TOXICOLOGICAL SCIENCES 92(2), 409–415 (2006)
doi:10.1093/toxsci/kfl021
Advance Access publication May 19, 2006
XRCC1 Protects against Particulate Chromate–Induced
Chromosome Damage and Cytotoxicity in
Chinese Hamster Ovary Cells
Eliza Grlickova-Duzevik, Sandra S. Wise, Ray C. Munroe, W. Douglas Thompson, and John Pierce Wise, Sr1
Wise Laboratory of Environmental and Genetic Toxicology, Maine Center for Toxicology and Environmental Health,
University of Southern Maine, Portland, Maine 04104-9300
Received January 13, 2006; accepted March 29, 2006
Water-insoluble hexavalent chromium compounds are wellestablished human lung carcinogens. Lead chromate, a model
insoluble Cr(VI) compound, induces DNA damage, chromosome
aberrations, and dose-dependent cell death in human and Chinese
hamster ovary (CHO) cells. The relationship between lead
chromate–induced DNA damage and chromosome aberrations is
unknown. Our study focus was on examining the role of XRCC1
in lead chromate–induced cytotoxicity and structural chromosomal aberrations in CHO cells. Three different cell lines were
used: AA8 (parental), EM9 (XRCC1 mutant), and H9T3 (EM9
complemented with human XRCC1 gene). Cytotoxicity was
significantly higher in EM9 cells when compared to AA8 and
H9T3 cells, indicating that XRCC1 is important for protecting
cells from lead chromate particles–induced cell death. The
frequency of damaged metaphase cells was not affected by
XRCC1 deficiency. However, the total amount of Cr(VI)-induced
chromosome damage was exacerbated by XRCC1 deficiency, and
the spectrum of damage changed dramatically. Chromatid and
isochromatid lesions were the most prominent aberrations induced
in all cell lines. XRCC1 was essential to reduce the formation of
chromatid lesions but not for isochromatid lesions. In addition,
XRCC1 deficiency resulted in a dramatic increase in the number
of chromatid exchanges, indicating that XRCC1 is involved in
protection from lead chromate–induced chromosome instability.
Key Words: chromate; particulate; XRCC1; chromosome instability; single-strand breaks.
Chromosome instability (CIN) is a common feature of lung
cancer cells (Masuda and Takahashi, 2002; Nakamura et al.,
2003). Hexavalent chromium (Cr(VI)) is a well-known human
lung carcinogen, and recent data indicate that Cr(VI) tumors
are characterized by CIN (Hirose et al., 2002; Takahashi et al.,
1
To whom correspondence should be addressed at Wise Laboratory of
Environmental and Genetic Toxicology, Maine Center for Toxicology and
Environmental Health, University of Southern Maine, 96 Falmouth Street,
PO Box 9300, Portland, ME 04104-9300. Fax: (207) 228-8057. E-mail: john.
[email protected].
2005). In particular, epidemiological and toxicological studies
indicate that the particulate Cr(VI) compounds are the potent
carcinogenic form inducing tumors in experimental animals
and neoplastic transformation of cultured cells (IARC, 1990;
Levy et al., 1986; Patierno et al., 1988). Studies of lead
chromate, a prototypical particulate Cr(VI) compound, show
that the particles dissolve outside the cell to produce lead (Pb)
cations and chromate anions, both of which enter the cell (Wise
et al., 2004). Once inside the cells, the chromate anions induce
chromosome and DNA damage, growth arrest, and apoptotic
cell death, while the lead ions do not appear to reach toxic
levels (Singh et al., 1999; Wise et al., 1993, 2002, 2004; Xu
et al., 1992). Particulate Cr(VI) compounds induce CIN
manifested as chromosomal aberrations (Wise et al., 1992,
2002), but the mechanisms involved in these effects are
unknown.
Particulate Cr(VI) is a potent clastogen inducing chromosomal aberrations in different species and cell types (DeFlora
et al., 1990; Wise et al., 1992, 2002). This clastogenic effect is
mediated by soluble extracellular Cr(VI) ions (Xie et al.,
2004). Previous data of soluble Cr(VI) in Chinese hamster
ovary (CHO) cells suggest a direct relationship between DNA
single-strand breaks (SSBs) and chromosome damage. For
example, pretreating CHO cells with vitamin E prior to
chromate treatment decreased both chromosome damage and
SSB but had no effect on DNA-protein cross-links (DPCs)
(Sugiyama et al., 1987, 1989a,b, 1991). Similarly, pretreating
cells with vitamin B2 prior to chromate treatment increased
both chromosome damage and SSBs but had no effect on DPCs
(Sugiyama et al., 1987, 1989a, 1992).
Based on these data, we hypothesize that efficient repair of
particulate Cr(VI)–induced SSB is necessary to protect cells
from Cr(VI)-induced chromosome damage and CIN. XRCC1
is a DNA repair protein involved in repairing both direct
SSB and indirect SSB generated indirectly during base excision repair (Caldecott, 2003). It serves as a scaffold connecting many of the other proteins involved in SSB repair
(Caldecott, 2003). XRCC1 is recruited to SSBs by poly(ADPribose)polymerase (PARP1) and then facilitates the enzymatic
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GRLICKOVA-DUZEVIK ET AL.
activities of several repair participants including proliferating
cell nuclear antigen (PCNA), DNA polymerase beta (POLb),
and polynucleotide kinase (PNK) (Caldecott, 2003; Fan et al.,
2004; Kubota et al., 1996; Vidal et al., 2001; Whitehouse et al.,
2001). XRCC1 protein is involved in the repair of Cr(VI)induced SSB (Christie et al., 1984), and thus, the purpose of
our study was to examine the importance of XRCC1 in preventing particulate chromate–induced CIN.
MATERIALS AND METHODS
Reagents and chemicals. Dulbecco’s minimal essential medium with
Ham’s F-12 medium (DMEM/F12) was purchased from Mediatech (Herndon,
VA). Cosmic calf serum was purchased from HyClone (Logan, UT).
Phosphate-buffered saline 13 (PBS), Gluta-MAX, penicillin/streptomycin,
sodium pyruvate, and trypsin/EDTA were purchased from Invitrogen Corporation (Grand Island, NY). Demecolchicine and sodium chromate were
purchased from Sigma-Aldrich (Milwaukee, WI). Tissue culture flasks, dishes,
and plastic tubes were purchased from Corning Inc (Acton, MA).
Cell lines and cell culture. Three cell lines were used in this study. CHO
AA8 cells were the parental cell line and served as a wild-type control. EM9
cells, derived from AA8 cells, are known to have a C / T substitution on
codon 221, forming termination codon in the XRCC1 gene, and are the SSB
repair–deficient cell line (Caldecott, 2003). H9T3 cells are EM9 cells
complemented with the human XRCC1 gene, which reverses the SSB repair
deficiency (Thompson et al., 1990). These cells lines were provided as
a generous gift from Larry Thompson. The status of XRCC1 in AA8, EM9,
and H9T3 was confirmed by Western blotting (data not shown). Cells were
cultured in DMEM/F-12 media supplemented with 15% Cosmic calf serum,
2mM L-glutamine, 100 U/ml penicillin, 100 lg/ml streptomycin, and 0.1mM
sodium pyruvate. Cells were maintained as subconfluent adherent monolayers
and subcultured at least twice a week.
Lead chromate preparation. Lead chromate (CAS # 7758-97-6, ACS
reagent minimum 98% purity) is used as a particulate, water-insoluble
chromium salt, and it was suspended in acetone as previously described (Wise
et al., 2002).
Cytotoxicity assay. Cytotoxicity was determined with a clonogenic assay
as previously described with few modifications (Wise et al., 2002). Briefly,
90,000 cells per well were seeded in six-well dishes and allowed to grow for
24 h. The cultures were then treated with lead chromate for 24 h. At the conclusion of treatments, cells were washed with PBS, detached from the dish with
trypsin/EDTA, and were reseeded at density of 200 cells/60-mm dish and allowed to grow for 5–6 days. Colonies were fixed with methanol and stained with
crystal violet. Four dishes were prepared for each treatment concentration
and a minimum of three experiments conducted for each cell line.
Chromosome preparations. Chromosome damage was evaluated as previously described with few modifications (Wise et al., 2002). Briefly, 200,000
cells per dish were seeded in 60-mm dishes and allowed to grow for 24 h. After
24 h of treatment with lead chromate, medium was removed, and cells were
harvested. Demecolchicine, 0.1 lg/ml, was added 1 h before the end of the
treatment time. At the end of treatment, medium was collected, and cells were
washed with PBS and detached from the dish with trypsin. Cells were
centrifuged for 5 min at 112 3 g at 4°C, and the cell pellet was treated with
trypsin/EDTA and resuspended in hypotonic 0.075M KCl solution for 10 min in
order to swell the cells and the nuclei. At the end of the hypotonic treatment,
cells were fixed in methanol:acetic acid (3:1) and dropped onto clean wet slides.
Slides were allowed to air-dry for 24 h at room temperature and then stained
with 5% Giemsa in Gurr’s buffer, dried, and coverslipped. We determined
a mitotic index to examine the possibility that there might be a difference in
expression of frequency of mitosis between the three cell lines after lead
chromate exposure and found no difference (data not shown).
Chromosome analysis and scoring criteria. Clastogenesis was measured
by the production of structural chromosomal aberrations, which were scored by
standard criteria (Wise et al., 1992). Gaps and breaks were pooled as ‘‘lesions’’
as previously described (Wise et al., 1992). This is because breaks can only be
unequivocally distinguished from gap lesions if the distal acentric fragment is
displaced. Thus, pooling aberrations avoids artificial discrepancies between
scorers due to different perceptions of the width of a gap relative to the width of
its chromatid. Accordingly, chromatid deletions and achromatic lesions were
pooled as chromatid lesions while isochromatid deletions and isochromatid
achromatic lesions were pooled as isochromatid lesions. One hundred metaphases per concentration were analyzed in each experiment. Data are presented
as the percent metaphases with damage, which reflects the number of
metaphases with at least one aberration, and as total chromosome damage,
which reflects the total amount of chromosome damage in 100 metaphases.
Determination of intracellular Cr ion levels. Intracellular Cr levels in
CHO cells were determined as previously described (Wise et al., 2004). Briefly,
cells were seeded at a density of 500,000 cells per dish in 100-mm dishes. Cells
were allowed to grow for 24 h and then treated with lead chromate for 24 h. At
the end of the treatment, cells were harvested immediately. Harvested cells
were treated with hypotonic solution followed by 2% SDS. Solution was
sheered through a needle seven times and then filtered through a 0.2-lm filter.
Intracellular Cr ion levels were then measured and calculated by inductively
coupled plasma mass spectrometry as previously described (Wise et al., 2004).
Data analysis. The t-tests and confidence intervals for individual comparisons were calculated using Satterthwaite’s approximation for unequal
variances. No adjustment for multiple comparisons was incorporated in the
analysis. In order to evaluate overall differences among cell types while
controlling for the effects of concentration, multiple linear regression analysis
was employed (SAS Institute Inc, 2004).
RESULTS
Intracellular Cr ion levels were measured in all three cells
lines. EM9 and H9T3 cells exhibited similar levels of Cr ions,
which were higher than the levels found in AA8 cells treated
with the same concentration (Fig. 1). Accordingly, the cytotoxicity and genotoxicity data are presented using both the
administered lead chromate concentration and the intracellular Cr ion level for all three cell lines.
Lead chromate induced concentration-dependent decreases
in relative survival of all three cell lines (Fig. 2A). It induced
only a modest amount of cell death in AA8 cells and an
increased amount in XRCC1-deficient EM9 cells. Complementing the EM9 cells with XRCC1 (H9T3 cells) restored the
cytotoxic response to normal levels. This pattern remained the
same when the data were corrected for differences in intracellular uptake levels (Fig. 2B). Thus, XRCC1 plays a role in
preventing lead chromate–induced cytotoxicity.
Lead chromate induced concentration-dependent increases
in clastogenicity in AA8, EM9, and H9T3 cells (Fig. 3). It
damaged a similar percentage of metaphases in all three cell
lines. Specifically, 0.1, 0.5, and 1 lg/cm2 damaged 10, 19, and
20 metaphases in AA8; 14, 22, and 25 metaphases in EM9; and
9, 17, and 20 metaphases in H9T3 cells, respectively (Fig. 3A).
XRCC1 PROTECTS AGAINST CHROMOSOME DAMAGE AND CYTOTOXICITY
411
FIG. 1. Intracellular Cr levels in AA8, EM9, and H9T3 cells after 24 h of
treatment with lead chromate. There is a concentration-dependent increase in
chromium levels in all three cell lines. Cr uptake was significantly higher in
EM9 and H9T3 cells compared to AA8 cells ( p < 0.05). Data represent mean of
at least three independent experiments ± SEM.
A similar pattern was seen after correcting for differential
uptake (Fig. 3B). Thus, XRCC1 deficiency does not appear to
affect the frequency of a metaphase incurring chromosome
damage after lead chromate exposure.
Lead chromate also induced concentration-dependent increases in total chromosome damage (Fig. 4). Total chromosome damage represents total frequencies of aberrations per
100 cells. There was a consistent increase in the amount of total
chromosome damage in EM9 cells compared to AA8 cells, and
complementing the cells with XRCC1 restored the wild-type
phenotype (Fig. 4A). Specifically, lead chromate concentrations of 0.1, 0.5, and 1 lg/cm2 induced 12, 22, and 25
chromosome aberrations in AA8 cells; 25, 49, and 52 in
EM9; and 9, 20, and 30 in H9T3 cells, respectively (Fig. 4A).
Adjusting for different uptake levels showed an even stronger
effect for XRCC1 deficiency (Fig. 4B). Thus, XRCC1 protects
cells from total particulate Cr(VI)–induced chromosome
damage.
We inspected the spectrum of chromosome damage to
determine which types of lesions were most affected by
XRCC1 deficiency. Chromatid lesions were the most frequently induced lesions in all three cell lines (Fig. 5). The
absence of XRCC1 (EM9 cells) increased the number of
chromatid lesions, and complementing the cells with XRCC1
(H9T3 cells) restored the damage levels to those observed in
wild-type cells (AA8). By contrast, levels of isochromatid
lesions were similar in all three cell lines and not affected by
XRCC1 deficiency (Figs. 6A and 6B). Chromatid exchanges
were much less frequent in all three cell lines but much more
prevalent in XRCC1-deficient EM9 cells compared to the wildtype AA8 and the XRCC1-complemented H9T3 cells (Fig. 7).
Thus, XRCC1 protects cells from lead chromate–induced
chromatid lesions and exchanges but does not appear to have
a role in preventing isochromatid lesions.
FIG. 2. Lead chromate induced cytotoxicity in the 24-h treated AA8, EM9,
and H9T3 cells. Lead chromate induces a concentration-dependent decrease in
relative survival in CHO cells. EM9 cells are more sensitive to lead chromate–
induced cytotoxicity than AA8 cells ( p < 0.05). Restoration of the XRCC1 gene
in H9T3 abolishes the hypersensitivity to lead chromate–induced cell death
(p < 0.05). Data represent mean of at least three independent experiments ±
SEM. (A) Data presented based on administered concentration. (B) Data presented based on intracellular ion concentrations.
DISCUSSION
Cr(VI)-induced tumors exhibit CIN, but the mechanisms are
unknown (Hirose, 2002). Particulate Cr(VI) compounds are
more potent lung carcinogens than soluble ones. These
compounds are complex genotoxicants inducing a spectrum
of DNA damage, but the mechanisms for repairing these
lesions are uncertain and their relationship to CIN is unknown.
In this study we show that XRCC1 is necessary for protecting
cells from lead chromate–induced chromosome damage. Inefficient repair in XRCC1-deficient cells leads to more
complex damage and CIN. Our data indicate that XRCC1
deficiency does not affect the frequency of cells that incur
Cr(VI)-induced damage, but it alters the total amount of
damage that occurs within a cell that does incur such damage.
These data are the first to consider genes involved in repair of
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GRLICKOVA-DUZEVIK ET AL.
FIG. 3. Percent metaphases with damage induced in AA8, EM9, and H9T3
cells treated for 24 h with lead chromate. Lead chromate induces similar
frequencies of damaged metaphases after 24 h of treatment with lead chromate
(p < 0.05). In control cells, the mean number of metaphases with damage was
6 ± 1.4 in AA8 cells, 18 ± 3.5 in EM9 cells, and 8 ± 3.2 in H9T3 cells. Control data for each cell line in each experiment were subtracted from results obtained for each treatment concentration. Data represent mean of at least three
independent experiments ± SEM. (A) Results based on administered concentration after 24 h of treatment. (B) Results based on measured Cr ion levels after
24 h of treatment.
particulate Cr(VI)–induced chromosome damage. They are
consistent with previous reports of functional polymorphisms
in XRCC1, which correlate with increased chromosome damage in exposed workers (Lei et al., 2002; Mateuca et al., 2005).
XRCC1 interacts with a number of proteins involved in SSB
repair, serving as a scaffold connecting many of the other
proteins involved in SSB repair such as PCNA, POLb, and
PNK (Caldecott, 2003; Fan et al., 2004; Kubota et al., 1996;
Vidal et al., 2001; Whitehouse et al., 2001). Thus, our data
strongly suggest that DNA SSBs are important lesions that
underlie chromatid lesions. This conclusion is consistent with
previous reports indicating that soluble Cr(VI)–induced SSBs
correlate positively with Cr(VI)-induced chromosome damages
(Bianchi et al., 1980; Levis and Majone, 1979; Sugiyama et al.,
1993). Our data also indicate that failure to repair these SSBs
FIG. 4. Total chromosome aberrations induced in AA8, EM9, and H9T3
cells treated for 24 h with lead chromate. Lead chromate induces more
aberrations in EM9 cells compared to AA8 cells ( p < 0.05). Restoration of the
XRCC1 gene in H9T3 cells reduces the chromosomal aberrations in the 24-h
treated cells ( p < 0.05). In control cells, there were 7 aberrations in AA8 cells,
25 in EM9 cells, and 10 in H9T3 cells. Control data for each cell line in each
experiment were subtracted from results obtained for each treatment concentration. Data represent mean of at least three independent experiments ± SEM.
(A) Data obtained after 24 h of treatment based on administered lead chromate
concentration. (B) Data obtained after 24 h of treatment based on intracellular
Cr ion levels.
leads to more complex chromosome damage such as chromatid
exchanges. Generation of chromatid exchanges may lead to an
increase in chromosome translocations, a hallmark of most
cancers (Cleary, 1991; Croce, 1986; Testa, 1990).
We also found that XRCC1-deficient cells are more sensitive
to the cytotoxic effects of particulate Cr(VI). These data
suggest that XRCC1 may also be involved in the pathway for
cell survival in cells exposed to lead chromate. These
observations are different than those observed for soluble
Cr(VI) and other metals, which found that EM9 cells were not
more sensitive to the cytotoxic effect of these compounds
(Christie et al., 1984; Thompson and West, 2000). One possible
explanation for this difference may be the fact that lead
chromate is a relatively insoluble Cr(VI) compound, and some
of the particles are internalized by phagocytosis (Wise et al.,
1993; Xie et al., 2004). Indeed, a previous study in human lung
cells found that the majority, but not all, of the cytotoxicity
XRCC1 PROTECTS AGAINST CHROMOSOME DAMAGE AND CYTOTOXICITY
413
FIG. 5. Chromatid lesions induced in AA8, EM9, and H9T3 cells after 24 h
of treatment with lead chromate. Chromatid lesions are more frequently
induced in lead chromate–treated EM9 cells than in parental AA8 cells ( p <
0.05). XRCC1 restoration in H9T3 cells protects cells from lead chromate–
induced chromatid lesions as these levels were not statistically different than
those in AA8 cells. In control AA8 cells there were 3 chromatid lesions, in EM9
cells 14 lesions, and in H9T3 control cells 8 lesions. Control data from each
experiment were subtracted from results obtained for treated cells. Data
represent mean of at least three independent experiments ± SEM. (A) Data
based on administered concentration after 24 h of treatment. (B) Data based on
intracellular Cr ion levels after 24 h of treatment.
FIG. 6. Isochromatid lesions in AA8, EM9, and H9T3 cells after 24 h of
treatment with lead chromate. Isochromatid lesions are induced at similar
frequencies in all three CHO cell lines ( p < 0.05). In control AA8 cells there
were 2 isochromatid lesions, in EM9 cells 5, and in H9T3 cells 1. Control data
from each experiment were subtracted from results obtained for treated cells.
Data represent mean of at least three independent experiments ± SEM. (A) Data
based on administered concentration after 24 h of treatment. (B) Data based on
intracellular Cr ion levels measured after 24 h of treatment.
induced by lead chromate was the result of partial extracellular
dissolution of the particles generating extracellular soluble
Cr(VI) ions; however, some of lead chromate’s cytotoxicity
was not related to chromium but rather to the internalization of
the particles (Holmes et al., 2005; Xie et al., 2004)
Our data also show that cells deficient in DNA repair may
have dramatically different intracellular levels of chemicals
from their parent cells (Fig. 1). These observations are
important because, while previous DNA repair studies have
tended to focus on radiation-induced effects, which do not
require cellular uptake, recent studies are beginning to consider the repair of lesions induced by chemicals such as
mitomycin C and psoralen. It is essential that these studies determine intracellular levels to ensure that differences observed in parent and DNA repair–deficient cells actually
reflect deficiencies in repair and not simply differences in
chemical uptake, particularly in instances when a single dose
is considered.
EM9 cells have been in culture for more than 25 years
(Caldecott, 2003). It is certainly possible that there are factors
in addition to XRCC1 that could play a factor in the greater
sensitivity of these cells to the cytotoxic and genotoxic effects
of particulate Cr(VI). For these endpoints, other factors such as
metabolic differences or defects in other repair pathways may
play a role. However, the fact that adjusting for differences in
Cr uptake and complementing the cells with XRCC1 did
correct the sensitivity with respect to total chromosome
damage (Fig. 4B), chromatid lesions (Fig. 5B), and chromatid
exchanges (Fig. 7B) does indicate that XRCC1 is the key factor
for these events.
In summary, we have shown for the first time that XRCC1 is
involved in protecting cells from particulate Cr(VI)–induced
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ACKNOWLEDGMENTS
We would like to thank Larry Thompson for the generous gift of the AA8,
EM9, and H9T3 cells. This work was supported by National Institute of
Environmental Health Sciences grant ES10838 (J.P.W.) and the Maine Center
for Toxicology and Environmental Health at the University of Southern Maine.
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