Skip Navigation


Mutagenesis Advance Access originally published online on September 29, 2005
Mutagenesis 2005 20(6):417-423; doi:10.1093/mutage/gei056
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
20/6/417    most recent
gei056v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (7)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Stearns, D. M.
Right arrow Articles by Lantz, R.C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Stearns, D. M.
Right arrow Articles by Lantz, R.C.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?


© The Author 2005. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org

Uranyl acetate induces hprt mutations and uranium–DNA adducts in Chinese hamster ovary EM9 cells

Diane M. Stearns*, Monica Yazzie, Andrew S. Bradley, Virginia H. Coryell, Jake T. Shelley, Adam Ashby, Craig S. Asplund and R.Clark Lantz1

Department of Chemistry and Biochemistry, Northern Arizona University, PO Box 5698, Flagstaff, AZ 86011-5698 and 1Department of Cell Biology and Anatomy, University of Arizona, Tucson, AZ, USA


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Questions about possible adverse health effects from exposures to uranium have arisen as a result of uranium mining, residual mine tailings and use of depleted uranium in the military. The purpose of the current study was to measure the toxicity of depleted uranium as uranyl acetate (UA) in mammalian cells. The activity of UA in the parental CHO AA8 line was compared with that in the XRCC1-deficient CHO EM9 line. Cytotoxicity was measured by clonogenic survival. A dose of 200 µM UA over 24 h produced 3.1-fold greater cell death in the CHO EM9 than the CHO AA8 line, and a dose of 300 µM was 1.7-fold more cytotoxic. Mutagenicity at the hypoxanthine (guanine) phosphoribosyltransferase (hprt) locus was measured by selection with 6-thioguanine. A dose of 200 µM UA produced ~5-fold higher averaged induced mutant frequency in the CHO EM9 line relative to the CHO AA8 line. The generation of DNA strand breaks was measured by the alkaline comet assay at 40 min and 24 h exposures. DNA strand breaks were detected in both lines; however a dose response may have been masked by U–DNA adducts or crosslinks. Uranium–DNA adducts were measured by inductively coupled plasma optical emission spectroscopy (ICP-OES) at 24 and 48 h exposures. A maximum adduct level of 8 U atoms/103 DNA-P for the 300 µM dose was found in the EM9 line after 48 h. This is the first report of the formation of uranium–DNA adducts and mutations in mammalian cells after direct exposure to a depleted uranium compound. Data suggest that uranium could be chemically genotoxic and mutagenic through the formation of strand breaks and covalent U–DNA adducts. Thus the health risks for uranium exposure could go beyond those for radiation exposure.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Questions about possible adverse health effects from environmental and occupational exposures to uranium have arisen as a result of uranium mining, residual mine tailings, and the use of depleted uranium in the military. Depleted uranium is uranium that has higher levels of 238U and lower levels of 235U and 234U relative to natural uranium. Over half of the US uranium reserves are believed to exist in the Four Corners area of the Southwestern United States (1Go). It is estimated that over 300 tons of depleted uranium were used during Gulf War I (2Go), but estimates for Gulf War II have not yet been reported. The impact of these high levels of environmental uranium on exposed populations is of growing concern.

The adverse health effects from occupational and experimental uranium exposures that have been established most significantly include lung cancer, from exposure to 222radon that is produced through the radioactive decay of 238U (3Go,4Go), and chemically induced kidney toxicity (5Go); however, bladder damage (6Go), birth defects (7Go) and chromosomal aberrations (8Go) have also been reported. Thorough epidemiological data for health effects from either environmental exposures to uranium tailings or military exposures to depleted uranium are currently lacking due to insufficient study of both Native American populations other than miners, and the short time span since initial military exposures to DU weapons and munitions. However, evaluation of DU-exposed veterans is in progress (9Go).

Previous work has shown that depleted uranium as uranyl acetate (UA) produced DNA strand breaks in the presence of vitamin C, which suggested a chemical rather than radiological mechanism (10Go). The purpose of the current study was to measure the potential for depleted uranium as UA to be toxic in mammalian cells. Depleted uranium was used because, besides being the commercially available form of uranium, it provides less likelihood for chemical effects to be masked by radioactivity. A soluble form of U(VI) was tested here because of an interest in environmental exposure through drinking water; however, it is not assumed that insoluble uranium provides no risk environmentally. Based on previous work in vitro (10Go) it was expected that DNA strand breaks may be formed in cultured cells, and it was presumed that if strand breaks were occurring then those lesions could be relatively easily repaired. For this reason experiments were carried out in both the parental CHO AA8 line and the CHO EM9 line, which has reduced levels of the XRCC1-DNA ligase III complex (11Go), and is therefore sensitive to DNA strand breaks (12Go). It was hypothesized that if UA caused direct DNA damage in CHO cells then it should be more cytotoxic and mutagenic in the repair-deficient EM9 line than the parental AA8 line. Results supported these hypotheses; however, the DNA damage produced in CHO cells was not limited to strand breaks, but included U–DNA adducts.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Reagents and chemicals
Depleted uranium as uranyl acetate dihydrate [6159-44-0] (UA), with a U234/U238 activity ratio of 0.12 (10Go), was obtained from Spectrum Chemical Mfg Corp. (Gardena, CA). 2-Amino-6-mercaptopurine (6-thioguanine) [154-42-7] (Sigma Chemical Co., St Louis, MO) was used as received.

General cell culture conditions
Chinese hamster ovary AA8 and EM9 cells were obtained from the American Type Culture Collection (Manassas, VA). Cells at passage 3 were thawed from cryopreservation, cultured in {alpha}MEM (Sigma Chemical Co.) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), antibiotic/antimycotic (100 U/ml penicillin, 100 µg/ml streptomycin and 0.25 µg/ml amphotericin B) (Sigma) and 1 mM glutamine (Gibco-BRL, Rockville, MD). Cells were maintained at 37°C in a 5% CO2/air humidified incubator calibrated with a Fryrite analyzer (Bacharach Co., Pittsburgh, PA).

Cytotoxicity measurements
Cytotoxicity, as decreased cell survival, was determined by measuring colony-forming ability in the CHO AA8 and CHO EM9 lines. Cytotoxicity measurements were carried out after incubation with UA for 24 h. Cells were seeded at 8 x 105 cells per 100 mm plate, allowed to adhere for over ~20 h, and treated with sterile-filtered aqueous solutions of UA (0–300 µM) for 24 h. Upon completion of exposure, cells were trypsinized, quantified on a Z1 Coulter Particle Counter (Beckman Coulter, Inc., Miami, FL) and reseeded at 200 cells (AA8) or 300 cells (EM9) per 60 mm dish in quadruplicate. After 7 days all dishes were stained with crystal violet and the colonies were counted. Cell survival was calculated as percent colonies in treated dishes relative to untreated controls. Plating efficiency was 86% for the AA8 line and 56% for the EM9 line.

Mutagenicity measurements
The HPRT assay was carried out following published procedures (13Go) with minor modifications. Cells were exposed to UA at 100–300 µM for 24 h as described above. Cells were harvested and analyzed for cell survival with a 7 day colony formation assay as described above. From the same cell populations harvested for cell survival measurements after 24 h treatment times, approximately 1 x 106 cells were reseeded in 100 mm dishes and that amount was passaged every 3 days until day 9 post-treatment. This expression time was found in previous studies (14Go) to be optimum (data not shown), and is consistent with recommendations for this assay (13Go). After this total 10-day expression time cells were seeded again for colony-forming ability as described above, and 2.5 x 105 cells were seeded in quadruplicate in 100 mm dishes and incubated in medium containing 11 µg/ml of 6-thioguanine (6-TG) for 7–8 days for mutant selection. Data are expressed as mutants per 106 surviving cells, calculated from the observed 6-TG-resistant colonies and the 10-day clonogenic values. Average induced mutant frequency and average mutant increase above background were calculated from the differences and ratios of individual experiments. Experiments were repeated 4–7 times.

Single cell gel electrophoresis (comet assay)
The ability of UA to produce DNA strand breaks in CHO AA8 and CHO EM9 cells was measured by the alkaline comet assay following recommended procedures (15Go,16Go). Briefly, CHO AA8 or CHO EM9 cells were seeded at 8 x 105 cells/ml and exposed to UA after cell attachment. Cells were treated with sterile aqueous solutions of 50–300 µM UA for 40 min or for 24 h at 37°C. As a positive control for strand breaks and oxidative damage, cells were treated with 40 or 80 µM H2O2 for 40 min at 37°C. Untreated cells served as a negative control. Cells were harvested by scraping in dim light, pelleted and placed on ice. Microgels were prepared in duplicate on MGE slides (Erie Scientific Inc., Portsmouth, NH) in four layers as recommended (16Go). All slides were subjected to lysis solution (2.5 M NaCl, 100 mM EDTA tetrasodium salt, 10 mM Tris, pH 10, 1% sodium lauroyl sarcosine, 1% Triton X-100) for 2 h at 4°C.

One set of duplicate slides for each dose was incubated with Escherichia coli formamidopyrimidine-DNA glycosylase (FPG) using the FLARE Assay kit (Trevigen, Inc., Gaithersburg, MD) after the 2 h lysis step, in order to detect the presence of oxidative damage. Slides were rinsed in 1x FLARE buffer for 15 min, and the FPG enzyme, at 50 U in 200 µl, was added to the slides for 30 min at 37°C. These slides were then combined with other duplicate slides for the remainder of the assay.

All slides were then subjected to unwinding in alkaline buffer (300 mM NaOH, 1 mM EDTA, 0.2% DMSO, 0.1% 8-hydroxyquinoline, pH >13) for 20 min, followed by electrophoresis for 15 min on a horizontal electrophoresis unit (MGE, Techniport, Inc.) at 250 mA and 4°C with buffer recirculation of 100 ml/min.

After electrophoresis, slides were neutralized in 1 M ammonium acetate in ethanol for 15 min at ambient temperature, followed by incubation in 1 mg/ml spermine in 66% ethanol for 15 min at ambient temperature. Slides were air-dried in the dark before staining. Slides were prestained for 1 min with a 60 µl volume of 5% sucrose and 1 mM monosodium phosphate. Slides were stained 30 min prior to analysis with 200 µl of a 1:10 000 dilution of SYBR® Green (Molecular Probes, Eugene, OR) followed by 50 µl of Vectashield (Vector laboratories, Inc., Burlingame, CA).

Slides were analyzed for DNA damage on an Olympus BX51 epifluorescence microscope equipped with an LAI Comet Assay Analysis System (Loats Associates, Inc., Westminster, MD) at 40x magnification. Tail moment (tail length x percentage of DNA in tail) was measured in 50 cells for each treatment, positive and negative controls, and independent experiments were repeated in triplicate or quadruplicate. The average of 50 cells was calculated for each treatment, and reported data represent the mean ± SEM of the individual averages of the 3–4 independent experiments.

Measurement of uranium/DNA-P binding by ICP-OES
CHO AA8 or CHO EM9 cells were seeded in duplicate 100 mm dishes at 5 x 105 or 7 x 105 cells per plate, respectively, and were either allowed to grow for 48 h and treated with UA at final concentrations of 0–300 µM for 24 h or were allowed to grow for 24 h and treated with 0–300 µM UA for 48 h. At 5 min before harvesting one set of duplicate dishes was treated with 0–300 µM UA to serve as a ‘zero time point control’ to measure background or membrane-bound uranium that could be carried along in harvested cells to artificially interact with DNA during DNA extraction. Values of U–DNA for zero time points were not statistically different from untreated cells (data not shown). Cells were washed three times with PBS, harvested with trypsin, and cell suspensions from duplicate treatments were combined, pelleted and stored at –4°C until DNA was extracted. Cells were subsequently thawed and lysed in 10 mM Tris, 5 mM EDTA, 5% SDS, 0.2 M NaCl. RNA was removed by incubating samples in lysis buffer containing 2 U of pancreatic RNase A (Sigma) at 37°C for 30 min. DNA was extracted with 25:24:1 phenol:CHCl3:isoamyl alcohol, separated by centrifugation in Light Phase Lock GelTM tubes, twice precipitated with isopropyl alcohol, washed with 75% and 100% ethanol, air-dried, and digested in 200 µl of 20% HNO3 and 50 µl of 30% H2O2 by heating for 1 h at 80°C. Samples were then diluted to a final volume of 3 ml containing 0.1 p.p.m. ytterium as an internal standard. The digested samples were then assayed for uranium and phosphorous on a PerkinElmer Optima 4300DV inductively coupled plasma optical emission spectrometer (ICP-OES) with a meinhard nebulizer and a cyclonic spray chamber. The plasma operated at 1300 W with a sample introduction rate of 1.50 ml/min. The plasma, auxiliary, and nebulizer flow rates were 15, 0.2, and 0.80 l/min, respectively. The emission wavelengths used for uranium and phosphorous were 385.958 and 213.617 nm, respectively. The metal-nucleotide binding ratios were calculated as the moles of uranium per mole of phosphorous in the sample. The limits of detection were 1.805 p.p.b. (7.58 x 10–9 M) for uranium, and 9.322 p.p.b. (3.01 x 10–7 M) for phosphorous. The limits of quantitation were 6.402 ppb (2.69 x 10–8 M) for uranium, and 20.71 ppb (6.69 x 10–7 M) for phosphorous.

Statistics
Statistical significance was evaluated by ANOVA using the Tukey post hoc test. Differences were considered significant at P < 0.05. Statistical outliers were verified by Grubbs' test (extreme studentized deviate method), P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
It was initially hypothesized that if UA caused DNA strand breaks in vitro (10Go) then UA should be more toxic in the strand break-sensitive CHO EM9 line than in the parental CHO AA8 line. This hypothesis was tested by measuring the cytotoxicity and mutagenicity of UA in CHO AA8 and EM9 cells.

The cytotoxicity of UA was measured by a colony formation assay. Cell survival of UA decreased with increasing doses in both CHO AA8 and CHO EM9 cells after 24 h exposures (Figure 1). UA was more cytotoxic in the EM9 line than in the AA8 line, with the 200 µM dose producing 12 and 37% cell death in the AA8 and EM9 lines, respectively, and the 300 µM dose producing 26 and 45% cell death in the AA8 and EM9 lines, respectively. The observation that UA was more cytotoxic in the repair-deficient EM9 line than the parental AA8 line supported the initial hypothesis and was interpreted to suggest that UA caused DNA damage in CHO cells.



View larger version (11K):
[in this window]
[in a new window]
  		Fig. 1.. Cytotoxicity of UA in CHO-AA8 (open circles) and CHO-EM9 (closed circles) cells after 24 h exposures. Cells were treated and assayed for 7–8 day colony formation as described in the text. Data represent mean ± SEM for n = 8–11 independent experiments. The asterisk indicates statistical significance between equivalent doses in the AA8 and EM9 lines at P < 0.05.

 
The mutagenicity of UA was measured at the hprt locus by a selection of cells resistant to 6-thioguanine (6-TG). After 24 h treatment and a 9 day recovery time to allow for expression of the mutant phenotype, survival of UA-treated cells recovered to >=98% in the AA8 line and >=85% in the EM9 line (Table I). UA was weakly mutagenic in the two CHO lines (Figure 2). The untreated AA8 cells averaged 5.1 spontaneous mutations (range 0–11), and the EM9 line averaged 5.3 spontaneous mutations (range 2–8) (Table I). The highest induced mutant frequency was observed in the EM9 line for the 200 µM and 300 µM doses, at 31.3 and 22, respectively, which were ~5-fold and ~4-fold higher than the frequency observed for equivalent doses in the AA8 line. These data were consistent with the interpretation that if UA caused direct DNA damage in CHO cells, then at least some, but perhaps not all, of the resulting DNA lesions were mutagenic.


View this table:
[in this window]
[in a new window]
  		Table I.. Cell survival and mutation induction in CHO AA8 and CHO EM9 cells treated with UA for 24 h

 


View larger version (12K):
[in this window]
[in a new window]
  		Fig. 2.. 6-Thioguanine-resistant cells obtained after 24 h exposure of CHO-AA8 (open diamonds) and CHO-EM9 (closed diamonds) cells to UA. Methods are described in the text. Each data point refers to an individual experiment.

 
It was hypothesized that if UA was mutagenic in repair-deficient cells then DNA damage should be occurring either by direct uranium–DNA interactions or by generation of reactive oxygen species. This hypothesis was tested by measuring DNA damage as strand breaks, oxidative damage and uranium–DNA adducts in CHO EM9 and AA8 cells exposed to UA.

The presence of DNA strand breaks was measured by the alkaline comet assay in cells exposed to 50–300 µM UA for 40 min and 24 h. Exposure to 40 and 80 µM H2O2 for 40 min at 37°C served as the positive control. Hydrogen peroxide showed an increase in tail moment with increasing dose in both cell lines (Figure 3A and B). In the AA8 line post-treatment with FPG significantly increased the tail moment for both H2O2 doses (P <= 0.01), suggesting that oxidative damage was present, as expected (Figure 3A). However, FPG did not produce a significant increase in H2O2-induced tail moment in the EM9 line (Figure 3B). The mean tail moments for CHO EM9 cells exposed to 40 or 80 µM H2O2 were slightly lower than those in the AA8 line. The mean tail moments for CHO EM9 cells exposed to H2O2 with FPG post-treatment were significantly lower than equivalent exposures in the AA8 line for both 40 and 80 µM doses (P < 0.02). These results are consistent with a study that found less H2O2-induced DNA strand breaks in the EM9 line relative to the AA8 line (17Go).



View larger version (27K):
[in this window]
[in a new window]
  		Fig. 3.. Analysis of DNA damage induced by UA and H2O2 by the comet assay. (A) CHO AA8 cells exposed to H2O2 or UA for 40 min at 37°C. (B) CHO EM9 cells exposed to H2O2 or UA for 40 min at 37°C. (C) CHO AA8 cells exposed to UA for 24 h. (D) CHO EM9 cells exposed to UA for 24 h. Cells were analyzed for strand breaks, or were post-treated with FPG and analyzed for oxidative damage. Data represent mean tail moment ± SEM for n = 3–4 independent experiments. Within each experiment 50 cells were scored for each dose.

 
Exposure to UA at 40 min resulted in significant increases in tail moments relative to untreated controls for all doses (50–300 µM) in both cell lines (P < 0.05); however, no dose response was apparent in either cell line (Figure 3A and B). There was no significant difference in tail moments between lines for equivalent UA doses, nor did FPG have a significant effect on increasing tail moment for any dose of UA in either line.

Exposure to UA at 24 h in the AA8 and EM9 lines produced similar increases in tail moment relative to untreated controls and a similar lack of clear dose response in both lines (Figure 3C and D). Treatment with UA plus post-treatment exposure to FPG yielded no significant increase in tail moments relative to treatments with UA alone in either cell line. These results for both 40 min and 24 h exposures could mean that UA did not produce any formamidopyrimidines, and could thus be interpreted to suggest that oxidative damage did not occur in UA-treated cells under these conditions. Data could also be interpreted to suggest that DNA strand breaks did occur in UA-exposed cells; however, the dose response in the comet assay could have been masked by the presence of another lesion, for example DNA crosslinks (vide infra).

Uranium is a metal that forms bonds with biological molecules; thus general uranium-DNA adducts could represent another potential class of DNA lesions. The ability of UA to produce uranium–DNA adducts was therefore measured by ICP-OES in both cell lines. Cells were exposed to 0–300 µM UA for 24 and 48 h. DNA was extracted and precipitated from washed, harvested cells. After digestion in HNO3/H2O2, concentrations of uranium and phosphorous were measured by ICP-OES and ratios of uranium to DNA-P were calculated. Experiments were carried out with and without RNaseA to compare uranium binding to DNA versus total nucleic acid. Data showed that uranium–DNA adducts existed on the order of a few U atoms per thousand nucleotides, and increased with increasing dose and increasing exposure time for 24 and 48 h treatments in both cell lines (Figure 4).



View larger version (25K):
[in this window]
[in a new window]
  		Fig. 4.. Measurement of uranium–DNA binding in CHO AA8 (open bars) versus CHO EM9 (closed bars) cells exposed to UA for 24 or 48 h by ICP-OES. Uranium was found bonded to DNA after incubation with 2U RNase A (+RNase A) and without (–RNase A). Data represent mean ± SEM for n = 4–9 experiments.

 
The effect of RNase A on adduct levels was only significant in the AA8 line at the highest dose tested. In the AA8 line, at the 24 h exposure there were 2.4-fold more (P < 0.0001) U–DNA adducts in samples exposed to RNase A relative to samples for which RNA was not degraded (Figure 4). At the 48 h exposure there were 2.5-fold more (P < 0.001) U–DNA adducts in RNase-treated samples. In the EM9 line there was no difference between uranium adducts in DNA versus total nucleic acid.

In general, there was no significant difference in U–DNA adducts between the AA8 and EM9 lines at either exposure times. In samples not exposed to RNase A, the EM9 line showed 1.7-fold higher U-nucleic acid adducts than the AA8 line after 48 h; however, this difference was not seen after the 24 h exposure, nor was it evident in samples exposed to RNase A.


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Measurements of UA-induced DNA toxicity were carried out in both the repair-proficient CHO AA8 line and the repair-deficient CHO EM9 line. The compromised DNA repair in the EM9 line is due to expression of 4-fold lower DNA ligase III{alpha} and ~10-fold lower X-ray repair cross-complementing gene I protein (XRCC1) relative to the parental line (11Go). Ligase III{alpha} catalyzes the rejoining of the DNA phosphodiester backbone and is thus involved in base excision repair and repair of DNA strand breaks (18Go). The XRCC1 protein has no catalytic activity but forms complexes with ligase III{alpha}, as well as with poly(ADP-ribose) polymerase 1 (PARP-1), PARP-2 and several other DNA repair proteins (19Go). The observation that UA was more cytotoxic in the CHO EM9 line than the CHO AA8 line, coupled with the assumption that the only difference between the two lines was their ability to repair DNA damage, was inferred to suggest that UA caused direct DNA damage that was differentially repaired in these two lines.

The lack of a significant difference between the two cell lines in terms of DNA strand break production was unexpected; however, strand breaks were estimated by mean tail moment in the comet assay, and the presence of other forms of damage may have interfered in the migration of DNA in that assay. For example, if U–DNA adducts existed in the form of crosslinks, or if crosslinks that did not contain uranium were present, those lesions could decrease tail migration, as has been observed for platinum and other crosslinking agents (20Go).

Comparisons can also be made between the two lines in terms of cytotoxicity. The 2- to 3-fold increased cytotoxicity of UA in the EM9 versus AA8 lines places UA in the same category as chemicals known to cause DNA strand breaks and DNA crosslinks, for example H2O2 (17Go), chromate and mercuric chloride (21Go), UVA light (22Go) and near visible and blue light (23Go). The observation that monofunctional alkylating agents ethyl methanesulfonate and methyl methanesulfonate were >10-fold more cytotoxic in the EM9 versus AA8 lines (24Go) is consistent with the interpretation that UA may not form monofunctional adducts or apurinic/apyrimidinic (AP) sites as predominant lesions.

The current observation of UA-induced hprt mutations is consistent with previous reports of soluble uranyl causing cellular and genetic damages in mammalian cells. Uranyl nitrate caused cell death, micronuclei formation, chromosomal aberrations and sister chromatid exchange in CHO cells (25Go). It caused an increase in dicentric chromosomes (26Go), oxidative damage in the presence of H2O2 (27Go) and micronuclei formation (28Go) in human osteoblast cells. Uranyl chloride increased sister chromatid exchange and transformed human osteoblast cells (29Go) and induced apoptosis in mouse J774 macrophages (30Go). The current work has shown that UA will induce mutations in the DNA repair-deficient CHO EM9 line. This is the first report of mutations produced by direct exposure to UA, but it is also consistent with previous work showing mutations in the Ames Salmonella reversion assay with exposure to urine from rats with imbedded DU pellets (31Go) and a slight increase in hprt mutations in lymphocytes of Gulf war veterans with imbedded DU shrapnel (9Go).

UA appears to be a weak mutagen compared to other chemicals that have been tested in CHO cells by this assay, producing an average induced mutant frequency (AIMF) of 31/106 surviving cells in the EM9 line (Table I). Other agents induce much higher AIMF in repair-proficient cells. Methamphetamine showed an AIMF of 13/106 in the CHO K1 line (32Go). The dietary supplement chromium picolinate produced an AIMF of 58/106 surviving cells in the AA8 line (14Go). 60Co {gamma} rays produced 116/106 surviving cells in the AA8 line (33Go). Stronger responses were observed with the alkylating agents ethyl methanesulfonate (20 mM, 1 h) and N-ethyl-N-nitrosourea (1.5 mM, 1 h), both exposures producing ~1000 mutants/106 surviving cells (34Go). The alkylating agent N-n-butyl-N-nitrosourea (2 mM, 1 h) was less mutagenic, producing ~410 mutants/106 survivors (35Go). However, the low mutant frequency of UA at the hprt locus measured in the current study may underrepresent UA mutagenicity since this assay would not detect mutants harboring multilocus mutations. Large multilocus mutations may inactivate essential genes neighboring hprt, causing lethality in those mutant cells since there is no homologous X chromosome to supply the essential gene. This interpretation is consistent with the observation that multiexon deletions were a dominant mutation in UA-exposed CHO EM9 cells (36Go).

The purpose of the current study was to begin to acquire mechanistic information. Therefore, the exposure levels in these experiments are higher than those found in drinking water. There is also evidence that cell lines may differ in their sensitivity to uranyl ion. The CI50 of uranyl nitrate and UA in kidney cells was found to be 500–650 µM for 24 h exposures in rat, human and porcine kidney proximal tubule cell lines (37Go,38Go). However, results from a short-term MTT assay cannot be directly compared to the colony formation assay in the current study. The human osteoblast (HOS) line appears to be more sensitive to uranyl ion than the CHO line, with a 24 h exposure to 100 µM uranyl chloride producing a 0.1 survival fraction by clonogenic assay (29Go) versus 87% survival for this dose of UA in the current study. However, this interpretation must be tempered by the possibility that the 238U/235U isotopic ratios could vary with the different forms of depleted uranium used in these studies, with more 235U causing more radiological toxicity. Also different relative concentrations of components in the cell culture medium for these different lines, for example carbonate or phosphate, could influence uranyl speciation, affecting uptake or bioactivity.

Uranium has generally been considered to be DNA damaging through its radioactivity, specifically through release of alpha and beta particles during its radioactive decay; however, chemical mechanisms may also exist. Combinations of depleted uranium as UA and ascorbate were found to produce DNA strand breaks in plasmid DNA that were greater than those for either UA or ascorbate alone, and were observed in the absence of ascorbate-induced reduction of uranyl ion (10Go), which suggested a direct chemical mechanism for uranium, ascorbate and DNA interactions because the half-lives for decay of uranium isotopes would not be changed by the addition of ascorbate.

Heavy metals in general have been considered to cause DNA damage through indirect mechanisms of free radical generation and oxidative stress. For example nickel, copper, iron and chromium are believed to either undergo electron transfer reactions with biological reducing agents or have their redox potentials altered by chelation with biomolecules, producing a metal complex that reacts with O2 or H2O2 to generate HO· or other reactive oxygen species (39GoGoGo–42Go). However, data from the current study and our previous work (10Go) suggest that, at least in the absence of added hydrogen peroxide, direct uranium(VI)–DNA interactions are more important than free radical mechanisms. If free radical generation were a major pathway under the current conditions, then it would have been expected that oxidative damage would have been detected by the comet assay (Figure 3).

Another mechanism by which metals damage DNA is by a direct covalent interaction. This pathway is known to be important for chromium (43Go), and it may occur for uranium as well. Uranium has been known to interact with DNA in vitro (44GoGo–46Go); however, to our knowledge this is the first report of U–DNA adducts recovered from cultured cells. The current experiments found that uranium covalently bonded to DNA; however, at this time data cannot distinguish between simple uranium–DNA adducts and uranium-containing DNA–protein crosslinks or uranium-containing DNA–DNA crosslinks. The observation of modest differences in adduct levels between these two cell lines is consistent with the interpretation that the CHO EM9 line is depleted in XRCC1-ligase complex, and is therefore less sensitive to crosslinks than strand breaks. Current work is in progress to measure U–DNA adducts in crosslink-sensitive lines.

The current experiments did show evidence of DNA strand breaks in CHO cells exposed to UA (Figure 3). This is consistent with other studies reporting chromosomal aberrations in CHO cells exposed to uranyl nitrate (25Go) and in mouse germ cells exposed to enriched uranyl fluoride (47Go). However, because strand breaks were not the only DNA lesion observed, it is not yet clear whether the strand breaks detected in the comet assay were caused by direct action of uranium on DNA, for example DNA hydrolysis catalyzed by uranium coordinating to the DNA phosphate backbone (10Go), or were indirect intermediates of DNA excision repair. Nevertheless, the lack of oxidative damage in the comet assay coupled with the presence of U–DNA adducts suggests that uranium is acting through a chemical rather than a radiological mechanism.

In conclusion, depleted uranium as UA was found to be genotoxic and mutagenic in CHO cells. The presence of U–DNA adducts lends further support to the hypothesis that uranium is chemically genotoxic. This possibility of direct U–DNA interaction should be considered when extrapolating potential risks for people exposed to uranium in the absence of measurable radioactivity, for example in soil and drinking water, and in DU-containing shrapnel.


   Acknowledgments
 
Funding for the study was provided by NIH grants CA096320 (DMS) and CA096281 (RCL). The ICP-OES and comet assay equipment were purchased with funding from the Arizona Board of Regents Biotechnology and Human Welfare Program (DMS). MY was supported by the NIH Minority Student Development Program grant GM56931.


   Notes
 
* To whom correspondence and reprint requests should be addressed. Tel: +1 928 523 4460; Fax: +1 928 523 8111; Email: Diane.Stearns{at}nau.edu


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

    1. Churchill,W. (1996) From a Native Son: Selected Essays in Indigenism, 1985–1995. South End Press, Cambridge, MA, p. 27.

    2. Durakovic,A. (2003) Undiagnosed illnesses and radioactive warfare. Croatian Med. J., 44, 520–532.[ISI][Medline]

    3. Mulloy,K.B., James,D.S., Mohs,K. and Kornfeld,M. (2001) Lung cancer in a nonsmoking underground uranium miner. Environ. Health Perspect., 1009, 305–309.

    4. Gilliand,F.D., Hunt,W.C., Pardilla,M. and Key,C.R. (2000) Uranium mining and lung cancer among Navajo men in New Mexico and Arizona, 1969–1993. J. Occup. Environ. Med., 42, 278–283.[ISI][Medline]

    5. Chen,J., Meyerhof,D.P. and Tracy,B.L. (2004) Model results of kidney burdens from uranium intakes. Health Phys., 86, 3–11.[ISI][Medline]

    6. Pinney,S.M., Freyberg,R.W., Levine,G.E., Brannen,D.E., Mark,L.S., Nasuta,J.M., Tebbe,C.D., Buckholz,J.M. and Wones,R. (2003) Health effects in community residents near a uranium plant at Fernald, Ohio, USA. Int. J. Occup. Med. Environ. Health, 16, 139–153.[Medline]

    7. Shields,L.M., Wiese,W.H., Skipper,B.J., Charley,P. and Benally,L. (1992) Navajo birth outcomes in the Shiprock uranium mining area. Health Phys., 63, 542–551.[ISI][Medline]

    8. Au,W.W., Lane,R.G., Legator,M.S., Whorton,E.B., Wilkinson,G.S. and Gabehart,G.J. (1995) Biomarker monitoring of a population residing near uranium mining activities. Environ. Health Perspect, 103, 466–470.[ISI][Medline]

    9. McDiarmid,M.A., Engelhardt,S., Oliver,M. et al. (2004) Health effects of depleted uranium on exposed Gulf War veterans: a 10-year follow-up. J. Toxicol. Environ. Health A, 67, 277–296.[ISI][Medline]

    10. Yazzie,M., Gamble,S.L., Civitello,E.R. and Stearns,D.M. (2003) Uranyl acetate causes DNA single strand breaks in vitro in the presence of ascorbate (vitamin C). Chem. Res. Toxicol., 16, 524–530.[CrossRef][ISI][Medline]

    11. Caldecott,K.W., Tucker,J.D., Stanker,L.H. and Thompson,L.H. (1995) Characterization of the XRCCa-DNA ligase III complex in vitro and its absence from mutant hamster cells. Nucleic Acid Res., 23, 4836–4843.[Abstract/Free Full Text]

    12. Thompson,L.H. and West,M.G. (2000) XRCC1 keeps DNA from getting stranded. Mutat. Res., 459, 1–18.[ISI][Medline]

    13. O'Neill,J.P. and Hsie,A.W. (1979) The CHO/HGPRT mutation assay: experimental procedures. In Hsie,A.W., O'Neill,J.P. and McElheny,V.K. (eds), Banbury Report 2. Mammalian Cell Mutagenesis: The Maturation of Test Systems. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 55–69.

    14. Stearns,D.M., Silveira,S.M., Wolf,K.K. and Luke,A.M. (2002) Chromium(III) tris(picolinate) is mutagenic at the hypoxanthine (guanine) phosphoribosyltransferase locus in Chinese hamster ovary cells. Mutat. Res., 513, 135–142.[ISI][Medline]

    15. Tice,R.R., Agurell,E., Anderson,D. et al. (2000) Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ. Mol. Mutagen., 35, 206–221.[CrossRef][ISI][Medline]

    16. Singh,N.P. (2000) Microgels for estimation of DNA strand breaks, DNA-protein crosslinks and apoptosis. Mutat. Res., 455, 111–127.[ISI][Medline]

    17. Cantoni,O., Murray,D. and Meyn,R.E. (1987) Induction and repair of DNA single-strand breaks in EM9 mutant CHO cells treated with hydrogen peroxide. Chem. Biol. Interact., 63, 29–38.[CrossRef][ISI][Medline]

    18. Martin,I.V. and MacNeill,S.A. (2002) ATP-dependent DNA ligases. Genome Biol., 3, 3005.1–3005.7.

    19. Leppard,J.B., Dong,Z., Mackey,Z.B. and Tomkinson,A.E. (2003) Physical and functional interaction between DNA ligase IIIalpha and poly(ADP-Ribose) polymerase 1 in DNA single-strand break repair. Mol. Cell Biol., 23, 5919–5927.[Abstract/Free Full Text]

    20. Pfuhler,S. and Wolf,H.U. (1996) Detection of DNA-crosslinking agents with the alkaline comet assay. Environ. Mol. Mutagen., 27, 196–201.[CrossRef][ISI][Medline]

    21. Christie,N.T., Cantoni,O., Evans,R.M., Meyn,R.E. and Costa,M. (1984) Use of mammalian DNA repair-deficient mutants to assess the effects of toxic metal compounds on DNA. Biochem. Pharmacol., 33, 1661–1670.[CrossRef][ISI][Medline]

    22. Churchill,M.E., Peak,J.G. and Peak,M.J. (1991) Correlation between cell survival and DNA single-strand break repair proficiency in the Chinese hamster ovary cell lines AA8 and EM9 irradiated with 365-nm ultraviolet-A radiation. Photochem. Photobiol., 53, 229–236.[ISI][Medline]

    23. Churchill,M.E., Peak,J.G. and Peak,M.J. (1991) Repair of near-visible- and blue-light-induced DNA single-strand breaks by the CHO cell lines AA8 and EM9. Photochem. Photobiol., 54, 639–644.[ISI][Medline]

    24. Thompson,L.H., Brookman,K.W., Dillehay,L.E., Carrano,A.V., Mazrimas,J.A., Mooney,C.L. and Minkler,J.L. (1982) A CHO-cell strain having hypersensitivity to mutagens, a defect in DNA strand-break repair, and an extraordinary baseline frequency of sister-chromatid exchange. Mutat. Res., 95, 427–440.[ISI][Medline]

    25. Lin,R.H., Wu,L.J., Ching,H.L. and Lin-Shiau,S.Y. (1993) Cytogenic toxicity of uranyl nitrate in Chinese hamster ovary cells. Mutat. Res., 319, 197–203.[CrossRef][ISI][Medline]

    26. Miller,A.C., Xu,J., Stewart,M., Brooks,K., Hodge,S., Shi,L., Page,N. and McClain,D. (2002a) Observation of radiation-specific damage in human cells exposed to depleted uranium: dicentric frequency and neoplastic transformation as endpoints. Radiat. Prot. Dosim., 99, 275–278.[Abstract]

    27. Miller,A.C., Stewart,M., Brooks,K., Shi,L. and Page,N. (2002b) Depleted uranium-catalyzed oxidative DNA damage: absence of significant alpha particle decay. J. Inorg. Biochem., 91, 246–252.[CrossRef][ISI][Medline]

    28. Miller,A.C., Brooks,K., Stewart,M., Anderson,B., Shi,L., McClain,D. and Page,N. (2003) Genomic instability in human osteoblast cells after exposure to depleted uranium: delayed lethality and micronuclei formation. J. Environ. Radioact., 64, 247–259.[CrossRef][ISI][Medline]

    29. Miller,A.C., Blakely,W.F., Livengood,D. et al. (1998a) Transformation of human osteoblast cells to the tumorigenic phenotype by depleted uranium-uranyl chloride. Environ. Health Perspect., 106, 465–471.[ISI][Medline]

    30. Kalinich,J.F., Ramakrishnan,N., Villa,V. and McClain,D.E. (2002) Depleted uranium-uranyl chloride induces apoptosis in mouse J774 macrophages. Toxicology, 179, 105–114.[CrossRef][ISI][Medline]

    31. Miller,A.C., Fuciarelli,A.F., Jackson,W.E., Ejnik,E.J., Emond,C., Strocko,S., Hogan,J., Page,N. and Pellmar,T. (1998b) Urinary and serum mutagenicity studies with rats implanted with depleted uranium or tantalum pellets. Mutagenesis, 13, 643–648.[Abstract/Free Full Text]

    32. Li,J.-H., Hu,H.-C., Chen,W.-B. and Kin,S.-K. (2003) Genetic toxicity of methamphetamine in vitro and in human abusers. Environ. Mol. Mutagen., 42, 233–242.[CrossRef][ISI][Medline]

    33. Diamond,A.M., Dale,P., Murray,J.L. and Grdina,D.J. (1996) The inhibition of radiation-induced mutagenesis by the combined effects of selenium and the aminothiol WR-1065. Mutat. Res., 356, 147–154.[CrossRef][ISI][Medline]

    34. Op het Veld,C.W., van Hees-Stuivenberg,S., van Zeeland,A.A. and Jansen,J.G. (1997) Effect of nucleotide excision repair on hprt gene mutations in rodent cells exposed to DNA ethylating agents. Mutagenesis, 12, 417–424.[Abstract/Free Full Text]

    35. Bol,S.A., van Steeg,H., van Oostrom,C.T., Tates,A.D., Vrieling,H., de Groot,A.J., Mullenders,L.H., van Zeeland,A.A. and Jansen,J.G. (1999) Nucleotide excision repair modulates the cytotoxic and mutagenic effects of N-n-butyl-N-nitrosourea in cultured mammalian cells as well as in mouse splenocytes in vivo. Mutagenesis, 14, 317–322.[Abstract/Free Full Text]

    36. Coryell,V.H. and Stearns,D.M. (2005) Molecular analysis of hprt mutations generated bin Chinese hamster ovary EM9 cells by uranyl acetate, by hydrogen peroxide and spontaneously. Molec. Carcinogen., in press.

    37. Carrière,M., Avoscan,L., Collins,R., Carrot,F., Khodja,H., Ansoborlo,E. and Gouget,B. (2004) Influence of uranium speciation on normal rat kidney (NRK-52E) proximal cell cytotoxicity. Chem. Res. Toxicol., 17, 446–452.[CrossRef][ISI][Medline]

    38. Mirto,H., Barrouillet,M.P., Henge-Napoli,M.H., Ansoborlo,E., Fournier,M. and Cambar,J. (1999) Influence of uranium(VI) speciation for the evaluation of in vitro uranium cytotoxicity on LLC-PK1 cells. Hum. Exp. Toxicol., 18, 180–187.[Abstract/Free Full Text]

    39. Kawanishi,S., Hiraku,Y., Murata,M. and Oikawa,S. (2002) The role of metals in site-specific DNA damage with reference to carcinogenesis. Free Radic. Biol. Med., 32, 822–832.[CrossRef][ISI][Medline]

    40. Schumann,K., Classen,H.G., Dieter,H.H., Konig,J., Multhaup,G., Rukgauer,M., Summer,K.H., Bernhardt,J. and Biesalski,H.K. (2002) Hohenheim consensus workshop: copper. Eur. J. Clin. Nutr., 56, 469–483.[CrossRef][ISI][Medline]

    41. Toyokuni,S. (1996) Iron-induced carcinogenesis: the role of redox regulation. Free Radic. Biol. Med., 20, 553–566.[CrossRef][ISI][Medline]

    42. Shi,X., Chiu,A., Chen,C.T., Halliwell,B., Castranova,V. and Vallyathan,V. (1999) Reduction of chromium(VI) and its relationship to carcinogenesis. J. Toxicol. Environ. Health B Crit. Rev., 2, 87–104.[CrossRef][ISI][Medline]

    43. O'Brien,T.J., Ceryak,S. and Patierno,S.R. (2003) Complexities of chromium carcinogenesis: role of cellular response, repair and recovery mechanisms. Mutat. Res., 533, 3–36.[Medline]

    44. Zobel,C.R. and Beer,M. (1961) Electron stains. I. Chemical studies on the interaction of DNA with uranyl salts. J. Biophys. Biochem. Cytol., 10, 335–346.

    45. Drynov,I.D., Poletaev,A.I., Kharitonenkov,I.G. and Klimenko,S.M. (1974) The interaction of uranyl acetate with DNA. Mol. Biol., 8, 27–33.[Medline]

    46. Jeppesen,C. and Nielsen,P.E. (1989) Uranyl mediated photofootprinting reveals strong E. coli RNA polymerase—DNA backbone contacts in the +10 region of the DeoP1 promoter open complex. Nucleic Acids Res., 17, 4947–4956.[Abstract/Free Full Text]

    47. Hu,Q.Y. and Zhu,S.P. (1990) Induction of chromosomal aberrations in male mouse germ cells by uranyl fluoride containing enriched uranium. Mut. Res., 244, 209–214.[CrossRef][ISI][Medline]

Received on January 27, 2005; revised on August 11, 2005; accepted on August 14, 2005.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Toxicol SciHome page
G. C.-T. Jiang, K. Tidwell, B. A. McLaughlin, J. Cai, R. C. Gupta, D. Milatovic, R. Nass, and M. Aschner
Neurotoxic Potential of Depleted Uranium Effects in Primary Cortical Neuron Cultures and in Caenorhabditis elegans
Toxicol. Sci., October 1, 2007; 99(2): 553 - 565.
[Abstract] [Full Text] [PDF]

Home page
 Toxicol SciHome page
C. Thiebault, M. Carriere, S. Milgram, A. Simon, L. Avoscan, and B. Gouget
Uranium Induces Apoptosis and Is Genotoxic to Normal Rat Kidney (NRK-52E) Proximal Cells
Toxicol. Sci., August 1, 2007; 98(2): 479 - 487.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
20/6/417    most recent
gei056v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (7)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Stearns, D. M.
Right arrow Articles by Lantz, R.C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Stearns, D. M.
Right arrow Articles by Lantz, R.C.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?