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. Author manuscript; available in PMC: 2008 Dec 1.
Published in final edited form as: Mutat Res. 2007 Jun 19;625(1-2):145–154. doi: 10.1016/j.mrfmmm.2007.06.003

Homologous Recombination Repair Protects Against Particulate Chromate-induced Chromosome Instability in Chinese Hamster Cells

Megan M Stackpole 1, Sandra S Wise 1,2, Eliza Grlickova Duzevik 1, Ray C Munroe 1, W Douglas Thompson 2,3, John Thacker 4, Larry H Thompson 5, John M Hinz 5, John Pierce Wise Sr 1,2,3,*
PMCID: PMC2230547  NIHMSID: NIHMS34500  PMID: 17662313

Abstract

Particulate hexavalent chromium [Cr(VI)] compounds are well-established human carcinogens. Cr(VI)-induced tumors are characterized by chromosomal instability (CIN); however, the mechanisms of this effect are unknown. We investigated the hypothesis that homologous recombination (HR) repair of DNA double strand breaks protect cells from Cr(VI)-induced CIN by focusing on the XRCC3 and RAD51C genes, which play an important role in cellular resistance to DNA double strand breaks. We used Chinese hamster cells defective in each HR gene (irs3 for RAD51C and irs1SF for XRCC3) and compared with their wildtype parental and cDNA-complemented controls. We found that the intracellular Cr ion levels varied among the cell lines after particulate chromate treatment. Importantly, accounting for differences in Cr ion levels, we discovered that XRCC3 and RAD51C cells treated with lead chromate had increased cytotoxicity and chromosomal aberrations, relative to wild-type and cDNA-complimented cells. We also observed the emergence of high levels of chromatid exchanges in the two mutant cell lines. For example, 1 ug/cm2 lead chromate induced 20 and 32 exchanges in XRCC3- and RAD51C-deficient cells, respectively, whereas no exchanges were detected in the wildtype and cDNA-complemented cells. These observations suggest that HR protects cells from Cr(VI)-induced CIN, consistent with the ability of particulate Cr(VI) to induce double strand breaks.

Keywords: chromate, particulate, XRCC3, RAD51C, chromosome instability, double strand break, homologous recombination

1. Introduction

Particulate hexavalent chromium Cr(VI) compounds are well-established human lung carcinogens. Chromosome instability (CIN) is a hallmark of lung cancer and recent data extend these observations to include Cr(VI)-induced tumors [1–4]. Particulate Cr(VI) compounds are considered to be the more potent carcinogenic form of Cr(VI), associated with a greater risk of lung cancer in exposed workers, and an increased incidence of tumors in experimental animals and neoplastic transformation of cultured cells [5–7].

Particulate Cr(VI) has been shown to cause CIN manifested as aneuploidy and chromosome aberrations [8]. The physicochemical mechanism indicates that the Cr(VI) particles partially dissolve outside the cells, releasing the chromate anion and a cation [9]. The chromate anion readily enters the cell via facilitated diffusion and is immediately reduced to trivalent chromium. The products of this reduction then induce DNA damage, growth arrest and cytotoxicity, while the cation (e.g. lead) has no apparent effect [9–13]. Soluble and particulate Cr(VI) compounds (lead chromate, potassium dichromate, calcium chromate, and chromium oxide, sodium chromate) induce cytotoxicity, mutation to 6-thioguanine resistance, and anchorage independence in cultured diploid human fibroblasts at uM concentrations, but Cr(III) compounds only cause cytotoxicity and weak mutagenicity at 100–1,000 fold higher concentrations (14–15). However, while the physicochemical mechanism is understood, the genetic mechanisms that protect cells from particulate Cr(VI)-induced CIN, manifested as structural chromosomal aberrations, are not characterized.

Particulate Cr(VI) has been shown to cause DNA single-strand and double-strand breaks, DNA adducts, and DNA-protein crosslinks. Several studies utilizing vitamin pretreatments have shown that adducts and crosslinks do not correlate with chromosomal aberrations [16–19]. DNA single strand breaks are very short-lived after particulate Cr(VI) exposure and thus do not seem like likely candidates for inducing chromosome damage [13]. By contrast, particulate Cr(VI)-induced DNA double strand breaks are more persistent and are known to cause chromosomal aberrations. Thus, we hypothesize that DNA double strand break repair mechanisms play an important functional role in protecting cells from particulate Cr(VI)-induced CIN. Importantly, we have found previously that the DNA double strand break repair pathway of non-homologous end joining (NHEJ), a key mechanism used by the cell to repair DNA double strand breaks, plays no role in protecting the cell from particulate Cr(VI) induced chromosomal damage [20].

Homologous recombination (HR) repair utilizes the presence of the sister chromatid as a template to repair DNA double strand breaks. HR has 3 major steps: Presynapsis (strand stabilization), synapsis (strand invasion and branch migration) and postsynapsis (Holliday junction formation and resolution). Briefly, during HR repair, DNA double strand breaks, particularly those arising during DNA replication, are converted to 3' single-stranded DNA tails, which are bound by RPA. Rad52, along with the five Rad51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3) interact with RPA and promote binding of RAD51 to the single-stranded DNA and stabilization of the nucleoprotein filament. Subsequently, the RAD51-bound single-stranded DNA invades a homologous molecule in a reaction stimulated by RAD54. This invasion leads to Holliday junction formation which is thought to be resolved by a complex containing XRCC3 and RAD51C [21–22].

A recent study has reported increased nuclear RAD51 foci (indicative of formation of RAD51 nucleoprotein filaments and the induction of HR repair after exposure to soluble Cr(VI) [23]. The study also found that cells deficient in XRCC3 or BRCA2 (another protein necessary for RAD51 filament formation) were hypersensitive to the cytotoxic effect of Cr(VI). However, the relative amount of chromium uptake in each of these cell lines was not considered and thus it is unclear if these data reflect true sensitivity or perhaps a simple difference in chromium uptake. In addition, studies of chromosome aberrations, which reflect the events associated with the carcinogenic effects of Cr(VI) were not performed in these cell lines.

XRCC3 is thought to participate in the resolvase step of HR repair and RAD51C is thought to participate in both the resolvase step and in presynapsis [24–25]. It is unknown if either of these genes are is involved in preventing Cr(VI)-induced CIN. Thus, the purpose of our study was to examine the importance of HR repair in preventing particulate chromate-induced CIN by examining the impact of XRCC3 or RAD51C deficiency on the amount and spectrum of particulate Cr(VI)-induced chromosome damage.

2. Materials and Methods

2.1 Chemical and Reagents

Lead chromate, colcemid and potassium chloride (KCl) were purchased from Sigma-Aldrich (St. Louis, MO). Sodium dodecyl sulfate (SDS) was purchased from American Bioanalytical (Natick, MA). Giemsa stain was purchased from Biomedical Specialties (Santa, Monica, CA). Crystal violet, methanol and acetone were purchased from J.T. Baker (Phillipsburg, NJ). Gurr’s buffer, Trypsin-EDTA, sodium pyruvate, penicillin/streptomycin, and L-glutamine were purchased from Invitrogen Corp. (Carlsbad, CA). Dulbecco’s minimal essential medium and Ham’s F-12 (DMEM/F-12) 50:50 mixture were purchased from Mediatech Inc. (Herndon, VA). Cosmic calf serum was purchased from Hyclone (Logan, UT). Tissue culture dishes, flasks and plasticware were purchased from Corning Inc. (Acton, MA).

2.2 Cells and Cell Culture

The cell lines used in this study are listed in Table 1. We used the well-characterized XRCC3-deficient Chinese hamster ovary (CHO) cell line model system of irs1SF [26], and the XRCC3 cDNA complimented 1SFwt8 [27] cell lines to examine the role of XRCC3. The effect of Cr (VI) on the CIN of the wildtype parental AA8 CHO cells, from which the irs1SF cells were derived, has been characterized previously [28].

Table 1.

Chinese Hamster Cell Lines Used to Study Homologous Repair

Cell Phenotype RAD51C XRCC3
Wild-type cell V79 AA8
Gene Deficient irs3 irs1SF
Gene Restored irs3#6 1SFwt8
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To study the RAD51C gene, we used the model Chinese hamster lung (CHL) fibroblast cell lines: V79, irs3 and irs3#6. The irs3 cell line, derived from the parental V79 cells, is deficient in RAD51C gene, and are used with their cDNA-complimented cell line irs3#6 [29–30].For further information, we have previously published the details and validation of these cells elsewhere [27–30].

All cells were routinely cultured in DMEM/F-12 supplemented with 10% Cosmic calf serum, 2 mM L-glutamine, 100 units/ml penicillin, 100μg/ml streptomycin, and 0.1 mM sodium pyruvate. Cells were maintained at 37 °C as adherent subconfluent monolayers. This was done by subculturing at least twice weekly using 0.25% trypsin/1 mM EDTA solution.

2.3 Chemical Preparation

Lead chromate (CAS #7758-97-6; ACS reagent minimum 98% purity) was used as a model for particulate chromium salt. It was suspended in acetone and administered to cells as previously described [8, 31].

2.4 Cytotoxicity Assay

A clonogenic assay was used to measure cytotoxicity. Briefly, this was done by treating logarithmically growing cells with lead chromate for 24 h. Cells were then reseeded to a colony forming density and grown for 6 to 7 days to allow colony formation. Each dish was fixed with 95% EtOH, stained with crystal violet and colonies were counted [30–31]. Each treatment group contained four dishes with at least three independent experiments per cell line.

2.5 Chromosome Preparations

Determination of chromosomal damage was measured as previously described [32–33]. Briefly, logarithmically growing cells were treated with lead chromate for 24 h. Cells were then harvested, resuspended in hypotonic solution and fixed with Carnoy’s fixative, dropped onto slides, stained with Giemsa, and cover slips were added. Background damage levels were subtracted from each data point as previously described [32–33]. Chromosomes were analyzed using our published methods [8]. Gaps and breaks were pooled as ‘lesions’ as previously described [8]. This was done because breaks can only be unequivocally distinguished from gaps 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. One hundred metaphases were scored for each experiment and all experiments were repeated at least three times for each cell line.

Additionally, a mitotic index was performed without addition of colchicine. There was no difference in the frequency of mitotic cells between the wild-type, gene deficient or gene complemented cells after lead chromate exposure (data not shown).

2.6 Intracellular Chromium Ion Measurement

Analysis of the intracellular chromium ion levels was completed using an inductively coupled plasma- optical emission spectrometer (ICP-OES) as previously described [33]. Briefly, logarithmically growing cells were treated with lead chromate for 24 h. Cells were rinsed with PBS, followed by the addition of hypotonic and then 2% SDS. This solution was then sheared through a needle seven times and filtered through a 0.2 um filter. Each experiment was repeated at least three times for each cell line.

2.7 Data analysis

The relationships between dose and cell type and each of the dependent variables were assessed using ANOVA and linear regression models. Indicator variables for dose levels and cell types, as well as product variables for the joint effects of dose level and cell type, were included as independent variables in the models. Differences were evaluated using t-tests and 95% confidence intervals. The relationship between dose and intracellular concentration was quantified using a separate regression analysis for each cell type. The resulting values and variance-covariance matrix were then used to estimate the differences among cell types at concentrations of 100, 500, 1000, and (for cytotoxicity only) 5000 [33]. All analyses were conducted using the SAS software package.

3. Results

3.1 XRCC3-deficient cells have lower intracellular ion uptake of chromium (Cr), but RAD51C-deficient cells have greater uptake compared to parental cells

As a first step we measured intracellular Cr ion levels in all six cell lines used in this study to detect any differences in the amount of Cr entering the cells after lead chromate exposure. Cr ion levels were significantly lower in the irs1SF (XRCC3-deficient) cells than AA8 (wildtype) and 1SFwt8 (XRCC3-cDNA complemented cells) (Figure 1A). For example, after a 24 h exposure to 1 ug/cm2 lead chromate, intracellular Cr ion levels were 230 uM in irs1SF cells compared to 817 uM in AA8 and 700 uM in 1SFwt8 cells.

Figure 1. Intracellular Ion Uptake of Chromium.

Figure 1

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Cells deficient in XRCC3 have less Cr uptake whereas cells deficient in RAD51C have more Cr uptake than wildtype and cDNA-complemented cells. A) Intracellular chromium ion concentrations of AA8 (wildtype), irs1SF (XRCC3-deficient) and 1SFwt8 (XRCC3-complemented). B) Intracellular chromium ion concentrations of V79 (wildtype), irs3 (RAD51C-deficient) and irs3#6 (RAD51C-complemented). Data represent the average of at least 3 independent experiments ± standard error of the mean

By contrast, Cr ion levels were higher in irs3 (RAD51C-deficient) cells relative to their parental (V79) or RAD51C-cDNA complemented (irs3#6) cells (Figure 1B). For example, after a 24 h exposure to 1 ug/cm2 lead chromate, intracellular Cr ion levels were 1,431 uM in irs3 cells compared to 664 uM in V79 and 528 uM in irs3#6 cells. Accordingly, because of the differential Cr uptake among the cell lines we considered administered concentrations and intracellular ion levels for each experimental endpoint.

3.2 Lead chromate induced higher cytotoxicity in XRCC3- and RAD51C-deficient cells versus parental cells

Lead chromate induced a clear concentration-dependent decrease in the relative survival of the irs1SF XRCC3-defective cells and their controls, (Figure 2A). Correcting for intracellular Cr levels show that the XRCC3-deficient cells are significantly more sensitive to the cytotoxic effects of lead chromate (Figure 2B). For example at an intracellular Cr level of 100 uM, the XRCC3-deficient cells show about 70% relative survival compared to 85% survival in the wildtype (V79) (p value: <0.0001). The XRCC3 cDNA-complimented cells show a decrease in sensitivity to the cytotoxic effects of chromium relative to the deficient cells.

Figure 2. Cytotoxic Effects of Lead Chromate.

Figure 2

Figure 2

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Cells deficient in XRCC3 and RAD51C are more sensitive to the cytotoxic effects of particulate Cr(VI). A) The cytotoxicity of lead chromate in AA8 (wildtype), irs1SF (XRCC3-deficient) and 1SFwt8 (XRCC3-complemented) based on administered concentration. B) The cytotoxicity of lead chromate in AA8 (wildtype), irs1SF (XRCC3-deficient) and 1SFwt8 (XRCC3-complemented) based on the measured intracellular Cr concentration. C) The cytotoxicity of lead chromate in V79 (wildtype), irs3 (RAD51C-deficient) and irs3#6 (RAD51C-complemented) based on the measured administered concentration. D) The cytotoxicity of lead chromate in V79 (wildtype), irs3 (RAD51C-deficient) and irs3#6 (RAD51C-complemented) based on intracellular Cr concentration. Data represent the average of at least 3 independent experiments ± standard error of the mean.

RAD51C-deficient cells also showed increased sensitivity to cytotoxicity by exposure to Cr. Lead chromate caused a concentration-dependent decrease in the relative survival of all three CHL cell lines tested (V79, irs3 and irs3#6; Figure 2C), but correcting for intracellular Cr levels show that the RAD51C-deficient cells are more sensitive to the cytotoxic effects of lead chromate (Figure 2D). For example at an intracellular Cr level of 500 uM the RAD51C-deficient cells show about 63% relative survival compared to 83% survival in the parent (V79) (p value: <0.0001). RAD51C-complemented cells were restored to wild-type levels of sensitivity (p value: <0.1444).

3.3 Lead chromate increased frequency of metaphases with chromosome damage in XRCC3- and RAD51C-deficient cells

Lead chromate induced a concentration-dependent increase in the percentage of metaphases with at least 1 broken chromosome in all three XRCC3-related cell lines (Figure 3A). For example, 1 ug/cm2 lead chromate induced chromosomal aberrations in 20% of XRCC3-deficient metaphases compared to 8% in parental (p value: <0.0345). XRCC3-complemented cells were restored to wild-type (p value: <0.3961). Correcting for differences in intracellular Cr levels showed an even greater effect (Figure 3B). For example, at an intracellular Cr level of 100 uM, XRCC3-deficient cells had 17% metaphases with damage compared to 3% in the parental (p value: <0.0019). XRCC3 cDNA-complemented cells were restored to wild-type levels of chromosome damage (p value: <0.5839).

Figure 3. Percent of Metaphases with Damaged Chromosomes.

Figure 3

Figure 3

Figure 3

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Lead chromate induced a greater percentage of metaphases with chromosomal damage in cells deficient in XRCC3 and RAD51C. A) The clastogenicity of lead chromate in AA8 (wildtype), irs1SF (XRCC3-deficient) and 1SFwt8 (XRCC3-complemented) based on administered concentration. B) The clastogenicity of lead chromate in AA8 (wildtype), irs1SF (XRCC3-deficient) and 1SFwt8 (XRCC3-complemented) based on intracellular Cr concentration. C) The clastogenicity of lead chromate in V79 (wildtype), irs3 (RAD51C deficient) and irs3#6 (RAD51C-complemented) based on administered concentration. D) The clastogenicity of lead chromate in V79 (wildtype), irs3 (RAD51C-deficient) and irs3#6 (RAD51C-complemented) based on intracellular Cr concentration. Data represent the average of at least 3 independent experiments ± standard error of the mean. Background (control) levels have been subtracted for each data point, AA8 control 1.7 ±0.9, irs1SF control 7.6±2.3, 1SFwt8 control 3.3±2.4, V79 control 2.3 ±1.3, irs3 control 10±2.6, irs3#6 control 2.6±1.2

Lead chromate also induced a concentration-dependent increase in the percent of damaged metaphases in all three RAD51C-related cell lines (Figure 3C). For example, 1 ug/cm2 lead chromate induced chromosomal aberrations in 45% of RAD51C-deficient metaphases compared to 8% in parental (p value: <0.0001). RAD51C-complemented cells showed 4% of metaphases with aberrations, similar to the parental cells (p value: <0.3833). Correcting for differences in intracellular Cr levels showed the same trend (Figure 3D). For example, at an intracellular Cr level of 500 uM, RAD51C-deficient cells had 20% metaphases with damage compared to 5% in the parental (p value: <0.0001). RAD51C-complemented cells were restored to wildtype (p value: <0.6171).

Next, we assessed total chromosomal damage, which reflects the total number of aberrations in 100 metaphases and provides a measure of cells with more than 1 chromosome aberration. As expected, we found that lead chromate induced a concentration-dependent increase in total amount of chromosome damage in the XRCC3-deficient irs1SF cells and their control cell lines (Figure 4A). For example, 1 ug/cm2 lead chromate induced 28 chromosomal aberrations in 100 XRCC3-deficient metaphases compared to 8 in parental (p value: <0.0603). XRCC3-complemented cells had 6 chromosomal aberrations which were similar to the wild-type (p value: <0.5902). Due to the reduced uptake of Cr by the irs1SF cells, correcting for differences in intracellular Cr levels showed an even greater effect (Figure 4B). At an intracellular Cr level of 100 uM, XRCC3-deficient cells had 20 aberrations in 100 metaphases with damage compared to 3 in the parental (p value: <0.0213). XRCC3 gene-complemented cells were restored to wild-type levels of total chromosomal aberrations per Cr dose (p value: <0.7773).

Figure 4. Total Metaphases with Damaged Chromosomes.

Figure 4

Figure 4

Figure 4

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Lead chromate induced a greater amount of total chromosome damage in100 metaphases of cells deficient in XRCC3 and RAD51C. A) The clastogenicity of lead chromate in AA8 (wildtype), irs1SF (XRCC3-deficient) and 1SFwt8 (XRCC3-complemented) based on administered concentration. B) The clastogenicity of lead chromate in AA8 (wildtype), irs1SF (XRCC3-deficient) and 1SFwt8 (XRCC3-complemented) based on intracellular Cr concentration. C) The clastogenicity of lead chromate in V79 (wildtype), irs3 (RAD51C-deficient) and irs3#6 (RAD51C-complemented) based on administered concentration. D) The clastogenicity of lead chromate in V79 (wildtype), irs3 (RAD51C-deficient) and irs3#6 (RAD51C-complemented) based on intracellular Cr concentration. Data represent the average of at least 3 independent experiments ± standard error of the mean. Background (control) levels have been subtracted for each data point. AA8 control 2.0 ±1.0, irs1SF control 8.3±3.0, 1SFwt8 control 3.6±2.7, V79 control 2.3 ±1.3, irs3 control 11±2.9, irs3#6 control 2.6±1.2

Lead chromate also induced a concentration-dependent increase in the total chromosome damage in RAD51C-deficient irs3 cell line and its controls (Figure 4C). For example, 1 ug/cm2 lead chromate induced 68 chromosomal aberrations in 100 RAD51C-deficient metaphases compared to 10 in parental (p value: <0.0001). RAD51C-complemented cells showed 5 aberrations per 100 metaphase spreads, similar to the wildtype cells(p value: <0.6033). Correcting for differences in intracellular Cr levels showed the same trend (Figure 4D). For example, at an intracellular Cr level of 500 uM, RAD51C-deficient cells had 38 aberrations in 100 metaphases with damaged compared to only 5 in the parental cells (p value: <0.0001). RAD51C-complemented cells were restored to wildtype levels of aberrations (p value: <0.8065).

3.4 Lead chromate induces chromatid exchanges in XRCC3- and RAD51C-deficient cells

Finally, we considered effects of XRCC3 and RAD51C deficiencies on the spectrum of chromatid-type damage. Lead chromate caused an overall increase in chromatid lesions in both Rad51 paralog-deficient cell lines studied compared to the corresponding wildtype and gene-complemented cells (Figure 5). However, even more remarkable was that lead chromate induced a high level of chromatid exchanges in XRCC3- and RAD51C-deficient cells. For example, 1 ug/cm2 lead chromate caused 20 and 48 chromatid exchanges in 100 metaphases for XRCC3-deficient and RAD51C-deficient cells compared to 0 chromatid exchanges in 100 metaphases for the wildtype (p value: <0.0057, <0.0001, respectively). Complemented cells of each mutant were restored to wild-type levels (p value: <0.9203, <1.0000, respectively)

Figure 5. Spectrum of Chromosome Damage.

Figure 5

Figure 5

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Total chromatid lesions and exchanges induced by lead chromate in 100 metaphase cells deficient in XRCC3 and RAD51C. A) The spectrum of damage for AA8 (wildtype), irs1SF (XRCC3-deficient) and 1SFwt8 (XRCC3-complemented). B) The spectrum of damage for V79 (wildtype), irs3 (RAD51C deficient), irs3#6 (RAD51C-complemented). Data represent the average of at least 3 independent experiments ± standard error of the mean and background (control) has been removed. For chromatid lesions:AA8 control 1.7 ±0.9, irs1SF control 5.3±2.0, 1SFwt8 control 2.6±2.7, V79 control 2.3 ±1.3, irs3 control 7.3±1.8, irs3#6 control 1.6±1.2. For chromatid exchanges: AA8 control 0, irs1SF control 3.0±1.2, 1SFwt8 control 0, V79 control 0, irs3 control 2.6±1.8, irs3#6 control 0.

4. Discussion

Chromosome instability (CIN) is a hallmark of chromium-associated lung cancer. However, the mechanisms of these carcinogenic rearrangements involved, and the cellular DNA damage signaling and repair pathways acting to prevent such mutagenic events are not well defined [3]. Cr(VI) is a well-established human lung carcinogen and previous studies show that Cr(VI) chromium is most potent in particulate form. Particulate Cr(VI) induces DNA strand breaks, Cr-DNA adducts, Cr-DNA crosslinks and mutation to 6-thioguanine resistance in diploid human fibroblasts [8,14–17, 12,35,36], however how these lesions are repaired and their relationship to Cr(VI)-induced CIN are uncertain.

In this study we show that the homologous recombination repair pathway plays a role in protecting cells from particulate Cr(VI)-induced CIN. Specifically, the Rad51 paralogs XRCC3 and RAD51C function to prevent simple and complex chromosome aberrations induced by particulate Cr(VI). Deficiency in either of these repair proteins also increased the Cr(VI)-induced frequency and total amount of chromosomal damage and caused a dramatic shift in spectrum of aberrations. This conclusion is supported by published data showing that Cr(VI)-induced DNA double strand breaks are preferentially formed after the S-phase of the cell cycle, likely during DNA replication, when HR repair is the most active [37–39]. Importantly, the role of HR in dealing with Cr(VI)-associated lesions is consistent with our recent data showing that a major alternative pathway for the repair of double strand breaks, NHEJ, does not protect cells from particulate Cr(VI)-induced CIN [20]. These data are the first to consider genes involved in HR repair and Cr(VI)-induced chromosome damage. They are consistent with previous reports of functional polymorphisms in XRCC3, which correlate with increased chromosome damage in metal-exposed workers and with increased risk of lung cancer [40–41].

Both genes participate in the repair of DNA double strand breaks. XRCC3 is thought to participate in the resolvase step and RAD51C in both the resolvase step and in presynapsis of HR repair [24–25]. Thus, our data strongly suggest that DNA double strand breaks, associated with DNA replication, underlie Cr(VI)-induced chromatid aberrations, particularly chromatid exchanges. Generation of chromatid exchanges may lead to an increase in chromosome translocations, a hallmark of lung cancer [42–44]. Other published data suggest that Cr(VI)-induced DNA double strand breaks probably result from replication fork collapse during the repair of single strand breaks, crosslinks or adducts [26], consistent with our previous report showing that a deficiency in single strand breaks also leads to more complex chromosomal aberrations [33]. Any unrepaired double strand breaks then manifest as chromatid aberrations and exchanges in mitosis. The proximate genotoxicant for particulate and soluble Cr(VI) is the same (chromate anion) and thus, the mechanism of formation of the double strand break should also be the same though it has not yet been demonstrated [45].

We also found that both XRCC3- and RAD51C-deficient cells are more sensitive to the cytotoxic effects of chromium. These data are consistent with our previous reports showing that cells deficient in Ku80, FANCG and XRCC1 are also sensitive to the cytotoxic effects of particulate Cr(VI) [33, 28, 20]. They are also consistent with a previous study of soluble chromate showing that XRCC3- and BRCA2-deficient cells were more sensitive to its cytotoxicity [23]. Considered altogether they indicate that after particulate Cr(VI) exposure the DNA repair machinery can interact with cell death pathways and delay apoptosis to allow time for repair.

In this study, and in previous reports using DNA-repair defective cells, we find that DNA repair-deficient cells, including those deficient in Ku80, XRCC1, and FANCG, exhibited large differences in the levels of intracellular genotoxic chemicals compared to their parental and gene complemented control cells [33, 20, 28]. Such differences in uptake can potentially reverse the conclusions of the relative sensitivities of a cell strain to an agent based only on administered dose. These data observations are significant as recent studies consider the repair of chemically-induced lesions including mitomycin C, psoralen and Cr(VI), but fail to account for differences in chemical uptake. A recent study measuring the Cr(VI)-induced toxicity of HR repair-deficient cells to the parent cells failed to consider the differential uptake of the compound among the cells in the study, thus potentially compromising their conclusions [23].

In summary, we show for the first time a role for the HR repair associated genes, XRCC3 and RAD51C in protecting cells from particulate Cr(VI)-induced CIN. Thus, implicating HR repair to be the primary repair pathway for resolving Cr(VI)-induced DNA double strand breaks. Further work will focus on understanding the link between HR repair in prevent Cr(VI)-induced neoplastic transformation and translocations.

Acknowledgments

We would like to thank David Kirstein and Christy Gianios, Jr for technical assistance. We also would like to thank Dr. Samantha Langley-Turnbaugh for the use of the ICP-OES. The research described in this paper has been funded in part by the NIEHS grant ES10838 (J.P.W) and the Maine Center for Toxicology and Environmental Health at the University of Southern Maine.

Footnotes

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