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Depleted Uranium Induces Neoplastic Transformation in Human Lung Epithelial Cells Hong Xie,†,‡,§ Carolyne LaCerte,†,‡ W. Douglas Thompson,‡,§ and John Pierce Wise, Sr.*,†,‡,§ Wise Laboratory of EnVironmental and Genetic Toxicology, Maine Center for Toxicology and EnVironmental Health, and Department of Applied Medical Sciences, UniVersity of Southern Maine, 96 Falmouth Street, P.O. Box 9300, Portland, Maine 04104-9300 ReceiVed October 1, 2009
Depleted uranium (DU) is commonly used in military armor and munitions, and thus, exposure of soldiers and noncombatants is frequent and widespread. Previous studies have shown that DU has both chemical and radiological toxicity and that the primary route of exposure of DU to humans is through inhalation and ingestion. However, there is limited research information on the potential carcinogenicity of DU in human bronchial cells. Accordingly, we determined the neoplastic transforming ability of particulate DU to human bronchial epithelial cells (BEP2D). We observed the loss of contact inhibition and anchorage independent growth in cells exposed to DU after 24 h. We also characterized these DUinduced transformed cell lines and found that 40% of the cell lines exhibit alterations in plating efficiency and no significant changes in the cytotoxic response to DU. Cytogenetic analyses showed that 53% of the DU-transformed cell lines possess a hypodiploid phenotype. These data indicate that human bronchial cells are transformed by DU and exhibit significant chromosome instability consistent with a neoplastic phenotype. Introduction Depleted uranium (DU) is a byproduct of uranium enrichment. It is a heavy metal with weak radioactivity. DU is used in numerous military applications, including munitions and armor, as well as in civilian industry, primarily for radiation shielding and aircraft balance control (1). The use of DU munitions and armor is likely to increase over the coming years both in the U.S. military and in other countries; thus, the potential for exposure is widespread and increasing (1). In addition to military-related exposures, 54 of the EPA National Priority List sites have unacceptably high levels of uranium contamination (2). Soldiers and civilians who live near battle zones also have a high risk for exposure to high levels of DU. Numerous studies have reported that DU causes neurological, reproductive, genotoxic, and leukemogenic effects (3). Currently, there is little known about the carcinogenicity of DU (1, 3). Epidemiological data concerning DU’s carcinogenicity are conflicting (4, 5). Some studies show carcinogenic risk, and others show limited or no risk. All of these studies are hampered because of a variety of factors including small sample size, lack of information on DU exposure, young study cohorts, and insufficient time to assess long latency outcomes (1). By contrast, experimental data are mostly positive. Animal studies show that DU can induce mutations in oncogenes and cause soft tissue sarcomas in muscle tissue, and that inhaled DU particles induce lung tumors and increase DNA damage and inflammation (6-8). Cell culture studies indicate that DU can induce neoplastic transformation, genomic instability, and DNA
strand breaks (9-12). Miller et al. reported that DU transformed human osteoblast (HOS) cells to a tumorigenic phenotype (9). Thus, while the epidemiological studies are inconsistent, the laboratory studies indicate a clear carcinogenic effect for DU (1). A major target for DU carcinogenicity is the lung. DU containing metal undergoes combustion, releasing large quantities of very small uranium oxide dust particles into the environment. The dust particles are sufficiently small to be inhaled deep into the lungs, leading to the exposure of lung cells (1). Little is known about the effect of DU on lung cells. Currently, only two studies have considered the potentially carcinogenic effects of DU in human bronchial cells. One study found that DU induces neoplastic transformation of human bronchial epithelial cells (BEAS-2B cells) but only used a single dose (13) and did not characterize the cytogenetic effects. The other study found that DU particles only weakly induce chromosomal aberrations in human bronchial fibroblasts at highly cytotoxic doses (14). Epithelial tumors are believed to develop through a multistep process involving genomic instability (15). Lung cancers are typically characterized by genomic instability including massive chromosome loss or gain, which is also associated with aggressive neoplasms (16). Accordingly, in this study we investigated the ability of DU to cause genomic instability and induce a morphological transformation of normal human lung epithelial cells that is consistent with a neoplastic phenotype.
Materials and Methods * To whom correspondence should be addressed. Wise Laboratory of Environmental and Genetic Toxicology, Maine Center for Toxicology and Environmental Health, 478 Science Building, University of Southern Maine, Portland, Maine 04104. E-mail:
[email protected]. † Wise Laboratory of Environmental and Genetic Toxicology. ‡ Maine Center for Toxicology and Environmental Health. § Department of Applied Medical Sciences.
Chemicals and Reagents. Uranium trioxide was purchased from Strem Chemicals (Newburyport, MA). LHC8, 0.25% trypsinEDTA, trypsin neutralizing solution, and Gurr’s buffer solution were purchased from Invitrogen. Colcemid and potassium chloride were purchased from Sigma Chemical (St. Louis, MO). Acetone, crystal violet, and methanol were purchased from J.T. Baker (Philipsburg,
10.1021/tx9003598 2010 American Chemical Society Published on Web 12/10/2009
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NJ). Tissue culture flasks, dishes, and all plastic ware were purchased from Corning Inc. (Acton, MA). Cells and Cell Culture. A human bronchial epithelial cell line, BEP2D, was used in all experiments. These cells were immortalized using the human papillomavirus 18 (17) and have a near diploid karyotype. They are anchorage-dependent and do not grow in soft agar. Cells were routinely cultured in serum-free LHC8 medium (Invitrogen) at 37 °C with 5% CO2 in a humidified incubator. Cells were maintained as adherent subconfluent monolayers and fed with fresh medium every two or three days. All experiments were performed on logarithmically growing cells less than 55 passages. Cells were checked for mycoplasma every month. Preparation of Depleted Uranium Compounds. Uranium Trioxide (CAS# 1344-58-7, ACS reagent minimum 99.8% purity) was used as a particulate DU compound. Suspensions of uranium trioxide particles were prepared in acetone and administered to cells as previously described (14). Transformation of BEP2D Cells. Cell Contact Inhibition Assay. Transformation studies were done according to our published methods (18). Briefly, 500,000 cells were seeded in 5 mL of medium into five 60 mm dishes for each treatment concentration and allowed to grow for 48 h. Cells were then treated for 24 h with UO3 (0, 0.25, 2.5, and 25 µg/cm2). After exposure, cells were washed twice with Hepes buffered saline solution (HBS) and trypsinized with 0.25% trypsin-EDTA. Then, 100,000 cells from each treated dish were reseeded into two 60 mm dishes (21 cm2 surface area), resulting in ten 60 mm dishes per treatment concentration. Cell growth was monitored regularly for cell morphology changes and focus formation. Foci were isolated with ring cloning cylinders and expanded. At the end of the experiment, dishes were stained with crystal violet, and the number of foci was counted and recorded. Anchorage Independence Assay. Anchorage-independent growth was measured by growth in soft agar according to our published methods (18). Briefly, control and foci cells were suspended in 0.35% agar, plated onto a 0.6% base layer in a 60 mm dish at a density of 5 × 104, and grown for 5 weeks (19). Cultures were examined microscopically 24 h after plating to confirm the absence of large clumps of cells. The number of soft agar colonies was detected by 5% 4-nitro-blue-tetrazolium chloride staining. Characterization of Cell Lines Derived from DU-Induced Foci. Forty-one isolated foci were cloned, expanded, and established into cell lines. Five of the DU-transformed cell lines that were isolated from each UO3 concentration were chosen to be characterized. The selection criteria were positive soft agar growth and having different focus morphology. Plating efficiency, cytotoxic response to UO3, and chromosome instability (CIN) were determined for each of these DU-induced transformed cell lines. All of the characterizations were compared to their parental cell lineBEP2D cells. Plating Efficiency Assay. To investigate potential differences in plating efficiencies of the BEP2D cell line and DU-transformed cell lines in culture, we seeded four 100 mm dishes coated with fibronectin at a colony forming density of 1000 cells/dish. The cells were stained with crystal violet, and the numbers of colonies were counted. Plating efficiency was determined by dividing the average number of colonies obtained per plate by the number of cells plated. There were four dishes per experiment, and each experiment was repeated at least three times. Clonogenic Cytotoxicity Assay. Cytotoxic response to UO3 of focus cells was determined by a clonogenic assay as previously described (14). Five focus-derived cell lines from each concentration were selected for this assay. There were four dishes per treatment group, and each experiment was repeated at least three times. The relative survival of colony results was calculated as a percentage of the control. Aneuploidy Assay. Metaphases were prepared according to our published methods (14). Chromosome number was determined by counting the number of chromosomes in solid stained metaphases
Xie et al.
Figure 1. DU induces loss of cell contact inhibition in human lung epithelial cells. This figure is a representative picture of foci from each DU treatment concentration. (A) Normal BEP2D cells. (B, C, and D) Foci produced in BEP2D cells after exposure to UO3 for 24 h. These foci grew in large masses on the top of the monolayer with complex borders. Magnification is equal to 100×.
Table 1. Depleted Uranium Induces Focus Formation and Anchorage Independence in BEP2D Cells UO3 concentration (µg/cm2)
foci frequency (# of foci/ # of dishes)
# of foci isolated for testing
% of tested foci growth in soft agar
0 0.25 2.5 25
3/30 23/30 24/30 21/30
0/3 13/23 21/24 17/21
0 67 100 75
on the basis of our published methods (14). For trypsin Giemsa banding of chromosomes, the slides were aged at 90 °C in an oven for 1 h, immersed in 0.025% trypsin for 5-8 s, stained with 4% Gurr-Giemsa solution for 5 min, dried, and mounted in permount. Cells were grouped on the basis of chromosome number into three categories: less than 44 chromosomes, 44-48 chromosomes, or greater than 48 chromosomes. A minimum of 100 metaphases was analyzed for each cell line. Statistical Analysis. Student’s t-test was used to calculate p-values to determine the statistical significance of the difference in means. No adjustment was made for multiple comparisons. Student’s t-test Web site: http://www.physics.csbsju.edu/stats/ t-test_bulk_form.html. To calculate the p value using the Student’s t-test Web site, we used: http://www.graphpad.com/ quickcalcs/pvalue1.cfm. One way ANOVA was used to compare the means of the cytotoxic response between the BEP2D cell
Figure 2. DU induces anchorage independent growth of human lung epithelial cells. This figure is a representative image of soft agar colonies. Foci were ring cloned, subcloned, and tested for growth in soft agar. Colonies grew on soft agar as visualized by 5% 4-nitro-bluetetrazolium chloride staining (arrow).
Depleted Uranium Induces Neoplastic Transformation
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Table 2. Plating Efficiency of DU-Transformed Cell Lines
Results
% of Plating Efficiencya DU concentration (µg/cm2) 0 normal BEP2D foci 1 foci 2 foci 3 foci 4 foci 5
0.25
2.5
25
31 ( 0.4 38 33 28 32 34
( ( ( ( (
0.4b 2.5 0.9b 5.6 5.2
35 30 42 33 53
( ( ( ( (
3.3 7.2 1.3b 0.7b 6.1b
19 40 37 40 35
( ( ( ( (
4.1b 3.7b 5.8 4.4b 7.1
a % of plating efficiency ) average number of total colonies/initial seeding density of 1000 cells × 100. b Statistical differences from normal BEP2D (p < 0.05).
line and the DU-induced transformed cell lines. A p value of
