[ RadSafe ] DU Proven Deadly To Human Bronchial Cells

Brennan, Mike (DOH) Mike.Brennan at DOH.WA.GOV
Thu May 10 12:55:13 CDT 2007

Thanks, Pete.  That makes the experiment more reasonable, but still it
has fatal flaws that make it meaningless for concluding anything about
DU exposure in the field, which from the title is clearly what was

-----Original Message-----
From: Peter Fear [mailto:FEARP at upstate.edu] 
Sent: Thursday, May 10, 2007 10:16 AM
To: Brennan, Mike (DOH); radsafe at radlab.nl
Subject: RE: [ RadSafe ] DU Proven Deadly To Human Bronchial Cells

The amounts are micrograms and not grams.


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

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

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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