 methanol/acetic acid. Cells were dried,
aged, and stained with Giemsa. The number of chromosomes from
metaphases in each colony was counted. All available colonies were scored
up to 50 colonies.
Mitotic stage analysis. A monolayer of cells was seeded onto chamber
slides and treated with 0 or 0.5 Ag/cm2 lead chromate for 96 or 120 hours.
Cells were fixed in situ with 20:1 methanol/acetic acid, aged overnight, and
stained with 5% Giemsa for 5 minutes. Mitotic figures were analyzed under
light microscopy. Mitotic figures were scored, as defined in Yih et al. (26),
considering stage (prophase, metaphase, anaphase, and telophase) or
abnormal appearance, such as metaphase with lagging chromosomes
(defined as containing some chromosomes that are not lined along the
metaphase plate like the rest of chromosomes), ball metaphase (where
chromosomes are all located in the center of the cell), c-metaphase
(chromosomes are located throughout the cell and are highly condensed
and still attached to its sister chromatid), anaphase with lagging chromosomes (one or more chromosomes are lagging behind the other chromosomes as they move to the two separate poles), disorganized anaphase
(chromosomes have separated from its sister chromatid and are scattered
Cancer Res 2006; 66: (8). April 15, 2006
throughout the cell in an unorganized fashion), and mitotic catastrophe
(chromosome are also very disorganized and located throughout the cell
but have a streaky appearance to them). One hundred metaphases per
concentration were scored.
Centrosome and microtubule analysis. A monolayer of cells was
seeded onto chamber slides and treated with 0, 0.1, 0.5, or 1 Ag/cm2 lead
chromate for 24, 96, or 120 hours. Cells were washed twice in a microtubule
stabilizing buffer (3 mmol/L EGTA, 50 mmol/L PIPES, 1 mmol/L
magnesium sulfate, 25 mmol/L potassium chloride), fixed with 20jC
methanol for 10 minutes, and rehydrated with 0.05% Triton X-100 for
3 minutes. Cells were then incubated in blocking buffer for 30 minutes
followed by an hour incubation with a primary anti-g-tubulin antibody
(Sigma, St. Louis, MO; T-6557). Cells were washed four times with PBS and
then incubated with Alexa Fluor 555 goat anti-mouse IgG secondary
antibody for an hour in the dark. Cells were washed four times in PBS and
then incubated with anti-a-tubulin/FITC–conjugated antibody for 1 hour in
the dark followed by four washes with PBS. A post-fix was done using 4%
paraformaldehyde followed by two PBS washes for 3 minutes each. DNA
was stained with TOPRO-3 for 30 minutes and then washed once with
water. Coverslips were mounted with Prolong, and cells were analyzed using
fluorescence microscopy. One hundred mitotic cells and 1,000 interphase
cells were analyzed per concentration.
Statistical analysis. Statistical analyses were done on the number and
percentage of cells having a particular abnormality. These values were
averaged across the replicates (typically three) for a particular condition
defined by dose and time. For each mean value, SEs were calculated based
on the unbiased estimate of variance. Differences among means were
evaluated using Student’s t test, with P < 0.05 taken as the criterion for
statistical significance. All analyses were conducted using the SAS software
package (27). Because all comparisons among means were considered to be
of substantive interest a priori, no adjustment for multiple comparisons was
incorporated into the analysis (28).
Results
Chronic exposure to lead chromate causes aneuploidy. To
determine if chronic exposure to lead chromate induces chromosomal instability, manifested as aneuploidy, we treated WTHBF-6
cells with varying concentrations of lead chromate for 24, 48, 72, 96,
or 120 hours. Chronic exposure to lead chromate induced a
concentration- and time-dependent increase in aneuploidy
(Fig. 1A). A 24-hour exposure to 0.1, 0.5, and 1 Ag/cm2 lead
chromate induced no aneugenic effect, whereas after 120 hours,
these concentrations induced 24%, 56%, and 60% aneuploid metaphases, respectively. Grouping the aneuploid cells based on
chromosome number into hypodiploidy (<46 chromosomes),
hyperdiploidy (between 47 and 91 chromosomes), and tetraploidy
(92 chromosomes) revealed two different patterns depending on
concentration.
One pattern occurred after chronic exposure to a very low
concentration of lead chromate and manifested as an increase in
tetraploid cells at 96 and 120 hours. Specifically, 0.1 Ag/cm2 lead
chromate induced tetraploidy in 5.4% and 8.4% of metaphases at
96 and 120 hours, respectively. For this pattern, there was no
increase in either hypodiploid or hyperdiploid cells.
A second pattern emerged at low to moderate concentrations of
lead chromate. Specifically, chronic exposure to these concentrations induced both tetraploidy, which steadily increased with
time, as well as hypodiploidy. For example, 1 Ag/cm2 lead chromate
induced tetraploidy in 5% of metaphases at 72 hours, which
increased to 15% by 120 hours (Fig. 1D). It also induced an increase
in the number of hypodiploid cells with 21% at 96 hours and 39% at
120 hours (Fig. 1B). Once again with this pattern, there was only a
slight increase in hyperdiploid cells (Fig. 1C).
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Lead Chromate Induces Chromosome Instability
Figure 1. Chronic exposure to lead chromate induces aneuploidy in human lung cells. Lead chromate induces a time- and concentration-dependent increase in
aneuploid cells. A, % metaphase cells with aneuploidy (anything >46 or <46 chromosomes). B, % hypodiploid metaphases (<46 chromosomes). C, % hyperdiploid
metaphases (47 and 91 chromosomes). D, % tetraploid metaphases (92 chromosomes). Columns, average at least three experiments; bars, SE. *, based on
271 analyzed metaphases. Statistical analysis. A, exposure to 0.5 Ag/cm2 lead chromate for 48 hours and 0.5 and 1 Ag/cm2 for 72 hours are statistically different from
control (P < 0.05). All concentrations are statistically different from control for 96 and 120 hours (P < 0.03). Exposure to 0.1 Ag/cm2 lead chromate for 24 hours is
statistically different from 48, 96, and 120 hours, and both 48 and 72 hours are statistically different from 96 and 120 hours (P < 0.04). Exposure to 0.5 Ag/cm2 lead
chromate for 24 hours is statistically different from 48, 72, and 120 hours, and 48 hours is statistically different from 96 and 120 hours (P < 0.05). All time comparisons
for 1 Ag/cm2 lead chromate are statistically significant (P < 0.035) except for 72 to 96 hours. B, exposure to 1 Ag/cm2 lead chromate for 72 hours and exposure to
0.5 and 1 Ag/cm2 for 96 and 120 hours are statistically different from control (P < 0.03). Exposure to 0.1 Ag/cm2 lead chromate for 24 hours is statistically different
from 48, 96, and 120 hours (P < 0.01). Exposure to 0.5 Ag/cm2 lead chromate for 24 hours is statistically different from 48 and 120 hours, and 48 hours is statistically
different from 120 hours (P < 0.02). Exposure to 1 Ag/cm2 lead chromate for both 24 and 48 hours are statistically different from 72, 96, and 120 hours (P < 0.05).
C, exposure to 0.5 Ag/cm2 lead chromate for 48 hours and 1 Ag/cm2 for 96 hours are statistically different from control (P < 0.01). All concentrations at 120 hours
are statistically different from control (P < 0.03). Exposure to 0.1 Ag/cm2 lead chromate for 72 hours is statistically different from 120 hours (P < 0.05). Exposure to
0.5 Ag/cm2 lead chromate for 48 hours is statistically different from 96 hours (P < 0.05). D, exposure to 1 Ag/cm2 for 96 hours and all concentrations at 120 hours are
significantly different from control (P < 0.03). Exposure to 0.5 Ag/cm2 lead chromate for 24 and 48 hours are statistically different from 96 and 120 hours (P < 0.05).
Exposure to 1 Ag/cm2 for 24 hours is statistically different from 72, 96, and 120 hours, and 48 and 96 hours are statistically different from 120 hours (P < 0.02).
Thus, chronic exposure to lead chromate induces tetraploidy
at low concentrations and both tetraploidy and hypodiploidy at
higher concentrations. These data suggest that lead chromate
induces aneuploidy via two different mechanisms: one that drives
the induction of tetraploidy and one that induces hypodiploid cells.
To determine if these aneuploid cells were viable, we assessed
their ability to form colonies. Figure 2A depicts a normal diploid
colony and a representative metaphase, whereas Fig. 2B depicts
an aneuploid colony and a representative metaphase with 92
chromosomes. We found that after 96 or 120 hours of exposure to
0.5 Ag/cm2 lead chromate, 7% and 14% of the colonies that formed
had an aneuploid complement of chromosomes compared with
0.9% and 0% in the controls, respectively (Fig. 2C). Therefore, lead
chromate induces a time- and concentration-dependent increase in
aneuploidy, and a portion of these aneuploid cells is able to grow
and form colonies.
Role of centrosome amplification in lead chromate–induced
aneuploidy. Centrosome amplification is known to induce
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chromosomal instability; therefore, we investigated the possible
role it plays in lead chromate–induced aneuploidy. We investigated
lead chromate’s effect on both interphase and mitotic cells,
focusing on 96 and 120 hours where the aneugenic effect was
the greatest (Fig. 1A). Normal interphase cells have 1 or 2 centrosomes (Fig. 3A). Aberrant interphase cells have >2 centrosomes as
illustrated by the lead chromate–treated cell in Fig. 3B. Lead
chromate induced increases in aberrant centrosome number in
interphase cells in a concentration- and time-dependent manner
(Fig. 3C). After a 24-hour exposure to lead chromate, there was no
centrosome amplification, but 96 and 120 hours of exposures
caused significant increases in the number of cells with >2
centrosomes (Fig. 3C). Specifically, at 120 hours, 0.1, 0.5, and
1 Ag/cm2 lead chromate induced centrosome amplification in 7%,
18%, and 21% of interphase cells, respectively. The majority of these
aberrant interphase cells had 3 or 4 centrosomes (Fig. 3C).
Normal mitotic cells have 2 centrosomes located on opposite
poles that produce bipolar spindles and organize the chromosomes
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Figure 2. Chronic exposure to lead chromate induces aneuploid colony
formation. A to B, depict diploid and aneuploidy colonies. A, left, normal diploid
colony at 10 from a control slide; right, representative diploid metaphase at
100 from the colony (circled metaphase ). B, left, aneuploid colony at 10
that formed from a cell that was treated with 0.5 Ag/cm2 lead chromate for
96 hours; right, representative aneuploid metaphase with 92 chromosomes from
the colony (circled metaphase ). C, effects of a 96- or 120-hour exposure to
0.5 Ag/cm2 lead chromate on the number of colonies with aneuploidy.
Columns, average of three experiments for 96 hours and two experiments for
120 hours; bars, SE. *, P < 0.02, statistically different from control.
along the metaphase plate, ensuring proper chromatid segregation
(Fig. 4A). In contrast, Fig. 4B to C depict aberrant mitosis with
numerous centrosomes, multipolar and disorganized spindle
assembly, and disorganized chromosome alignment and segregation after chronic lead chromate exposure. Lead chromate induced
centrosome amplification in a concentration- and time-dependent
manner in mitotic cells. For example, a 120-hour exposure to 0.1,
0.5, and 1 Ag/cm2 lead chromate–induced centrosome amplification in 3%, 32%, and 69% of mitotic cells, respectively (Fig. 4D).
The majority of aberrant mitotic cells had between 7 and 20
centrosomes (Fig. 4D).
Lastly, we investigated the effects of chronic exposure to
particulate lead to determine if lead causes centrosome amplification. We found that lead oxide, a particulate lead compound,
does not induce centrosome amplification in mitotic or interphase
cells (Fig. 3C and Fig. 4D). Specifically, a 120-hour exposure to 10
Ag/cm2 lead oxide, a concentration that is 10 higher than our
highest lead chromate concentration, induced centrosome ampli-
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fication in only 3% of interphase cells, and 1.5% of mitotic cells
(Fig. 3C and Fig. 4D). Therefore, particulate lead does not induce
centrosome amplification.
Chronic exposure to lead chromate disrupts normal mitotic
progression. Once we determined that lead chromate induces
aneuploidy and centrosome amplification, we wanted to investigate how these cells proceeded through mitosis. Given the strong
aneugenic and centrosome amplification response, we focused on
0.5 Ag/cm2 lead chromate at 96 and 120 hours. We categorized
normal mitoses into prophase, metaphase, anaphase, and telophase and placed abnormal mitoses into six categories, including
metaphase with lagging chromosomes, c-metaphase, ball metaphase, anaphase with lagging chromosomes, disorganized anaphase, and mitotic catastrophe (Fig. 5A). We found that 34% of
mitotic cells were abnormal after a 96-hour exposure to 0.5 Ag/cm2
lead chromate and 46% were abnormal after 120 hours of exposure
(Fig. 5B). The majority of aberrant mitoses after 96 hours were
in disorganized anaphase (6%), c-metaphase (7%), or mitotic
catastrophe (15%). After 120 hours of exposure, the percentage of
cells in mitotic catastrophe remained the same, but the percentage
of cells in disorganized anaphase increased to 24%. Therefore, lead
chromate induced a time-dependent increase in abnormal mitosis
with the majority of aberrant mitoses in mitotic catastrophe and
disorganized anaphase.
Many of the cells with extreme centrosome amplification had
very disorganized chromosomes similar to those seen in disorganized anaphase or mitotic catastrophe in the mitotic progression
analysis. Figure 6 shows a picture of a mitotic stage analysis cell
in disorganized anaphase stained with Giemsa (Fig. 6A) alongside
a centrosome analysis cell stained with TOPRO-3 (Fig. 6B). The
chromosomes of these two cells look highly similar, suggesting
that both cells are in disorganized anaphase. When we merged the
centrosomes and microtubules with the cell stained with TOPRO-3,
we found that the disorganized anaphase cell had 15 centrosomes
and highly disorganized microtubule formation (Fig. 6C). Therefore, it seems that the mitotic figures we categorized as aberrant
are most likely due to centrosome amplification.
Discussion
Hexavalent chromium is a well-known human lung carcinogen.
Solubility plays a key role in Cr(VI) carcinogenicity, with the
particulate Cr(VI) compounds being the most potent. Chromosomal instability is a common feature of lung tumors; however, the
mechanism by which particulate Cr(VI) induces chromosomal
instability remains unknown (19).
This is the first study to show that particulate chromate induces
aneuploidy. One previous study found that a 30-hour exposure to
soluble potassium dichromate induced hypodiploid cells but not
hyperdiploid or tetraploid cells (29). In our study, hypodiploid cells
did not start to increase until after 48 hours of exposure. We
also observed tetraploidy after 72 hours of exposure (Fig. 1). Thus,
it seems that it takes longer for particulate chromate to induce
hypodiploid cells than soluble chromate. Longer exposures to
particulate chromate also induce tetraploid cells, which did not
occur after a soluble chromate exposure. The explanation for this
discrepancy is uncertain, but it may be due to solubility or cation
differences between particulate and soluble Cr(VI) compounds.
There is an interesting difference between exposure to very low
concentrations of lead chromate and low to moderate concentrations. All three concentrations induced an increase in tetraploid
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Lead Chromate Induces Chromosome Instability
cells, but only the low to moderate concentrations of lead
chromate also induced an increase in hypodiploid cells. This
suggests that there are different mechanisms for lead chromate–
induced hypodiploidy and tetraploidy. One important difference
between these concentrations is that the very low concentration
of lead chromate does not induce a significant increase in
centrosomes amplification, whereas the low to moderate concentrations of lead chromate induce a high percentage of mitotic cells
with centrosome amplification. Therefore, we hypothesize that
centrosome amplification is inducing hypodiploidy, but tetraploidy
is induced by a different mechanism possibly by effects on the
spindle assembly checkpoint. It is important to note that the
hypodiploidy occurs before tetraploidy at the two higher concentrations. Previous studies have suggested that tetraploidy is the first
outcome of centrosome amplification (29). Our data indicate that
this outcome is not the case for lead chromate–induced
centrosome amplification.
Another more recent report by Shi and King shows that
chromosome nondisjunction and the induction of binucleated
tetraploid cells can induce aneuploid cells and aberrant centrosome numbers (30). However, we do not believe that lead
chromate–induced aneuploidy and centrosome amplification
occurs via this mechanism for several reasons. First, we found no
increase in binucleated cells observed after chronic exposures to
lead chromate (data not shown). Second, chromosome nondisjunction and the induction of tetraploidy can account for cells with
4 centrosomes but may not explain the cells with 3 centrosomes
or >4 centrosomes that we also observe after longer exposures to
lead chromate. It might be argued that a subsequent division of
binucleated tetraploid cells can produce greater centrosome
numbers; however, if this occurred, it would require either a
tetraploid cell to fail to divide leading to a cell with 8 centrosomes
and 184 chromosomes or an aneuploid cell with <8 centrosomes
but between 46 and 92 chromosomes. In our aneuploid analysis, we
did not see any cells with 184 chromosomes and relatively no
increase in hyperdiploid cells. Lastly, centrosome dysfunction did
not correlate with tetraploidy, because we observed more cells
with aberrant centrosome number than cells with a tetraploid
complement of chromosomes. Therefore, we do not believe that
chromosome nondisjunction is a mechanism by which lead
chromate induces centrosome amplification.
Aneuploidy is considered a well-known hallmark of cancer;
however, whether it is an early-stage or late-stage event in
tumorigenesis is still debated. Our data are consistent with
pathology studies showing that the majority of lung tumors exhibit
aneuploidy (19). It also suggests that aneuploidy may be an early
Figure 3. Chronic exposure to lead
chromate induces centrosome amplification
in interphase cells. A to B, depict a
normal and aberrant interphase cell.
Blue (TOPRO-3), DNA; green (FITC),
microtubules; and red (Alexa 555),
centrosomes. A, a normal interphase cell
with two centrosomes or two red dots.
B, an aberrant interphase cell that has been
treated with 0.5 Ag/cm2 lead chromate
for 96 hours. It has five centrosomes.
C, centrosome amplification in interphase
cells increases in a time- and concentrationdependent manner after treatment with lead
chromate (LC ) but does not increase after
exposure to lead oxide (LO). *, average of at
least three independent experiments F SE
unless otherwise noted (100 mitotic cells
per experiment were analyzed). c, vehicle
control (acetone). b, P < 0.03, statistically
significant compared with 96 hours.
x, P < 0.03, statistically significant compared
with 120 hours. k, P < 0.002, statistically
significant compared with control.
**, P < 0.002, statistically significant
compared with 24 hours. cc, average of two
independent experiments F SE.
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event in tumorigenesis because we are seeing aneuploid cells after
only 2 to 3 days of lead chromate treatment. Other studies have
also shown that relatively short exposures to other metals induce
aneuploidy. Ochi et al. showed that both organic and inorganic
arsenic can induce aneuploidy after only 48 hours of exposures in
Syrian hamster embryo cells. Another study in normal human lung
fibroblasts showed that a 30-hour exposure to cadmium chloride,
potassium dichromate, or dimethylarsinic acid had an aneugenic
effect (31). Therefore, these data suggest that aneuploidy is an early
step in the development of cancer.
One mechanism by which aneuploidy occurs is via centrosome
amplification. Aberrant centrosome numbers can cause multipolar
spindle formation and unequal pulling of the chromosomes
resulting in chromosome misegregation and aneuploidy. This
study is the first to investigate and report that chronic exposure to
particulate chromate induces centrosome amplification. No studies
have considered soluble Cr(VI). There was no centrosome
amplification after a 24-hour exposure to lead chromate, which is
consistent with the aneuploid data showing no aneugenic effect
after 24 hours. However, after 96 and 120 hours, lead chromate
induced a concentration- and time-dependent increase in centrosome amplification in both mitotic and interphase cells, which is
also consistent with the aneuploid data showing a concentrationand time-dependent increase in aneuploid cells as well (Figs. 1A,
3C, and 4D). These data are consistent with reports showing that
organic arsenic and methylmercury are able to induce centrosome
amplification (22–25) and aneuploidy (24).
The mitotic stage analysis data are consistent with the
centrosome amplification data. The majority of aberrant mitoses
induced after chronic lead chromate exposure were in mitotic
catastrophe or disorganized anaphase (Fig. 5B), which was most
likely a direct result of centrosome amplification (Fig. 6). Mitotic
Figure 4. Chronic exposure to lead chromate
induces centrosome amplification in mitotic
cells. A to C, normal and aberrant mitotic cells.
Blue (TOPRO-3), DNA; green (FITC),
microtubules; red (Alexa 555), centrosomes.
A, a normal mitotic cell with two centrosomes,
well-organized bipolar spindles, and
chromosomes lined up along the metaphase
plate. B, this cell is an aberrant mitotic figure
with four centrosomes, multipolar spindle fiber,
and disorganized DNA. C, an aberrant mitotic
figure with 15 centrosomes and completely
disorganized spindle fibers and DNA. The cells
in (B) and (C) were treated with 0.5 Ag/cm2
lead chromate for 96 hours. D, centrosome
amplification in mitotic cells increases in a
time- and concentration-dependent manner
after treatment with lead chromate (LC ) but
does not increase after exposure to lead
oxide (LO). *, average of at least three
independent experiments F SE unless
otherwise noted (100 mitotic cells per
experiment were analyzed). c, vehicle
control (acetone). b, P < 0.04, statistically
significant compared to 96 hours.
x, P < 0.04, significant compared to 120 hours.
k, P < 0.007, statistically significant compared
to control. **, P < 0.007, statistically significant
compared to 24 hours. cc, based on 254
analyzed mitotic cells. bb, average of two
independent experiments F SE.
Cancer Res 2006; 66: (8). April 15, 2006
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Lead Chromate Induces Chromosome Instability
Figure 5. Chronic exposure to lead
chromate induces aberrant mitosis.
A, representative examples of normal and
aberrant mitotic figures. 1 to 4, normal
mitotic figures: 1, prophase; 2, metaphase;
3, anaphase; 4, telophase. 5 to 11,
abnormal mitotic figures: 5, aberrant
metaphase with lagging chromosomes
(circled ); 6, c-metaphase; 7, ball
metaphase; 8, anaphase with lagging
chromosome (circled ). 9 to 10, disorganized
anaphase. 11, mitotic catastrophe.
B, effects of a 96- or 120-hour exposure
to 0.5 Ag/cm2 lead chromate on the
number of aberrant mitoses. The percentage
of aberrant mitoses increases in a
time-dependent manner with 96 or
120 hours of 0.5 Ag/cm2 lead chromate
exposure inducing 34% and 46 % abnormal
mitoses.
catastrophe is a type of cell death that occurs during mitosis. Cells
with damaged DNA and/or aberrant spindle fibers combined with
abrogation of the G2-M and/or the spindle assembly checkpoints
often results in mitotic catastrophe (32). One study suggests that a
possible outcome of overduplication of centrosomes is mitotic
catastrophe (33). Cells that are able to escape mitotic catastrophe
result in asymmetrical cell division or mitotic slippage, leading to
the generation of aneuploid cells, which could possibly be an
important mechanism in tumorigenesis (32). Our data are
consistent with these findings.
The physicochemical mechanism of lead chromate–induced
centrosome amplification is unknown. It is established that lead
chromate partially dissolves outside the cell, releasing Pb and Cr
ions that then enter the cell (9). Once inside the cell, the Cr ions
induce the cytotoxic, growth-inhibiting, and genotoxic effects
observed after exposure to lead chromate and the lead ions have no
Figure 6. Aberrant mitotic figures are due
to centrosome dysfunction. A, disorganized
anaphase cell stained with Giemsa.
B, different disorganized anaphase cell
stained with TOPRO-3. Note that the
organization of the chromosomes in (A)
and (B) look very similar. C, the same
disorganized anaphase cell as in (B)
merged with staining for centrosomes and
microtubules. This cell has 15 centrosomes
and highly disorganized microtubule
formation.
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4047
Cancer Res 2006; 66: (8). April 15, 2006
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Research.
Cancer Research
apparent effect (14, 15, 17, 18). Thus far, the potential effects of
Cr ions on centrosome amplification have not been studied, but a
30-hour exposure to soluble chromate induced aneuploidy, which
suggests that Cr ions could have the capacity to cause centrosome
amplification, although that study did not consider it (31). On
the other hand, it is possible that lead ions could contribute to lead
chromate–induced centrosome amplification. Studies show that
lead ions do enter the cell after lead chromate exposure (17), and a
recent study by Bonacker et al. showed that soluble lead ions
disrupt microtubule function and formation (34). However, lead’s
effect on centrosome function is unknown. A third possibility is
that lead chromate–induced centrosome amplification could be
due to physical interactions with the lead chromate particles
themselves. Studies show that asbestos particles disrupt spindle
formation and induce aneuploidy (35), suggesting that particles
may also induce centrosome amplification. Thus, determining
the relative contribution of chromium ions, lead ions, or the
particles in lead chromate–induced centrosome amplification is an
important future direction.
The overall hypothesis for particulate chromate-induced carcinogenesis indicates that particles dissolve outside the cell and
enter the cell as their respective ions (9). Once inside the cell, the
chromate ions are reduced to Cr(III) through a series of redox
reactions releasing Cr(V), Cr(IV), and free radicals as intermediates
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Received 9/14/2005; revised 2/7/2006; accepted 2/21/2006.
Grant support: National Institute of Environmental Health Sciences grant ES10838
(J.P. Wise) and the Maine Center for Toxicology and Environmental Health at the
University of Southern Maine.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
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Chronic Exposure to Lead Chromate Causes Centrosome
Abnormalities and Aneuploidy in Human Lung Cells
Amie L. Holmes, Sandra S. Wise, Sarah J. Sandwick, et al.
Cancer Res 2006;66:4041-4048.
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