Long-term accumulation and microdistribution of uranium in thebone and marrow of beagle dog

J. D. T. ARRUDA-NETO{{*, M. V. MANSO GUEVARA{§, G. P. NOGUEIRA},

I. D. TARICANO{, M. SAIKI||, C. B. ZAMBONI||, L. V. BONAMIN{,

S. P. CAMARGO{, A. C. CESTARI{, A. DEPPMAN{, F. GARCIA{{, A. N. GOUVEIA{**,

F. GUZMAN§, O. A. M. HELENE{, S. A. C. JORGE{{{, V. P. LIKHACHEV{,

M. N. MARTINS{, J. MESA{, O. RODRIGUEZ§ and V. R. VANIN{

(Received 9 December 2002; accepted 27 April 2004)

Abstract.The accumulation and microdistribution of uranium in the bone and marrow of Beagle dogs were determined by both neutron activationand neutron-fission analysis. The experiment started immediately after the weaning period, lasting till maturity. Two animal groups werefed daily with uranyl nitrate at concentrations of 20 and 100 mg g21 food. Of the two measuring techniques, uranium accumulated alongthe marrow as much as in the bone, contrary to the results obtained with single, acute doses. The role played by this finding for theevaluation of radiobiological long-term risks is discussed. It was demonstrated, by means of a biokinetical approach, that the long-termaccumulation of uranium in bone and marrow could be described by a piling up of single dose daily incorporation.

1. Introduction

The toxicity of uranium, (U), as well as itsradiobiological effects, has been under study for atleast 50 years, including life-span studies in animals.Data on uptake through ingestion, gastrointestinalabsorption, biokinetics and chemical toxicity areabundant. However, the great majority of thesestudies were conducted in small animals followingsingle administration of acute dosages of uranylnitrate solutions (Sullivan 1980, Tandon et al. 1998,Ubios et al. 1998). In fact, experiments so far reportedhave dealt with alpha-emitting radionuclides—U,plutonium (Pu) and americium (Am)—administeredto adult animals, where micro-distributions in thefemoral shaft have been studied (Austin et al. 1998,1999, 2000, Austin and Lord 2000).

The U issue was also addressed in ICRP

(International Committee on Radiation Protection)Publication 69 (1995). It was argued that the uranylion apparently exchanges with Cazz at the surfaceof bone mineral crystals, the distribution of U amongdifferent bones and bone parts is similar to that ofcalcium, and U becomes diffusely distributed in bonevolume within several days after intravenous injection.

As shown by Austin et al. (1999, figure 2), asubstantial portion of U deposited in the bonemarrow (following a single dose) is lost to plasma,except for a persistent and small residue. This findingled us to conjecture how much U residue would bedeposited in the marrow, after a prolonged dailyingestion of U, starting from the early stages of theanimal skeleton development?

However, there is no information in the literatureabout the distribution of radionuclides in bonemarrow following chronic ingestion and spanningcrucial life periods starting after weaning and lastingtill maturity.

Other reasons for carrying out the present workare as follows:

(1) Daily intake of U through food and water maybe regarded as chronic ingestion, and some-times occurs in high concentrations, asdiscussed by Arruda-Neto et al. (1997).

(2) Additional U had entered some regions as aconsequence of military use of depleteduranium (DU) weapons by NATO (NorthAtlantic Treatise Organization) and othermilitary organizations since the 1990s.

International Journal of Radiation Biology ISSN 0955-3002 print/ISSN 1362-3095 online # 2004 Taylor & Francis Ltd

http://www.tandf.co.uk/journals

DOI: 10.1080/09553000410001723884

{Physics Institute, University of Sao Paulo, PO Box 66318,Sao Paulo, SP 05315-970, Brazil.{Laboratory of Toxicological Analysis/UNITOX, University

of Santo Amaro/UNISA, Sao Paulo, SP, Brazil.§High Institute of Nuclear Sciences and Technology,

Havana, Cuba.}Faculty of Veterinary Medicine/UNESP, Aracatuba, SP,

Brazil.||Institute for Energetic and Nuclear Research/IPEN-CNEN,

Sao Paulo, SP, Brazil.**Biological Sciences Institute, University of Sao Paulo, SP,

Brazil.{{Laboratory of Viral Inmunology, Butanta Institute, Sao

Paulo, SP, Brazil.{{Santa Cruz State University, Ilheus, Bahia, Brazil.

*Author for correspondence; e-mail: arruda@usp.br

INT. J. RADIAT. BIOL., AUGUST, 2004, VOL. 80, NO. 8, 567–575

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(3) Young children could receive greater exposureto DU when playing in or near DU impactsites. Typical hand-to-mouth activity couldlead to high DU ingestion from contaminatedsoil (WHO—World Health Organization2001).

(4) It is very likely that young animals were oftenin positive U balance (between uptake andexcretion) due to a build up of U in the growingskeleton.

Therefore, we decided to measure the distributionof U in the bones of beagle dogs, following aprolonged ingestion of U present in the food, startingafter weaning and lasting up to the young animal’spost-puberty period.

Also, a biokinetic approach was worked out andused for estimates of time-dependent U accumulationin bone and bone marrow.

2. Materials and methods

Seven 3-months old male beagle dogs from theUniversity of Santo Amaro kennel were fed daily withU-doped feed. The U content in the food corre-sponded to 100 mg (three dogs) and 20 mg (one dog) ofU g21 of food ingested daily. Three dogs received noU (the control group). To allow for a better control ofthe intake, U in the form of uranyl nitrate wasadministered as U-doped cookies made from thesame food, each containing 12 or 60 mg U.Considering the average amount of food ingesteddaily by each animal, the U contents of the cookiescorresponded, approximately, to 20 and 100 ppm U,respectively. Note that for the adult animal, 100 ppmU day21 is equivalent to an ingested activity of about15 Bq kg21 body weight, which is much less thandoses used in typical single injection experiments. TheU-doped cookies were always administered early inthe morning following an overnight fast.

Some procedures and tasks were carried outduring the experiment, such as the frequent evalua-tion of clinical conditions and laboratorial examina-tion. Thus, urine and faeces were collected andanalysed to check, for example, for occult blood, pHand protein concentration, which could have theirnormal levels altered by the high toxicity of U.

To obtain some information on U biokinetics inthe bones, the first toes (ergot) of the two foreleg pawswere removed by surgical procedures after 82 and180 days, respectively. Note that these toes (phalanxof digit I) are functionless and their removal did notcompromise the health of the animal.

The animals were sacrificed and necropsed after279 days. Femora and vertebra were removed,

cleaned with in 40% hydrogen peroxide and thendehydrated under infrared light. Bone samples wereanalysed by means of two techniques: neutron-induced fission (intended for U microdistributiondetermination) and neutron activation analysis(intended for quantification of U concentrations).

This experiment with beagle dogs was conductedin accordance with accepted ethical practice, and itwas approved by the Ethics Council of the Universityof Santo Amaro (where the experiments were carriedout). The animals received adequate care withappropriate veterinary supervision.

2.1. Uranium microdistribution

Slabs of femur samples belonging to 1-year-oldbeagle dogs, about 1.5 mm thick each, were obtainedfrom transversal cuttings of this bone at the epiphysisand mid-shaft (figure 1). The femur slabs weresandwiched between two foils of Makrofol E(Bayer), and each set was irradiated for 5 min bythermal neutrons with a flux of 1.261013 n cm22 s21,near the core of the Instituto de PesquisasEnergeticas e Nucleares (IPEN) reactor (IEA-R1,5 MW, pool type) (figure 1c).

The U located at and/or near the surface of thefemur slabs undergoes neutron-induced fission, andthe corresponding fission fragments hit one of theMakrofol foils producing a microscopic fission track.Therefore, the spatial distribution and the density offission tracks in the Makrofol foils correspond to the

(a) (b) (c)

Figure 1. (a) Schematic representation of a femur showingwhere the transversal cuttings were performed. Thesebone slabs taken near the femur head correspond to theepiphyses region. (b) Typical representation of theepiphysis x–y plane, referred to in the text as the surfaceof the femur slab sample or simply as the femurtransversal section. The results shown in figures 3 and 4represent microdistributions of uranium measured in thesex–y planes. (c) Irradiation/detection geometry. The femurslab is sandwiched between two Makrofol foils (M) parallelto the x–y plane, the set being irradiated with thermalneutrons (n).

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distribution and density of U at the surface of thefemur slabs, respectively.

To enlarge the size of these fission tracks, theMakrofol foils were etched by 1 M solutions of KOHat 65‡C for 7 min, and then the tracks were countedwith the use of a projection optical microscope allalong the whole foil surface, using scanning fields of0.1860.18 mm2. In this sense, the number of fissiontracks inside a scanning field is proportional to the Ucontent at this position in the surface of the femur

slab, encompassing an area of 0.1860.18 mm2. Theposition of each scanning field is determined by a pair(x, y) of coordinates (figure 1b).

According to our scanning coordinate convention,a scanning row corresponds to the sequence of all thescanning fields located at (x, y0), where y0 is fixed(figure 2).

Thus, the results for all scanning fields arepresented as three-dimensional figures, where theplane x–y corresponds to the surface of the femur slab(figure 1b), and in the z-axis we have the number offission tracks counted in each scanning field,expressed as ng U mm22 (for more details, seebelow) (figures 3 and 4).

2.2. Uranium concentration in bones

Bone samples (femora, vertebra and toes) werecalcinated during 2 h at 900‡C, then ground andhomogenized. Approximately 100 mg bone ashes(from each animal) were weighed and sealed inpolyethylene involucres. Standard aliquots of the Usolutions, with their exactly known concentrations,were pipetted onto 1 cm2 pieces of Whatman no. 40filter paper, and dried in a dessicator.

Bone ashes and U standards were irradiatedtogether inside a cadmium capsule in the centralcore of the IPEN reactor, at a flux of261013 n cm22 s21 for 8 h. The irradiated materialswere analysed by means of the three gamma-lines(106, 228 and 278 keV) of 239Np (U daughter),

Figure 2. Three typical scanning rows of femur transversalsections. The shaded elliptic areas indicate the location ofthe so-called ‘hot spot’, near the endosteal region; alsoobserved in mice (Austin et al. 1998, 1999). (a, b) Resultsfrom sections taken near the epiphysis; (c) section wastaken at the mid-shaft.

Figure 3. Microdistribution of uranium in a femur transversal section (see figure 1b) of beagles belonging to the 100-ppm group. Thecounts were integrated in scanning rows of 0.1860.18 mm2.

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formed in the reaction:

238U n, cð Þ239U ?

b{

23min

239Np ?b{

2:35d

239Pu:

These lines were detected and analysed with theuse of a high resolution 75 cm3 Ge detector(FWHMv1.87 keV for the 60Co 1.33 MeV gammaline) mounted inside a lead shield hood. The detectorwas operated with a 671 ORTEC amplifier in pile-uprejection mode.

2.3. Uranium concentration in the marrow

To obtain a direct, correction-free absolutemeasurement of the U content in the marrow, wealso performed neutron activation analysis (exactly asdescribed above) of the total marrow content fromfemora.

Thus, several femora were cracked longitudinally,that is, along the z-axis (figure 1a) without the use ofcutting instruments (only gloved hands), in order tomake sure that bone contamination would benegligible. The marrow was then removed from thebone cavity with the help of a spatula, carefully,without rubbing the endosteo. The material wasdried, homogenized and irradiated with neutronsinside a cadmium capsule.

3. Results

Figures 3 and 4 show the microdistribution of U(U topography) in femur transversal sections ofBeagles (figure 1) belonging to the 100- and 20-ppmgroups, respectively. Those were bone samples from1-year-old animals sacrificed after of 279 days. Theabsolute scale (ng U mm22) was set by normalizing

the relative scale, expressed as number of fissiontracks per mm2, with the absolute results obtainedfrom the neutron activation measurements in boneashes (see the Materials and methods). From such acomparison we determined that each fission trackrecorded in the Makrofol foils corresponded toapproximately 1022 ng U in the surface of thefemur slabs. The background associated with ourmeasurements of the U content in bones wasestimated, and found negligible, from bone samplesof animals belonging to the control group.

Typical scanning-rows are shown in figure 2. Thelength of the rows corresponds to linear dimensions ofthe femur transversal sections (figure 1b), encompass-ing from 50 to 60 scanning fields along the x-axisdirections. Since each field is 0.18 mm wide, theequivalent diameters of the femur sections range from9 to 11 mm, where the bone matrix is about 1.5 mmwide. The results in figure 2c were obtained from afemur transversal section located 2.5 cm below theepiphysis and are presented as histograms encom-passing four scanning fields each.

The number of fission tracks measured in somescanning areas was considerably higher than theaverage taken from all the scanning fields. Themarked eliptic areas in figure 2 indicate the locationof these so-called ‘hot spots’, almost all were near theendosteal region (they were also observed in mice;Austin et al. 1998, 1999). We selected as hot spots allthe scanning fields with the number of fission tracksexceeding the average by three times its standarddeviation (3s). We observed typically no more thanfive to six of such hot spots per femur slab surface.

Figure 5 shows the concentration of U in beaglesbones as a function of the elapsed time in the

Figure 4. Same as in figure 3 for the 20-ppm group.

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experiment; thus, t~0 corresponds to a 3-month-olddog. The data points corresponding to t~82 and180 days were obtained from the dogs toes (ergots),while for t~279 days we measured the U content ofnecropsed femora. All the results obtained with theanimals from the control group are compatible with aU concentration equal to zero (our detection limitcorresponds to a few ng g21). The results for the threedogs fed with 100 ppm U agree within uncertainties;therefore, we decided to take an average of the three datasets. Final results are shown in figure 5, where the errorbars represent the external standard deviations.

Finally, figure 6 shows all the results of volumetric,absolute scale measurements obtained by neutronactivation analysis.

4. Discussion

4.1. Role played by contamination, cancellous bone andtrabeculae

The possibility for substantial contamination of themarrow by bone fragments, occurring at the cuttingprocess of femur transversal sections, is excluded onthe grounds of the following evidence:

(1) Because of the careful cutting of the femur

sections, followed by several rounds of the

washing/cleaning processes, the remaining

bone fragments glued in the marrow surface

are randomly distributed over it. As a result,

they would manifest themselves as spikes of U

concentration, randomly distributed over anunderlying continuum of U belonging to themarrow.

(2) Microdistribution of U (e.g. figure 3) fluctuatesaround its average by amounts smaller than 2s(where s is the standard deviation of theaverage). Those few points exceeding theaverage by 3s (or more) were classified as‘hot spots’ (see above), and they are alllocalized near the endosteo.

(3) In another series of measurements, dedicatedto an intercomparison of several kinds of bones(e.g. vertebrae, cortical femur, femur head andphalanx), we undertook neutron activationmeasurements separately in bone and marrowsamples. Contamination is presumably negli-gible, since it was found that the total,volumetric contents of U in both marrowand femoral cortex are very similar (figure 6).

Since there is some cancellous bone in the regionwhere samples were taken (proximal femur; figure 1),

Figure 5. Uranium concentration in the bones of beagles as afunction of time (data points) determined for the 20 (+)and 100-ppm (e) groups. The shaded bands wereobtained from the biokinetic approach (details are inthe text); also shown in the insert are calculations for C(t),the concentration of uranium, up to t~3000 days. Theerror bars represent the external standard deviation.

Figure 6. Uranium concentration in several kinds of beaglebones determined for (a) 100 and (b) 20-ppm groups,respectively. The lettering in the horizontal axis is asfollows: V, vertebrae; M, marrow; FH, femur head; FC,femoral cortex; P, phalanx. Vertical arrows indicate thatthe marrow and femoral cortex formerly constituted thesame femur sample. The error bars represent the externalstandard deviations.

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we considered the possibility that the concentrationsof U measured in the marrow could just be assignedto cancellous bone. Note, in this regard, that if U isuniformly distributed in the bone volume (a reason-able assumption), then the U density (DU) measured(figure 3) is proportional to the bone mass density(Db), that is, DU3Db. By labeling the U densities atthe cortex and marrow regions, respectively as DU(1)and DU(2), we observe from our results thatDU(2)§DU(1), implying that Db(2)§Db(1), whichsimply could not be true because the marrow densityis smaller than the bone density. Additionally, withDb(2)§Db(1), there would be no room in the bonecavity to house even a very small amount of marrow.

Note in figure 6, for the two groups of animals(20 and 100 ppm U doping), that the total amount ofU in marrow is about 20% higher than in the femoralcortex (see histograms M and FC). This is consistentwith the fact that about 20% of the bone masscorresponds to trabecular bone (Berne and Levy1998). Actually, all pieces of information obtained inthis work are consistent with each other. For instance,the U content in the femur head (histogram FHin figure 6) is about 40% higher than in the cortexand for both animal groups. This is also consistentwith both the higher calcification of the femurhead, and the chemical affinity between uranyl ionsand calcium.

4.2. Uranium landscape in the femur

We are reporting, for the first time, results on long-term accumulation of U in bone and bone marrow ofbeagle dogs following chronic ingestion. All studies sofar reported deal with single-injection of radionuclidesand their biokinetics as a function of the post-injection (PI) time. The results displayed in figures 2–4and 6 are quite revealing: U is accumulated in thewhole marrow content as much as in the bone, whichis at variance with acute single-dose experiments. Inthis regard, Austin and collaborators (Austin et al.1999, 2000, Austin and Lord 2000) have conductedquite detailed microdistribution studies of radio-nuclides (239Pu, 241Am and 233U) in the mousefemoral shaft for PI times ranging from 1 to 448 days.They found a relatively smaller concentration ofradionuclides in marrow compared with bone. Also,macrophages containing radioactive deposits wereobserved migrating from the bone surface toward thecentral marrow, accumulating with PI time at thecentral venous sinus. 233U, although not accumulatedin macrophages, showed a small (4%) but diffusedeposition throughout the bone marrow at all PItimes suggesting this process is weakly time dependent(Austin et al. 1999, figure 2).

As discussed below, the long-term accumulation ofU in bone and marrow could be described as a timeintegrated, accumulation of single-dose retentionprocesses. In this sense, our results for the marrow(figures 2–4 and 6) are not that surprising.

The present findings regarding U microdistribu-tions are consistent with volumetric contents of U,measured by neutron activation analysis separately inmarrow and femoral cortex, and in other bones(figure 6). It is important to stress that we performedtwo kinds of independent measurements, employ-ing two distinct techniques: neutron-induced fissiontrack analysis (for microdistributions) and neutronactivation analysis for volumetric determination(figure 6). From the results of U content in thewhole marrow, it is quite obvious that microdistribu-tions obtained from femur transversal sectionstaken at different positions, as for example, in themid-shaft, should exhibit the same mean results.Figure 2c shows a result obtained away from theregion of trabecular bone. We cannot estimate howmuch trabecular bone is interfering in eachmicrodistribution, but from the volumetric results(figure 6) there is 20% more U in the marrow than inthe femoral cortex. If we make the unrealisticassumption that the trabeculae account for 40% ofthe total bone mass, the U mass in the marrowcorresponds to 80% of the U deposited in the cortex(which still is a lot).

4.3. Biokinetics of long-termed accumulation processes

Any kind of long-term accumulation process couldalways be interpreted by means of the well-knownproblem of ‘decay plus input’, provided that in thedecay phase of the process (clearance), after a singledose intake, a measurable residue persists for a long time.This is particularly true for bone (Stevens et al. 1980)and marrow (e.g. Austin et al. 1999, figure 2; see alsothe discussion below).

The concentration of U in bone or marrow as afunction of PI time following a single-dose, C(t), variesat a rate:

dC tð Þdt

~{lC tð Þ, ð1Þ

where l21 is the average clearance time. The solutionof equation (1) is the skeletal or marrow retentionequation:

C tð Þ~C0e{lt , ð2Þwith C0~fD0, where D0 is the single dose (injected,ingested or inhaled), f is the fraction of D0

accumulated at t~0 (days), and l is the clearanceconstant. Thus, l21 is the average clearance time.

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Stevens et al. (1980) found that l21<1800 daysfor hexavalent 233U in beagle femora, followingintravenous injection of about 100 kBq 233U kg21,corresponding to about 0.25 ppm 233U (valent 6).Note that the alpha activity in 233U is four orders ofmagnitude higher than in 238U.

Assuming that in addition to the removal of U(clearance) at a rate 2lC(t), there is a ‘leakage’ ofU into bone or marrow at a constant rate b (chronic,daily intake), then, the net rate is given by:

dC tð Þdt

~b{lC tð Þ, ð3Þ

whose solution is:

C tð Þ~ b

l1{e{lt� �

: ð4Þ

Note that

limt??

C tð Þ~b=l:Cm, ð5Þ

that is, for long times, the concentration converges toCm. In our case, b is the amount of U effectivelytransferred to bone or marrow every day(mg U day21). We could measure C(t) only for bone(figure 5), and for the marrow the information we gotis for the saturating concentration Cm (see below).

Our main approximation is to consider b equal toa constant, while we know that it could vary with thedevelopment of the animal. However, substantialvariation of b should occur mostly during the earlierstages of the life of the animal.

The curves shown in the inset to figure 5 wereobtained from equation (4) by using l21~1795.3 days, as obtained by Stevens et al. (1980).No attempt was made to fit the data; it was a directcalculation. We used the point at t~82 days in orderto get b. Thus we got b equal to 0.016 and0.029 mg U g21 day21 for the 20 and 100-ppm groups,respectively. This procedure generated an uncertaintyof about 5–7% in the calculations, represented by theuncertainty bands shown in figure 5. The agreementwith the experimental data is very good. Because ofthe long mean clearance time (l21 about 1800 days),the U accumulation C(t) is quite linear up to t about500 days. Saturation is predicted by our model asoccurring for tw3000 days (figure 5 insert).

Assuming that such a slow clearance of U alsoprevails in the marrow and that the residual amountof U in the marrow is similar to the amount of 233U,after a single dose (Austin et al. 1999, figure 2), it isquite evident that the marrow would accumulate asubstantial amount of U after a long-term dailyingestion process.

Another interesting aspect of the accumulation ofU in bone marrow is the higher efficiency of this

process at lower doses (also verified for other targetorgans; Arruda-Neto et al. 2001).

4.4. Dose distribution and hot spots

From the results presented in figures 3 and 4, weobtained the corresponding isodose areas (figure 7) bycalculating the dose due to one single a-particleemitted by U, delivered to the biological masscontained in a cylinder encompassing the ionizationtrack of this particle in the bone (more details are inArruda-Neto et al. 1997, Stevens et al. 1980).

Note from figure 7 that average doses deliveredto the epiphysis are in the ranges 2–4 and1–3 Gy mm22 year21 for the 100 and 20-ppm groups,respectively. The situation is worst at and/or near thehot spots.

It is important to stress that the hot spots weobserved are spikes of U sparsely distributed alongthe endosteo, which is distinct from the well-knownobservations of enhanced bone-seeking radionuclideson bone surfaces of humans (Salmon et al. 1994) andanimals (Salmon et al. 1995), following chronicexposure. Actually, Salmon et al., (1994, 1995)attributed these observations to a hypermineralizedlayer at endosteal surfaces. If true, our hot spotswould be an indication that such a hypermineralizedlayer has a few non-homogeneous harder points,where U would accumulate more.

Our results indicate that the doses delivered to thebone stem cells (in the bone marrow), and particularlynear the hot spots (see above) are intense enough toinduce neoplasia in individuals submitted to chronicingestion of U, over a prolonged (w3–5 years) time.This is made clear by the following points:

(1) For the high linear energy transfer (LET)alpha-radiation from U, the energy isabsorbed within a microscopic cylindricalvolume defined by the ionization track of thealpha-particle.

(2) Mean dose imparted to a cell nucleus traversedby a single-alpha-particle is 1 Gy, while the meandose imparted to the biological material insidethe alpha-particle ionization cylinder (diameterabout 20 nm and height about 30–40 mm) is76104 Gy (Mays et al. 1987). Thus, alpha-particle microdoses could be devastating ifimparted to radiosensitive cells.

5. Discussion of a real-life occurrence:depleted uranium in the environment

Earlier results suggested that the beagle may be anappropriate experimental animal from which one can

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extrapolate data to humans with reference to thepercentage of U, Th, and Pu found in their organs(Singh and Wrenn 1989). To the degree this is so, atleast qualitatively, members of the public submitted tochronic ingestion of U would accumulate U in theirmarrow as much as in the bones, regardless of the intakeamount, provided three conditions are fulfilled:(1) chronic, daily intake; (2) starting at the childhood,and (3) persisting till adult age. A likely scenario for allthese circumstances could be the areas affected by DU.

The main difficulties in estimating the overallDU radiobiological impact are related to: (1) howmuch DU is being ingested and/or inhaled, and (2)how the age dependency of the doses (see the ICRP1995 for a review).

Biokinetic models to estimate age-dependent dosesconsider the bone as the main target and, therefore, theyall lead to the conclusion that doses to the primitive stemcells (central marrow) are much lower than in proximityto the bone surfaces, but with the three conditions of ourstudy (see above) this is not true.

However, we do not know for sure how thedistribution of U in the marrow evolves with age. Inthis regard, the presence of fat in the bone marrow inthe form of fat cells plays an important role, becauseU dissolved in fat has an enhanced transit time in themarrow leading to an enhanced dose—details of thisprocess were published elsewhere (Richardson et al.

1991, Allen et al. 1995). On the other hand, these fatcells are not present in the foetus or in young animals,but the gastrointestinal tract of newborn animals isorders of magnitude more permeable to a number ofradionuclides, including U, than is that of the adults(Sullivan and Gorham 1982), which could enhancethe uranium input rate b (see equation 5).

Additionally, since we do not know how much DUis being incorporated from the environment by thelocal populations, radiobiological risks cannot beevaluated with the aid of our isodose areas (figure 7).Notwithstanding, we emphasize the point that evensmall quantities of ‘chronically’ ingested and/orinhaled U, depleted or not, is a matter of concernsince our findings show that this radionuclideaccumulates in the bone marrow.

Our findings are not applicable to the DU scenariofor operations involving an acute single-dose expo-sure, e.g. military personnel involved in the battle-field. More information and details on depleted U areavailable in two comprehensive reports of The RoyalSociety (2001, 2002).

6. Final remarks and conclusions

Our results address cum grano salis a few relevantquestions and provide important conclusions regard-ing long-term U incorporation:

(a) (b)

Figure 7. (a) Isodose areas obtained from the microdistribution of uranium relative to the 100-ppm group (figure 3), expressed as Graymm22 of the epiphysis x–y surface (figure 1b), but integrated over 1 year, that is, Gy6mm22 y21. (b) The same as in (a) for the20-ppm group.

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(1) U is accumulated through prolonged ingestionand distributed both in bone and bonemarrow. Saturation is reached only afterabout 10 years.

(2) Doses are imparted to the whole bone marrowvolume and, therefore, the primitive haema-topoietic stem cells, concentrated in the centralmarrow (Lord 1990), are subject to radioactiveburdens as intense as in the bone. This is aqualitative result since we do not know, asdiscussed above, how the incorporated U isredistributed in the marrow volume as afunction of time (particularly if the ingestionstarted in the childhood). For more informa-tion on cell radiosensitivity as well as radi-ological risks, see Hall (1994).

(3) From our biokinetic approach, we observe thatafter a chronic ingestion period of about 5years, for each ppm ingested (1 mg U g21 food),the accumulation in bone and bone marrow isequal to 1 mg U g21 bone (in terms of U mass).

Acknowledgement

Work was partially supported by FAPESP andCNPq, Brazilian agencies, and the Latin-AmericanPhysics Center/CLAF.

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