Establishment of an Animal Model to

Evaluate the Biological Effects of

Intramuscularly Embedded

Depleted Uranium Fragments

Carl Andrew Castro

Kimberly A. Benson

Victor Bogo

Eric G. Daxon

John B. Hogan

Henry M. Jacocks

Michael R. Landauer

Sharon A. McBride

Christina W. Shehata

Armed Forces Radiobiology Research Institute Technical Report 96-3

Approved for public release; distribution unlimited.
Establishment of an Animal Model to

Evaluate the Biological Effects of

Intramuscularly Embedded

Depleted Uranium Fragments

Carl Andrew Castro

Kimberly A . Benson

Victor Bogo

Eric G . Daxon

John B . Hogan

Henry M . Jacocks

Michael R . Landauer

Sharon A . McBride

Christina W . Shehata

Armed Forces Radiobiology Research Institute

Technical Report 96-3

Approved for public release ; distribution unlimited .

Establishment of an Animal Model to

Evaluate the Biological Effects of

Intramuscularly Embedded

Depleted Uranium Fragments

Carl Andrew Castro

Kimberly A . Benson

Victor Bogo

Eric G . Daxon

John B . Hogan

Henry M . Jacocks

Michael R . Landauer

Sharon A. McBride

Christina W . Shehata

Approved for public release : distribution unlimited .

July 1996

Armed Forces Radiobiology Research InstituteTechnical Report 96-3

8901 Wisconsin Avenue

Bethesda, Maryland 20889-5603
This and other AFRRI publications are available to qualified users from the Defense

Technical Information Center . Attention : OCP, 8725 John J . Kingman Road . Suite 0944 .

Fort Belvoir. VA 22060-6218 : telephone (703) 767-8274 . Others may contact the

National Technical Information Service . 5285 Port Royal Road, Springfield, VA 22161 :

telephone (703) 487-4650 . AFRRI publications are also available from university

libraries and other libraries associated kith the U .S . Governments Depository Library

System .
Contents

Introduction	 1

Methods	 5

Subjects and Experimental Design . . . 5

DU and Ta Pellets	 5

Surgical Procedures for Pellet Implantation . . . 5

Behavioral Measurements	 6

Urinary Sampling and Collection Procedures 6

Determination of Urinary Uranium Levels .. . . 6

Results	 7

Surgical Implantation	 7

Locomotor Activity and Grip Strength . . . . 7

Body Weights, Food and Water Consumption .

and Urinary Output	 8

Urinary Uranium Levels	 9

Discussion	 I I

Acknowledgements	 1 1

References	 13

Introduction

Natural uranium (U) consists of three isotopes : 238 U

(99.276%),

235 U (0.718%), and

234U

(0 .0056%) . During the uranium enrichment process two isotopic

mixtures are produced, "enriched uranium" and "depleted uranium" (DU) with different relative ratios

of the three isotopes. Enriched uranium contains a

higher percentage of the fissionable isotope

235U and

is used for nuclear reactor fuel and nuclear weapons .

DU has a lower

235U content . The DU used by the

U.S . military for kinetic energy penetrators is alloyed with titanium (0 .75% by weight) to increase

its tensile strength and to retard oxidation . Current

U.S . antitank weapons contain DU penetrators, and

most of the Abrams tanks are armored with DU .

During Operation Desert Storm, DU munitions were

fired by the Army and Air Force . Unfortunately,

during this conflict, a number of U .S . military personnel were wounded by DU fragments (Daxon,

1993 ; Daxon and Musk, 1993 ; GAO Report, 1993) .

Many of these fragments were not removed because

the surgical procedure would produce excessive tissue damage . Radiographs of injured soldiers show

multiple embedded fragments ranging in size from

1 mm to over 5 mm in diameter (see figures 1 and

Fig.] . Radiograph of the leg of a soldier wounded by a DU munition during the Persian Gulf War. This soldier also had DU fragments

in the feet and knees of both legs .

1
Technical Report TR96-3

0 1

Fig. 2. (A) Photograph of an actual DU fragment removed from a soldier wounded during the Gulf War . (B) Photograph of a

Ta pellet implanted in a rat . (C) Photograph of a DU pellet implanted in a rat .

2a). Indeed, fragments as large as 20 mm in diameter

have been noted in other patients . Bioassays taken

over a year after injury indicate that uranium was

present at levels up to 30 μg U/l urine, well in excess

of natural background (U .S . Army Environmental

Hygiene Agency Memorandum for Office of the

Surgeon General, 1994) .

Although the toxicity of embedded DU is unknown,

numerous studies have addressed the consequences

of inhalation, ingestion, and parenteral administration of other forms of uranium (Diamond, 1989 ; La

Touche et al ., 1987 ; Morrow et al ., 1982; Ortega et

al ., 1989a, b ; Wrenn et al ., 1989) . After uranium is

absorbed, it circulates in the blood as the uranyl ion,

forming uranium-carbonate and uranium-albumin

complexes . As the uranium-carbonate complex

passes through the kidney, it is filtered rapidly by the

glomeruli where 60% to 80% of the absorbed uranium is excreted in the first 24 hours after acute

exposure. The uranium that is not excreted is reabsorbed by the proximal tubules where it produces

2

BC

2

significant toxic effects . Uranium also enters the

bone, where it competes with calcium to form complexes with phosphate ions, thus becoming part of

the bone matrix (Cabrini et al ., 1984 ; Domingo et al .,

1992; Guglielmotti et al ., 1989 ; Neuman, 1950) .

This bone matrix then serves as both a long- and

short-term storage site from which uranium is slowly

released back into circulation (Kathren et al ., 1989;

Wrenn et al ., 1985) . The liver and muscle are other

major sites of uranium deposition, with a possible

long-term storage mechanism in the kidney (Kathren

et al ., 1989; Wrenn et al ., 1985) .

Acute morphological and biochemical changes of

the kidney result from uranium exposure (Diamond,

1989 ; Kocher, 1989 ; Leggett, 1989 ; Neuman, 1950) .

Changes in the glomerular epithelial architecture

(Kobayashi et al ., 1984) and cellular necrosis in the

proximal tubules near the corticomedullary junction

of the kidney have been reported in experimental

animals after acute uranium exposure (Brady et al .,

1989 ; Haley et al ., 1982; Haley, 1982) . In addition,
polyuria, enzymuria, glucosuria, and increased excretion of amino acids have been demonstrated (Diamond, 1989 ; Diamond et al ., 1989 ; Kocher, 1989 ;

Zalups et al ., 1988) . Acute renal failure can indeed

occur following exposure to high doses of uranium

(Neuman, 1950 ; Ubios et al ., 1994) . Even acute

environmental stressors such as restricted diets or

changes in housing conditions have enhanced uranium toxicity significantly (Andrews and Bates,

1987; Damon et al ., 1986) .

Few studies have addressed the chronic toxicity of

uranium, and the results available are conflicting

(U.S . Department of Health and Human Services,

1990) . Galibin and colleagues (1971) reported severe renal toxicity in rats that inhaled ammonium

diuranate (1 or 8 mg/m 3 ), a slightly soluble uranium

compound, for 128 days . Urine protein and blood

non-protein nitrogen were elevated . In the proximal

tubules, there were sloughed dead cells and abnormal regenerating cells . Although the total number of

tubules was reduced and the kidney exhibited an

increased amount of connective tissue, all the animals recovered . In contrast, Leach and colleagues

(1970; 1973) found no renal toxicity in rats repeatedly exposed to uranium dioxide dust (5 mg/m 3) for

a period of 12 months nor in dogs or monkeys

exposed for 5 years . Yet uranium concentrations in

the kidneys were as high as 1 .1 g U/g kidney wet

weight in the rat, 8 .3 g U/g kidney weight in the

dog, and 17 .0 g U/g kidney weight in the monkey .

Uranium concentrations at these levels have been

reported to cause acute renal toxicity (e .g ., Kathren

et al ., 1989) . Thus, the chronic effects of uranium

exposure remain for the most part unresolved (Diamond, 1989) .

Introduction

The threshold concentration of kidney uranium levels in humans that result in kidney chemical toxicity

is in dispute (Diamond, 1989 ; Kathren and Moore,

1986; Kocher, 1989 ; Stradling et al ., 1988) . While

the Nuclear Regulatory Commission has set the level

at 3 .0 μg U/g kidney weight for renal damage in

humans, there is evidence from both human and

animal reports that this level could be considerably

lower. For example, chronically exposed uranium

mill workers, whose kidney uranium levels probably

did not exceed 1 μg U/g kidney weight (Thun et al .,

1985), showed mild renal dysfunction with increased urinary excretion of be ta2-microglobulin and

various amino acids . In rats exposed subchronically

to low doses (cumulative dose : 0 .66 or 1 .32 mg/kg)

of uranyl fluoride, kidney uranium levels as low as

0.7 to 1 .4 μg U/g wet weight kidney produced cellular and tubular necrosis of the proximal tubule, proteinuria, and enzymuria (Diamond et al ., 1989) .

These changes in rat renal function, however, were

temporary, with complete recovery occurring within

35 days of exposure . These studies are important

because they indicate that renal injury can occur at

kidney uranium levels well below the 3 .0 g U/g

limit .

Currently, no research into the direct toxic effects of

embedded DU has been reported . The toxicity data

that exist for low-level chronic uranium exposure

used other routes of administration, and the results

are contradictory . The uranium levels in humans that

result in kidney toxicity are in dispute . For these

various reasons, it is necessary to determine the

health risks to the soldier resulting from long-term

exposure to DU fragments . The goal of this pilot

study was to establish an animal model that could be

used in future research to investigate the biological

effects of embedded DU .

3

Methods

Subjects and Experimental Design

Subjects were 12 naive Sprague-Dawley male rats

(8-10 weeks old) obtained from Charles River

Breeding Laboratories, Raleigh, N .C . On arrival,

rats were quarantined and screened for diseases and

were maintained in an AAALAC-accredited facility

in accordance with the Guide for the Care and Use

of Laboratory Animals (NIH Publication No. 86-23) .

Six rats were implanted with eight DU pellets (four

in each biceps femoris muscle of the lateral thigh),

and six rats were implanted with eight tantalum (Ta)

pellets . Rats were individually housed in plastic

Micro-Isolator cages with hardwood chips as bedding; during urine collection, rats were placed in

metabolic cages . Commercial rodent chow and acidified water (pH 2 .5, using concentrated HCl) were

provided ad libitum . Rats were on a 12-hour light/

dark cycle .

DU and Ta Pellets

DU pellets (1 mm in diameter x 2 mm in length) were

obtained from Oak Ridge National Laboratories,

Oak Ridge, Tenn . (see figure 2c) . The cylindrical

shape was chosen because it is the geometrical average of fragments left in soldiers wounded by conventional or DU munitions . The size of the pellets was

based on two considerations . First, the total DU

implanted was approximately 1 % of the total biceps

femoris muscle volume and did not seem to cause

undue discomfort to the animal . Second, the surface

area of 8 DU pellets of this size should result in

detectable urinary uranium levels . DU pellets consisted of 99 .25% DU and 0 .75% titanium by weight .

The uranium isotopes in DU were

235U (99.75%),

235U (0 .25%), and trace amounts of

234 U . This is the

same DU alloy used in U .S . military munitions .

Ta pellets (1 mm in diameter x 2 mm in length) were

obtained from Alfa Products, Ward Hill, Mass ., and

served as the heavy metal control (see figure 2b) . Ta

was selected because its density is similar to DU

density, 16 .6 g/cm3 for Ta versus 18 .8 g/cm 3 for DU

(Radiological Health Handbook, 1970), it is relatively inert in a biological medium (Johansson et al .,

1990), and it is commonly used in human orthopedic

reconstructive surgery (Hockley et al ., 1990) .

Surgical Procedures for Pellet Implantation

Before implantation surgery, the DU and Ta pellets

were cleaned by immersion in an industrial detergent, rinsed in absolute alcohol, sterilized by immersion in a 50% nitric acid solution for 3 minutes,

rinsed with sterile water, and then placed in acetone

to inhibit oxidation . These sterilization procedures

completely remove the oxide formation from the

surface of DU metal (Tonry, 1993), and the results

of an abbreviated sterility test of 10 Ta pellets using

either a thioglycollate medium or soybean-casein

digest medium detected no microorganisms .

Rats were administered atropine (0 .05 mg/kg i .m.)

before being anesthetized . Anesthesia was induced

with ketamine hydrochloride (50 mg/kg) in combination with xylazine hydrochloride (10 mg/kg)

given i .p . in a 0 .5-ml bolus, using a 25-gauge needle .

These injections were administered intraperitoneally to prevent irritating the site of implantation . The

surgical sites were then shaved and cleansed with

Betadine. Four pellets were implanted approximately 15 mm apart in each biceps femoris muscle on the

lateral side of each thigh . Using a scalpel blade,

incisions were made through the skin and approximately 10 mm deep into the muscle mass . The proximal incisions were 10 mm distal to the iliac crest and

were the implantation sites of the first pellets . Pellets

were secured in place with absorbable sutures

(Dexon 4-0) to prevent movement . Rats were closely

monitored following surgery until they were ambulatory . A veterinarian or a veterinary technician examined the surgical sites for signs of inflammation,

infection, and local DU toxicity daily for 2 weeks

5
Technical Report TR96-3

following surgery and weekly thereafter throughout

the study .

Behavioral Measurements

Locomotor activity and grip strength were assessed

on days 3 and 5 before surgical implantation and on

days 1, 3, 7, 14, 28, 60, and 120 after surgery . Locomotor activity was quantified using computerized

Digiscan activity monitors (Omnitech Electronics,

Columbus, Ohio) . Each monitor used an array of

infrared photodetectors spaced 2 .5 cm apart to determine horizontal locomotor activity, which was expressed as total distance traveled . Activity was

monitored for 1 h with measurements taken every 5

min (Landauer et al ., 1988) .

Immediately following locomotor activity testing,

the strength of both hindlimb and forelimb grips of

each animal was measured using a grip strength

apparatus (San Diego Instruments, San Diego,

Calif.) . In this test, the animal was required to grip a

rectangular wire mesh surface (12 x 7 cm) with its

forepaws and was then gently pulled back along a

platform until its grip was broken . The backward

motion was continued until the animal's hindpaw

gripped another rectangular wire mesh surface (12 x

10 cm). As with the forelimb grip, the animal was

gently pulled back until the hindlimb grip was broken. Readings on three push-pull strain gauges were

used to record the maximum strain required to break

both forelimb and hindlimb grips . This behavioral

test is used in many laboratories to assess muscular

weakness (Haggerty, 1989 ; Meyer et al ., 1979) .

Urinary Sampling and Collection Procedures

Urine samples were collected following behavioral

testing on days 1, 3, 7, 14, 28, 60, and 120 after

surgery and analyzed for uranium levels . Sampling

at these time points was necessary because signs of

nephrotoxicity in laboratory animals exposed to low

doses of uranium are frequently not detected until 3

to 5 days after exposure and may subside within 7

6

days (Diamond, 1989) . Urine samples were collected from rats in individual metabolic cages (23 .5

cm diameter x 12 cm high) where they had continuous access to food and water . Rats were acclimated

to the metabolic cages for 5 days before the study

began because naive rats exposed to these housing

procedures have shown a stress-induced increase in

uranium toxicity (Damon et al ., 1986) .

A 24-h urine sample was obtained from each rat, and

the volume was recorded. In addition, each animal's

body weight and food and water consumption were

recorded . Care was taken to prevent contaminating

the urine with food or feces. After collection, urine

was filtered to remove any debris and stored in

plastic containers at 4° C until analyzed . The metabolic cages were disinfected and decontaminated

between each animal use . During animal-handling

periods, overt signs of behavioral toxicity and the

overall appearance of the rats were recorded .

Determination of Urinary Uranium Levels

Urinary uranium levels were determined by alpha

spectrometric techniques (Martin Marietta Energy

Systems, Inc ., Oak Ridge, Tenn .) . An aliquot of the

sample was dissolved in nitric acid (HNO3 ) and

hydrogen peroxide (H202). The sample was then wet

ashed, and the uranium coprecipitated with calcium

oxalate . After dissolving the precipitate in HCl, the

uranium was further separated by ion exchange chromatography . The uranium was then eluted from the

column with a solution of dilute HCl to which titanous chloride had been added to reduce actinides

that may have been in an elevated oxidation state .

The final fraction of the eluate was treated first with

ascorbic acid to reduce any iron and then with hydrofluoric acid . The uranium isotopes were next

coprecipitated on neodymium fluoride. The neodymium was caught on a 0 .1-μm filter, which was

rinsed, dried, and then mounted on a planchet for

alpha spectrometry . The minimum detectable activities (MDA) for uranium in urine using these procedures were 1 .4 x 10-6 μg/l for234Uand 0.03μg/l for 238U.
Results

Surgical Implantation

Two rats assigned to the DU group and one rat

assigned to the Ta group did not survive implantation surgery. One of these rats expired during

surgery, and the other two within 6 h after surgery .

Necropsies indicated asphyxiation, suggesting

that the animals received too much anesthetic . The

other nine animals were alert and moving in the

metabolic cages within 2 h after surgery . Figure 3

is a radiograph of the left rear leg of a rat implanted

with four DU pellets ; the right rear leg was also

implanted with four DU pellets . The cylindrical

shape and size of the pellets are similar to DU

fragments observed in wounded soldiers (figure

1) .

Locomotor Activity and Grip Strength

The locomotor activity of rats implanted with DU

pellets was not significantly different from the activity of rats implanted with Ta, p >0.05 (figure 4) .

200

-Ta (n = 5)

o DU (n = 4)

0	'	'	'	'	'	'	'	

//

	I	 '

-5-3 2 4 613 29 60 120

time postimplantation (days)

Fig . 4. Locomotor activity of rats surgically implanted with DU

pellets expressed as percent of Ta control . Vertical bars represent the SEM (standard error of the mean).

Fig. 3. Radiograph of the left rear leg of a rat surgically implanted with four DU pellets (1 mm in diameter x 2 mm in length) .

7
Technical Report TR96-3

1 .25

front grip strength - force (kg)

U 1 • .00

O

.o 0 .75

0>

C

m

N 0 .50

0

° 0.25

C

O

w

0.00

1 .25- body weight (g)

Rear grip strength - force (kg) •

1 •.00 U

0

r 0 .75

rn food consumption (g) 0 .50

water consumption (ml)-

6,0

•

.25

` 0 .00

-3 3 5 7 13 30 60 120

time (day)

Fig. 5. (A) Forelimb grip strength of rats before and after receiving DU or Ta implants . Vertical bars represent the SEM. (B)

Hindlimb grip strength of rats before and after receiving DU or

Ta implants . Vertical bars represent the SEM .

Similarly, neither the forelimb nor the hindlimb grip

strength of the two groups was different, p >0 .05

(figures 5a and 5b) .

Body Weights, Food and Water

Consumption, and Urinary Output

The body weights of the rats embedded with DU

pellets were not different than the body weights of

rats embedded with Ta, p >0.05 (figure 6) . In fact,

700

01

07ID

3

00

.0

A

B

DU

O Ta

IF

600

500

400

300 (-

200

100 -

•Ta

V DU

01'

030 60 90 120

time (day)

Fig. 6. Body weights of rats before and after receiving DU or

Ta implants. Vertical bars represent the SEM .

8

O

E

C

Oa

3

50

40

0)

C

0

30

E

n

CC

o 20

0

10

of	1	1	1

030 60 90 120

time (day)

Fig. 7. Food consumption of rats before and aMer receiving DU

or Ta implants . Vertical bars represent the SEM .

the body weights in both groups remained relatively

stable for the first week following surgery and, as

expected, increased throughout the study as observed in normal rats .

The food and water consumption for the DU- and

Ta-implanted rats did not differ, p >0 .05 (figures 7

and 8) . There was, however, a trend toward a decrease in water consumption for the Ta group and an

increase in water consumption for the DU group .

There was a significant difference in the volume of

urinary output between the DU and Ta groups . On

the day of surgery, urine output for the Ta group

decreased but did not change for the DU group, p

70

60

50

40

E

0 30

U

m 20

10

Fig. 8 . Water consumption of rats before and after receiving DU

or Ta implants. Vertical bars represent the SEM .
40

35

30

urinary output (ml)

25

a

•

• 20

L,

c 15

10

5

0	I	1	1	I	I 0

30 60 90 120

time (day)

Fig. 9. Urinary output of rats before and after receiving DU or

Ta implants. Vertical bars represent the SEM .

<0.05 (figure 9) . This decrease in the urinary output

for the Ta group, however, was temporary and returned to baseline levels by day 3 after surgery .

Urinary Uranium Levels

Figure 10 illustrates mean uranium levels in the urine

of DU-implanted animals and the pooled value of the

uranium analysis for Ta-implanted animals after implantation surgery . Figure 11 provides the individual

urinary uranium levels of the four DU-implanted

rats . As expected, only background levels of uranium were detected in the Ta control group . In contrast, significant levels of uranium were detected within 24 h of DU implantation (mean = 28 .69 ± 10 .00,

300 urinary uranium levels (μg U/l)

time postsurgery (day)

Fig . 10. Time course of uranium levels detected in the urine of

rats implanted with either DU or Ta . Uranium concentration

detected in the Ta group is at background levels. Vertical bars for

the DU group (N = 4) represent the SEM . Urine for the Ta-implanted animals was pooled for uranium analyses.

range = 14 .21 to 56 .99 μg U/l). By day 7 following

surgery, uranium levels had increased nearly fourfold (mean =111 .86 ±41 .05, range = 56 .38 to 233 .91

μg U/l) and remained elevated at day 120 (mean =

204.56 ± 99 .73, range = 35 .01 to 458 .53 g U/1) .

D

500

o- 400

a

>

•

300

•200

E

m

•

100

•

0

•

- too

•Ta

o DU

Results

040 80 120

time postsurgery (day)

Fig. IL Individual time courses of uranium levels detected in

the urine of each rat implanted with DU . Data on Ta time courses

are the same as in figure 10.

9

Discussion

The purpose of this study was to develop an animal

model that could be used in future research to determine the health risks associated with DU fragment

injuries . It was especially important to establish procedures in which DU exposure would produce urinary uranium levels comparable to those observed

in soldiers wounded by DU munitions during the

Persian Gulf War . Measured by these criteria, this

initial study was successful . The average urinary

uranium level in the rat 24 h after DU implantation

was 28.69 μg U/l. This value is very close to the

urinary level of 30 g U/l reported for soldiers

wounded during the Persian Gulf War and assayed

1 year after injury . Unfortunately, no bioassays were

taken of any of the soldiers within the first year after

DU injury so no direct time course comparisons can

be made .

It should be emphasized that the urinary uranium

levels in the rat did not reach asymptote until day 7

following DU implantation surgery and remained

elevated throughout the study (figure 10) . Although

the data are preliminary, this finding has clinical

significance because it indicates that soldiers with

suspected DU fragment wounds should be monitored for uranium exposure for at least the first week

after injury and perhaps even longer . Certainly a

complete pharmacokinetic study should be conducted to definitively address this patient-monitoring

issue (Daxon, 1993) .

Although numerous studies have assessed the toxic

effects of other forms of uranium exposure (Diamond, 1989, and Kocher, 1989, for the latest reviews

of the literature), this is the first study that assessed

the effects of intramuscularly embedded DU . The rat

proved to be an excellent animal model for this

purpose. It tolerated the surgical procedures for pellet implantation relatively well, as measured by both

locomotor activity and grip strength (figures 4 and

5), both indices of quality of life for humans . Further,

the lateral thigh muscle of the adult rat is large

enough to implant at least four pellets (1 .0 mm

diameter x 2 mm length) into each leg (figure 3), with

the possibility of as many as ten pellets . Moreover,

the rat's lifespan of more than 18 months enables it

to be used in chronic toxicity studies (Brady et al .,

1989 ; Lang and White, 1994 ; Lumley et al ., 1992 ;

Lumley and Walker, 1986; Monro, 1993 ; Nohynek

et al ., 1993 ; Rao et al ., 1990) .

In conclusion, this study was successful in developing a rodent model that can be used to evaluate the

biological effects of intramuscularly embedded DU

fragments . However, the potential short-term and

long-term health risks associated with DU exposure

remain to be investigated . Certainly the behavioral,

physiological, biochemical, and histological consequences of embedded DU are research areas of immediate concern . Equally important is identification

of the health risks to the fetus exposed in utero to

DU from fragments embedded in the mother before

pregnancy (Angleton et al ., 1988 ; Bosque et al.,

1993 ; Domingo et al ., 1988a, b, c ; Paternain et al .,

1989). This latter research area is especially significant considering that the placenta does not prevent

cross-placental transfer of uranium (Durbin and

Wrenn, 1976; Sikov and Mahlum, 1968) . Moreover,

fetal toxicity often occurs in the absence of maternal

toxicity (e .g ., Price et al ., 1985) . Regardless of the

research strategy adopted, a coordinated interdisciplinary health hazard assessment is required to identify the potential medical risks that DU poses to our

soldiers wounded by this unconventional munition .

Acknowledgements

We thank Ms . Elizabeth L . Wampler for radiation safety advice, Major Rebecca A . Cockman-Thomas for performing surgical

implantations, Dr . G . David Ledney and Dr . Thomas B . Elliott for conducting and interpreting pellet sterility tests, Mr . William

E. Jackson III for statistical advice, and Ms . Modeste E . Greenville and Ms . Carolyn Wooden for publication assistance .

1 1
I
References

Andrews PM, Bates SB (1987) Effects of dietary

protein on uranyl-nitrate-induced acute renal

failure . Nephron 45 :296-301

Angleton GM, Benjamin SA, Lee AC (1988) Health

effects of low-level irradiation during development: Experimental design and prenatal and

early neonatal mortality in beagles exposed to

60 Co gamma rays . Radiation Research 115 :70-83

Bosque MA, Domingo JL, Llobet JM, Corbella J

(1993) Embryotoxicity and teratogenicity of uranium in mice following subcutaneous administration of uranyl acetate . Biological Trace Element Research 36 :109-118

Brady HR, Kone BC, Brenner RM, Gullans SR

(1989) Early effects of uranyl nitrate on respiration and K+ transport . Kidney International 36:

27-34

Cabrini RL, Guglielmotti MB, Ubios AM (1984)

Prevention of the toxic effect of uranium on bone

formation by tetracycline . Acta Odontologica

Latinoamericana 1 :61-63

Damon EG, Eidson AF, Hobbs CH, Hanh FF (1986)

Effects of acclimation to caging on nephric response of rats to uranium. Laboratory Animal

Science 36 :24-27

Daxon EG (1993) Protocol for monitoring Gulf War

veterans with imbedded fragments of depleted

uranium. AFRRI Technical Report TR93-2,

Armed Forces Radiobiology Research Institute,

Bethesda, MD

Daxon EG, Musk JH (1993) Assessment of the risks

from imbedded fragments of depleted uranium .

AFRRI Technical Report TR93-1, Armed Forces

Radiobiology Research Institute, Bethesda, MD

Diamond GL (1989) Biological consequences of

exposure to soluble forms of natural uranium .

Radiation Protection Dosimetry 26 :23-33

Diamond GL, Morrow PE, Partner BJ, Gelein RM,

Baggs RB (1989) Reversible uranyl fluoride

nephrotoxicity in the Long-Evans rat . Fundamental and Applied Toxicology 13 :65-78

Domingo, JL, Ortega A, Llobet JM, Paternain JL,

Corbella J (1989a) The effects of repeated parenteral administration of chelating agents on the

distribution and excretion of uranium . Research

Communications in Chemical Pathology and

Pharmacology 64 :161-164

Domingo JL, Ortega A, Paternain JL, Jacinto C

(1989b) Evaluation of the perinatal and postnatal

effects of uranium in mice upon oral administration . Archives of Environmental Health 44 :395-

398

Domingo JL, Paternain JL, Llobet JM, Corbella J .

(1989c) The developmental toxicity of uranium

in mice . Toxicology 55 :143-152

Domingo JL, Colomina MT, Llobet JM, Jones MM,

Singh PK (1992) The action of chelating agents

in experimental uranium intoxication in mice :

Variations with structure and time of administrations . Fundamental and Applied Toxicology 19 :

350-357

Durbin PW, Wrenn ME (1976) Metabolism and

effects of uranium in animals . In : Conference on

Occupational Health Experience with Uranium .

U .S . Energy Research and Development Administration, WA 470 C75c, Washington, D.C ., 68-

99

Galibin GP, Vlasov PA, Fedoseyeva LA (1971) Remote aftereffects of killing rats using ammonium

diurinate . In : Otdalennye Posledstviya

1 3
Technical Report TR96-3

Luchevykh Porazhenii, Moskalev UI (ed), pp

197-206, Atomizdat, Moscow ; English translation, AEC-TR-7387

GAO Report (1993) Army not adequately prepared

to deal with depleted uranium contamination .

GAO/NISAID-93-90

Guglielmotti MB, Ubios AM, Larumbe J, Cabrini

RL (1989) Tetracycline in uranyl nitrate intoxication: Its action on renal damage and U retention

in bone. Health Physics 57 :403-405

Haggerty GC (1989) Development of tier I neurobehavioral testing capabilities for incorporation

into pivotal rodent safety assessment studies .

Journal of the American College of Toxicology

8 :53-69

Haley DP (1982) Morphologic changes in uranyl

nitrate-induced acute renal failure in saline- and

water-drinking rats . Laboratory Investigation 46 :

196-207

Haley DP, Bulger RE, Dobyan DC (1982) The longterm effects of uranyl nitrate on the structure and

function of the rat kidney . Virchows Archives

[Cell Pathology] 41(1-2) :181-192

Hockley AD, Goldin JH, Wake MJC, Igbal J (1990)

Skull repair in children . Pediatric Neurosurgery

16 :271-275

Johansson CB, Hansson HA, Albrektsson T (1990)

Qualitative interfacial study between bone and

tantalum, niobium or commercially pure titanium. Biomaterials 11 :277-280

Kathren RL, Mclnroy JF, Moore RH, Dietert SE

(1989) Uranium in the tissues of an occupationally exposed individual . Health Physics 57 :17-21

Kathren RL, Moore RH (1986) Acute accidental

inhalation of U : A 38-year follow-up. Health

Physics 51 :609-619

1 4

Kobayashi S, Nagase M, Honda N, Hishida A (1984)

Glomerular alterations in uranyl acetate-induced

acute renal failure in rabbits . Kidney International 26 : 808-815

Kocher DC (1989) Relationship between kidney

burden and radiation dose from chronic ingestion

of U : Implications for radiation standards for the

public . Health Physics 57 :9-15

Landauer MR, Davis HD, Dominitz JA, Weiss JF

(1988) Long-term effects of radioprotector WR2721 on locomotor activity and body weight of

mice following exposure to ionizing radiation .

Toxicology 49 :315-323

Lang PL, White WJ (1994) Growth, development,

and survival of the Crl :CD(SD)BR stock and

CDF(F344)/CrIBR strain . Pathobiology of the

Aging Rat 2 :587-608

La Touche YD, Willis DL, Dawydiak 01 (1987)

Absorption and biokinetics of U in rats following

oral administration of uranyl nitrate solution .

Health Physics 53 :147-162

Leach LJ, Maynard EA, Hodge HC, Scott JK, Yuile

CL, Sylvester GE, Wilson HB (1970) A five year

inhalation study with uranium dioxide (U02 )

dust . I . Retention and biologic effect in the monkey, dog, and rat . Health Physics 18 :599-612

Leach LJ, Yuile CL, Hodge HC (1973) A five year

inhalation study with uranium dioxide (U0 2 )

dust . II . Postexposure retention and biologic effect in the monkey, dog, and rat . Health Physics

25 :239-258

Leggett RW (1989) The behavior and chemical toxicity of U in the kidney : A reassessment. Health

Physics 57 :365-383

Lumley CE, Parkinson C, Walker SR (1992) An

international appraisal of the minimum duration

of chronic animal toxicity studies. Human and

Experimental Toxicology 11 :155-162
Lumley CE, Walker SR (1986) A critical appraisal

of the duration of chronic animal toxicity studies .

Regulatory Toxicology and Pharmacology 6 :66-

72

U.S . Army Environmental Hygiene Agency, Aberdeen Proving Ground, MD, Memorandum for

Office of the Surgeon General, PSP, Subject :

Results of analyzing urine bioassay specimens

for uranium (Interim Report), 20 April 1994

Meyer OA, Tilson HA, Byrd WC, Riley MT (1979)

A method for the routine assessment of forelimb

and hindlimb grip strength of rats and mice .

Neurobehavioral Toxicology 1 :233-236

Monro A (1993) How useful are chronic (life-span)

toxicology studies in rodents in identifying pharmaceuticals that pose a carcinogenic risk to humans? Adverse Drug Reactions and Toxicology

Reviews 12(1):5-34

Morrow P, Gelein R, Beiter H, Scott J, Picano J,

Yuile C (1982) Inhalation and intravenous studies of UF6/UO2F in dogs . Health Physics 43 :

859-873

Neuman WF (1950) Urinary uranium as a measure

of exposure hazard . Industrial Medicine and Surgery 19:185-191

Nohynek GJ, Longeart L, Geffray B, Provost JP,

Lodola A (1993) Fat, frail and dying young:

Survival, body weight and pathology of the Charles River Sprague-Dawley-derived rat prior to

and since the introduction of the VAFR variant in

1988 . Human Experimental Toxicology 12 :87-

98

Ortega A, Domingo JL, Gomez M, Corbella J

(1989a) Treatment of experimental acute uranium poisoning by chelating agents . Pharmacology and Toxicology 64 :247-251

Ortega A, Domingo JL, Llobet JM, Thomas JM,

Paternain JL (1989b) Evaluation of the oral toxReferences

icity of uranium in a 4-week drinking study in

rats . Bulletin of Environmental Contamination

and Toxicology 42 :935-941

Paternain JL, Domingo JL, Ortega A, Llobet JM

(1989) The effects of uranium on reproduction,

gestation, and postnatal survival in mice. Ecotoxicology and Environmental Safety 17 :291-

296

Price CJ, Kimmel CA, Tyl RW, Marr MC (1985)

The developmental toxicity of ethylene glycol in

rats and mice . Toxicology and Applied Pharmacology 81 :113-127

Radiological Health Handbook, U .S . Department of

Health, Education, and Welfare, Public Health

Service, Ed ., Bureau of Radiological Health and

Training, Institute of Environmental Control Administration, p . 65, 1970

Rao GN, Haseman JK, Grumbein S, Crawford DD,

Eustis SL (1990) Growth, body weight, survival,

and tumor trends in F344/N rats during an elevenyear period . Toxicologic Pathology 18 :61-70

Sikov MR, Mahlum DD (1968) Cross-placental

transfer of selected actinides in the rat . Health

Physics 14 :205-208

Stradling GN, Stather JW, Gray SA, Moody JC,

Hodgson A, Cooke N (1988) The metabolism of

ceramic and non-ceramic forms of uranium dioxide after deposition in the rat lung . Human Toxicology 7(2) :133-139

Thun MJ, Baker DB, Steenland K, Smith AB,

Halperin W, Berl T (1985) Renal toxicity of

uranium mill workers. Scandinavian Journal of

Work, Environment and Health 11 :83-90

Tonry LL (1993) Solubility of depleted uranium

fragments within simulated lung fluid . Unpublished Dissertation, Boston University

1 5
Technical Report TR96-3

Ubios AM, Braun EM, Cabrini RL (1994) Lethality

due to uranium poisoning is prevented by ethane1-hydroxy-1,1-biphosphonate (EHBP) . Health

Physics 66:540-544

U.S . Department of Health and Human Services

(1990) Toxicological profile for uranium, TP-90-

29. U .S . Government Printing Office, Washington, DC

Wrenn ME, Durbin PW, Howard B, Lipszten J,

Rundo J, Still ET, Willis DL (1985) Metabolism

of ingested U and Ra . Health Physics 48 :601-633

1 6

Wrenn ME, Lipszten J, Bertelli L (1989) Pharmacokinetic models relevant to toxicity and metabolism for uranium in humans and animals . Radiation Protection Dosimetry 26 :243-248

Zalups RK, Gelein RM, Morrow PE, Diamond GL

(1988) Nephrotoxicity of uranyl fluoride in uninephrectomized and sham-operated rats . Toxicology and Applied Pharmacology 94 :11-22
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July 1996

3 . REPORT TYPE AND DATES COVERED

Technical Report

4 . TITLE AND SUBTITLE

Establishment of an Animal Model to Evaluate the

Biological Effects of Intramuscularly Embedded Depleted

Uranium Fragments

5 . FUNDING NUMBERS

PE : NWED QAXM

6 . AUTHOR(S)

Castro CA, Benson KA, Bogo V, Daxon EG, Hogan JB,

Jacocks HM, Landauer MR, McBride SA, Shehata CW

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Armed Forces Radiobiology Research Institute

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8901 Wisconsin Avenue

Bethesda, MD 20889-5603

TR96-3

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Uniformed Services University of the Health Sciences

4301 Jones Bridge Road

Bethesda, MD 20814-4799

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13 . ABSTRACT (Maximum 200 words)

During the Persian Gulf War, 36 U .S . soldiers were wounded by depleted uranium (DU) munitions . Based on

medical guidelines for conventional shrapnel injuries (nonradioactive), many DU fragments were left in soldiers .

Unfortunately, health risks associated with embedded DU were unknown, and an animal model to investigate

this did not exist . The purpose of this study was to develop an animal model to examine the health risks associated

with DU shrapnel injuries . Twelve rats were surgically implanted intramuscularly with 8 DU pellets (1 mm in

diameter x 2 mm in length) or 8 chemically inert tantalum (Ta) pellets of similar size . Urinary uranium levels

were measured on days 1, 3, 7, 14, 28, 60, and 120 after implantation of DU pellets . Physiological and behavioral

parameters, including locomotor activity, forelimb and hindlimb grip strength, food and water consumption, and

urinary output, were measured 5 and 3 days before surgery and on days 1, 3, 7, 14, 28, 60, and 120 after surgery .

Urinary uranium levels for Ta-implanted rats remained at background levels . In contrast, the average urinary

uranium level for DU-implanted rats was significantly elevated on day 1 (28 .69 μg U/l) after implantation and

remained elevated until day 120 (204 .56 μg U/l). There was no significant difference between DU- and

Ta-implanted rats in any behavioral or physiological measures . Results indicate that the rat is an appropriate

animal model for evaluating biological effects of embedded DU fragments .

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