[ RadSafe ] DU Proven Deadly To Human Bronchial Cells

[John R Johnson]

Thanks Bob

Now that the information is "out there", I'm going for lunch-)!

John
***************
John R Johnson, PhD
CEO, IDIAS, Inc.
Vancouver, B. C.
Canada
(604) 222-9840
idias at interchange.ubc.ca

----- Original Message ----- 
From: "Bob Cherry" <bobcherry at satx.rr.com>
To: <radsafe at radlab.nl>
Sent: Thursday, May 10, 2007 11:18 AM
Subject: RE: [ RadSafe ] DU Proven Deadly To Human Bronchial Cells


> Physicists love to do "back of the envelope (order of magnitude)
> calculations":
>
>>From the ATSDR: "People eat about 1-2 micrograms (0.6-1.0 picocuries) of
> natural uranium every day with their food and take in about 1.5 micrograms
> (0.8 picocuries) of natural uranium for every liter of water they drink, 
> but
> they breathe in much lower amounts."
>
> Reference man has a mass of 70 kg.
>
> The article indicates concentrations on the order of 10 ppm.
>
> Daily intake related to body mass looks to be about 0.01 ppm, three to 
> four
> orders of magnitude less than the concentration in the study.
>
> I don't have the information handy but I seem to remember that reference 
> man
> always has about 0.1 microgram of uranium in his body for a concentration 
> of
> about 0.001 ppm = 1 ppb.
>
> In any event the study apparently does not represent realistic
> concentrations, even for someone hit by friendly fire.
>
> Lunch time is over. Back to work.
>
> Bob C
>
> -----Original Message-----
> From: radsafe-bounces at radlab.nl [mailto:radsafe-bounces at radlab.nl] On 
> Behalf
> Of Peter Fear
> Sent: Thursday, May 10, 2007 12:16 PM
> To: Mike.Brennan at DOH.WA.GOV; radsafe at radlab.nl
> Subject: RE: [ RadSafe ] DU Proven Deadly To Human Bronchial Cells
>
> The amounts are micrograms and not grams.
>
> Pete
>
>
> Peter Fear
> Health Physics Technologist
> SUNY Upstate Medical University
> Radiation Safety Office
> 636 UH
> 750 E. Adams St.
> Syracuse, NY 13210
>
> Phone: (315)464-6510
> FAX:     (315)464-5095
> fearp at upstate.edu
>
>
>
>>>> "Brennan, Mike  (DOH)" <Mike.Brennan at DOH.WA.GOV> 05/10/07 12:50 PM >>>
> It is a shame they went through all of this work without doing anything
> that supports or refutes the hypothesis that DU is a human health hazard
> of particular note, as the experiment was not done with DU, but rather
> with natural uranium, or U-Nat, which has a much higher specific
> activity.  Beyond that, the absurdly high concentrations (unless, as Bob
> suggests, a prefix was left out) make the results suspect.  5g/cm of
> salt would be hard on cells, too.
>
> That being said, I don't think there is anyone who thinks that breathing
> DU is good for you.  But consider:  The most likely people to be exposed
> to high concentrations of breathable DU particles are those breathing
> smoke from a fire containing (and started by) a DU projectile.  In turn,
> the people most likely to be breathing that smoke is the crew of an
> armored vehicle that has just been hit by said projectile.  Given that
> at that moment it is the Foreign Policy of the United States of America
> to try to kill those people (otherwise, we shouldn't be shooting at
> them), their long term health risks are not high on the priority list.
> Given their immediate environment includes (1) a burning vehicle loaded
> with fuel, plastics, ammunition, and various explosives, (2) people who
> are not only shooting at them, but hitting them, and are likely to do it
> again, and (3) a level of overall stress that just can't be good for
> you, DU is not even near the top of their health risks.  They should
> live so long that DU is their number one concern.
>
> I would welcome some well designed studies on DU.  To date, however,
> every article I've seen about DU has had at least one flaw so serious as
> to invalidate the conclusions.  This is one more.
>
> -----Original Message-----
> From: radsafe-bounces at radlab.nl [mailto:radsafe-bounces at radlab.nl] On
> Behalf Of Roger Helbig
> Sent: Thursday, May 10, 2007 2:38 AM
> To: radsafelist
> Subject: [ RadSafe ] DU Proven Deadly To Human Bronchial Cells
>
> 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.
>
> Roger Helbig
>
> ----- 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
>
>
>
> Abstract:
>
> 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.
>
>
>
>
> ------------------------------------------------------------------------
> --------
>
>
> Introduction
> 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.
>
>
>
> Results
> 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.
>
>
> More-
> http://pubs.acs.org/cgi-bin/sample.cgi/crtoec/asap/html/tx700026r.html
>
>
>
>
>
>
> ------------------------------------------------------------------------
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