[ RadSafe ] DU Proven Deadly To Human Bronchial Cells

[Bob Cherry]

I do not have the time nor the expertise to assess this paper, so this is is
merely my observation from the information in the note below.

--------
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.
-----------

Those are pretty high concentrations. (Did a "micro" symbol get lost? Even
so, a microgram per milliliter in bronchial tissue is still pretty high for
any foreign substance, I would guess.) Virtually anything at those
concentrations are "deadly to human bronchial cells" and probably any other
cells. I doubt that concentration is anywhere close to the concentrations in
our war veterans, even the friendly fire victims.
	
Bob C

-----Original Message-----
From: radsafe-bounces at radlab.nl [mailto:radsafe-bounces at radlab.nl] On Behalf
Of Roger Helbig
Sent: Thursday, May 10, 2007 4:38 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