[ RadSafe ] DU Proven Deadly To Human Bronchial Cells
rhelbig at california.com
Thu May 10 04:37:56 CDT 2007
That's the subject line as circulating on a Yahoo list Global Police State by someone who calls themselves Neomulder. The question is what exactly has this study proven and are the people doing the study genuinely studying DU or is this like the literature study that was made by the professor from Tufts who fell under the spell of the Traprock Peace Center .. the Maine location is very close to where the Military Toxics Project used to operate and they were closely tied to Traprock who sponsored trips by Moret and Rokke and Rokke was in nearby New Hampshire last year. Does anyone on the list care to comment further? Thank you.
----- Original Message -----
From: arthur cottrell
To: Global_Police_State at yahoogroups.com
Sent: Tuesday, May 08, 2007 8:47 PM
Subject: [Global_Police_State] DU Proven Deadly To Human Bronchial Cells
----- Original Message -----
From: Neo Mulder
Subject: DU Proven Deadly To Human Bronchial Cells
Particulate Depleted Uranium Is Cytotoxic and Clastogenic to Human Lung Cells
Sandra S. Wise, W. Douglas Thompson, AbouEl-Makarim Aboueissa, Michael D. Mason, and John Pierce Wise, Sr.*
Wise Laboratory of Environmental and Genetic Toxicology, University of Southern Maine, 96 Falmouth Street, Portland, Maine 04104-9300, Maine Center for Toxicology and Environmental Health, University of Southern Maine, 96 Falmouth Street, Portland, Maine 04104-9300, Department of Applied Medical Science and Department of Mathematics and Statistics, University of Southern Maine, 96 Falmouth Street, Portland, Maine 04104-9300, and Institute for Molecular Biophysics, Department of Chemical and Biological Engineering, University of Maine, Orono, Maine 04469
Received January 18, 2007
Depleted uranium (DU) is commonly used in military armor and munitions, and thus, exposure of soldiers and non-combatants is potentially frequent and widespread. DU is considered a suspected human carcinogen, affecting the bronchial cells of the lung. However, few investigations have studied DU in human bronchial cells. Accordingly, we determined the cytotoxicity and clastogenicity of both particulate (water-insoluble)
and soluble DU in human bronchial fibroblasts (WTHBF-6 cells). We used uranium trioxide (UO3) and uranyl acetate (UA) as prototypical particulate and soluble DU salts, respectively. After a 24 h exposure, both UO3 and UA induced concentration-dependent cytotoxicity in WTHBF-6 cells. Specifically, 0.1, 0.5, 1, and 5 g/cm2 UO3 induced 99, 57, 32, and 1% relative survival, respectively.
Similarly, 100, 200, 400, and 800 M UA induced 98, 92, 70, and 56% relative survival, respectively. When treated with chronic exposure, up to 72 h, of either UO3 or UA, there was an increased degree of cytotoxicity. We assessed the clastogenicity of these compounds and found that at concentrations of 0, 0.5, 1, and 5 g/cm2 UO3, 5, 6, 10, and 15% of metaphase cells exhibit some form of chromosome damage. UA did not induce chromosome damage above background levels. There were slight increases in chromosome damage induced when we extended the UO3 treatment time to 48 or 72 h, but no meaningful increase in chromosome damage was observed with chronic exposure to UA.
Uranium (U) is a naturally radioactive metal that consists of three isotopes: 235U, 234U, and 238U. The nuclear industry has used refined U for many years for energy production. The refinement of U results in the production of large quantities of depleted uranium (DU) consisting primarily of 238U (1). DU retains the same chemical properties of natural uranium; however, it is much less radioactive. These properties, high density and pyrophoricity in particular, have made DU ideal for military applications of armor-plating and armor-piercing munitions. Explosions and fires involving these DU products result in DU dust, which leads to significant inhalation of DU particles (2).
These small DU particles, (<10 m) can be inhaled deeply into the lung, leading to longer retention and thus longer exposure.
DU is now becoming a major international concern as a possible health hazard and carcinogen (1-4). Little is currently known about DU mechanisms of effect, but reported data indicate that it may cause lung cancer (1-4), embryotoxicity and teratogenicity (5), reproductive and developmental damage (6), genomic instability (7), and single strand DNA breaks (8). Given the widespread use of uranium for military application and the present worldwide deployment of the United States military, it is imperative that we investigate the carcinogenicity and genotoxicity of DU.
It is difficult to address the issue of DU exposure in humans. Most of the epidemiologic data with regard to human exposure to U that show increases in cancer morbidity and mortality are associated with either radon or other chemical confounders (4). Chromosomal analysis performed on blood samples from war veterans exposed to DU 10 years prior shows aberrations typical of exposure to ionizing radiation (9). However, many experts suggest that because of DU's low specific activity, it does not pose a significant radiologic risk.
Only a few studies have considered the genotoxic and carcinogenic potential of DU. Animal studies using rodents embedded with DU fragments were found to induce mutations in several key oncogenes, to induce serum mutagenicity, and to cause soft tissue sarcomas in muscle tissue (10-12). DU particles inhaled by rats showed increased DNA damage and inflammatory effects (13). Studies in human osteosarcoma cells indicate that DU can induce transformation (14) and cause cytotoxicity, genomic instability, and micronuclei formation (7). Soluble DU caused micronuclei formation, sister chromatid exchanges, DNA adducts, hprt mutations, and chromosomal aberrations in Chinese hamster ovary (CHO) cells (15, 16). However, while these papers provide some evidence that DU is genotoxic and potentially carcinogenic, they do not focus on the target cells, and the genotoxic effect was not strong.
The major route of exposure to DU is through inhalation of particles (1, 4). Thus human bronchial cells (HBC) are a primary target of DU's effects; however, the effects of DU in the lung are poorly characterized (17). Only two studies have considered the interaction of uranium and HBC (18, 19). One study found that insoluble DU induced neoplastic transformation of HBC with chronic exposures (18). The other reported that uranium ore dust induced lipid peroxidation and micronuclei formation (19); however, the chemical analysis of that ore dust revealed no actual uranium content. No studies have considered the clastogenicity of DU in HBC. Accordingly, the purpose of this study was to improve our current understanding of DU by studying the clastogenicity of both particulate and soluble DU in human bronchial cells.
Materials and Methods
Chemicals and Reagents. Uranium trioxide was purchased from Strem Chemicals (Newburyport, MA). Uranyl acetate was purchased from Electron Microscopy Sciences (Fort Washington, PA). Colcemid and potassium chloride (KCl) were purchased from Sigma Chemical (St. Louis, MO). Giemsa stain was purchased from Biomedical Specialties Inc. (Santa Monica, CA). Crystal violet, methanol and acetone were purchased from J. T. Baker (Phillipsburg, NJ). D-MEM/F-12 was purchased from Mediatech Inc. (Herndon, VA). Cosmic calf serum (CCS) was purchased from Hyclone (Logan, UT). Gurr's buffer, trypsin-EDTA, sodium pyruvate, penicillin-streptomycin, and L-glutamine were purchased from Invitrogen Corporation (Grand Island, NY). Tissue culture dishes, flasks, and plasticware were purchased from Corning Inc. (Acton, MA).
Cells and Cell Culture. WTHBF-6 cells, a clonal cell line derived from normal human bronchial fibroblasts that ectopically express human telomerase, were used in all experiments. These cells have a similar doubling time (24 h) and clastogenic and cytotoxic responses to metals compared to those of their parent cells (20). Ectopically expressing telomerase can induce a variety of phenotypes in mass cultured cells from normal to genomically unstable cells (21); thus, this cell line was subcloned from a mass culture and chosen as a model system because it reflects the normal phenotype (20). After more than 1000 population doublings, these cells retain a normal diploid karyotype (data not shown). Cells were maintained as subconfluent monolayers in DMEM/F-12 supplemented with 15% CCS, 2 mM L-glutamine, 100 U/mL penicillin/100 g/mL streptomycin, and 0.1 mM sodium pyruvate and incubated in a 5% CO2 humidified environment at 37 C. CCS is a synthetic serum supplemented with iron and growth factors. The levels of iron in CCS reflect physiological concentrations and as such are higher than levels seen in bovine serum. Cells were fed three times a week and subcultured at least once a week using 0.25% trypsin/1 mM EDTA solution. All experiments were performed on logarithmically growing cells, and cell densities relative to surface area were kept the same across experimental assays.
Preparation of DU Compounds. Uranyl acetate
(CAS# 541-09-3, ACS reagent minimum 99.6% purity)
was used as a model soluble DU compound. Solutions of UA were prepared by weighing out the desired amount and dissolving it in double distilled water. Dilutions were made for appropriate treatment concentrations and then filter sterilized through a 10 mL syringe with a 0.2 m filter.
Uranium trioxide (CAS# 1344-58-7, ACS reagent minimum 99.8% purity)
was used as a model particulate form of uranium. Suspensions of UO3 particles were prepared by rinsing twice in double distilled water to remove any water soluble contaminants and then twice in acetone to remove any organic contaminants. Air dried particles were weighed, placed in acetone (for sterilization) in a borosilicate scintillation vial, and homogenized for 3-5 min. The mean size distribution of the particles was 400 nm as measured with Zetasizer 3000 HS (Malvern Instruments, Worcestershire, UK). The particles were kept in suspension using a vortex mixer and diluted into appropriate suspensions for specific treatments. Dilutions were also maintained as a suspension using a vortex mixer, and treatments were directly dispensed into cultures from these suspensions. Control groups were treated with equivalent amounts of acetone to account for this vehicle.
Positive controls were treated with soluble (sodium chromate) or particulate (lead chromate) hexavalent chromium compounds. These solutions and suspensions were prepared as reported in previous studies (20, 22-24).
Cytotoxicity Assays. Cytotoxicity was determined using published methods (20) for a clonogenic assay, which measures a reduction in plating efficiency in treatment groups relative to the controls. Briefly, 90,000 cells were seeded in 2.3 mL of medium into each well of a 6 well tissue culture plate and allowed to grow for 48 h. The cultures were then treated for 24, 48, and 72 h with either UO3 or UA. After the respective exposure time, the treatment medium was collected
(to include any loosely adherent mitotic cells);
the cells were rinsed twice with phosphate buffered saline (PBS); and then removed from the dish with 0.25% trypsin/1 mM EDTA. The trypsinized cells were added to the collected medium to stop the trypsin and centrifuged at 1000 rpm for 5 min. The resulting pellet was resuspended in 10 mL of medium, counted with Coulter Multisizer III, and reseeded at colony forming density
(1000 cells per 100 mm dish in 5 mL of media)
. The colonies were allowed to grow for 10 days, fixed with 100% methanol, stained with crystal violet, and the colonies counted. There were four dishes per treatment group, and each experiment was repeated at least three times.
Chromosome Preparation. Cells were prepared for chromosome analysis using published methods (20). Briefly, cells were seeded at 500,000 cells per 100-mm dish in 13 mL of media and allowed to grow for 48 h. The cultures were treated for 24, 48, and 72 h with UO3 or UA. One hour before the end of the treatment time, 0.1 g/mL colcemid was added to arrest the cells in metaphase. At the conclusion of treatment, medium was collected (to include any loosely adherent mitotic cells), the cells rinsed with phosphate buffered saline, and then removed from the dish with 0.25% trypsin/1 M EDTA. The trypsinized cells were added to the collected medium to stop the trypsin and centrifuged at 1000 rpm for 5 min. The supernatant was removed, and the pellet was resuspended in 10 mL of 0.075 M potassium chloride (KCl) hypotonic solution for 17 min to swell the cells and the nuclei. At the end of the hypotonic time, 1 mL of methanol/acetic acid fixative (3:1) was added and mixed with the hypotonic solution to condition the cells. The cells were centrifuged a second time for 5 min at 1000 rpm. Again, the supernatant was aspirated, the pellet was resuspended, and 10 mL of methanol/acetic acid fixative (3:1) was added. This cell suspension was kept at room temperature for 20 min, and then the fixative was changed twice. Finally, the cells were dropped on a clean, wet slide and uniformly stained using a 5% Giemsa stain in Gurr's buffer. Each experiment was repeated at least three times.
Chromosome Scoring Criteria. Clastogenesis was measured by the production of chromosomal aberrations, which were scored by standard criteria (22). Aberrations were pooled as described by Wise et al. (22). This is because deletions can only be unequivocally distinguished from achromatic lesions if the distal acentric fragment is displaced. Thus pooling aberrations avoids artificial discrepancies between scorers because of the different perceptions of the width of an achromatic lesion relative to the width of its chromatid. Accordingly, chromatid deletions and achromatic lesions were pooled as chromatid lesions, whereas isochromatid deletions and isochromatid achromatic lesions were pooled as isochromatid lesions. One hundred metaphases per data point were analyzed in each experiment.
Statistical Analysis. The 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. Interval estimates of differences are 95% confidence intervals, based also on Student's t distribution.
Uranyl acetate induced a time- and concentration-dependent cytotoxicity in WTHBF-6 cells after treatment (Figure 1). Uranium trioxide also induced a time- and concentration-dependent cytotoxicity in WTHBF-6 cells (Figure 2). UO3 did not fully dissolve in our tissue culture conditions. If complete dissolution had occurred, the concentrations for UO3 would be 2.1, 4.2, 21, and 42 g/mL, and for UA, they would be 42, 85, 170, and 339 g/mL.
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