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Radiographic visualization of the deposition of radioberyllium in the rat.

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RADIOGRAPHIC VIStJALIZATIOK O F THE DEYOSITION O F RADIOBERYLLIUM IN THE RL4T1
C. T. KAPLOR AND C. D. VAN C L E A V E
Department of Anatomy, University of iVortk Carolina, Chapel Hill
TWENTY-NINE FIGURES
The experiments described in this paper were part of a
study aimed a t ascertaining in the rat the areas of concentration of tracer amounts of radioberyllium within organs
known to be principally involved in beryllium poisoning. It
has been reported (Scott, '48; Aldridge et al., '49) that acute
beryllium poisoning in laboratory animals was accompanied
by a inidzonal necrosis of hepatic cells, necrosis of the distal
parts of the convoluted tubules of the kidney, and changes in
the hematopoietic system leading to anemia and leucocytosis.
I t has, accordingly, been predicted (Crowley et al., '49) that
these areas would contain the greatest concentrations of beryllium?.
Both radiographic and wet-ash assay methods of determining distribution of Be7 have been used; the latter confirm
grossly the deposition in organs concerned, extend the observations to organs in which the activity was too slight to be
detected photographically, and follow the process of elimination of the isotope. The methods and results of the wet-ash
assays will appear in a separate paper (Van Cleave and
Kaylor, '53).
A new method of radioautography was necessary for photographic detection of radioactive beryllium, inasmuch as thc
usual autographic methods are based on the emission of alpha
or beta particles. Be7 is a pure gamma emitter. Gamma
'Work performed under eontrnct No. AT-(40-1)-266
Energy Commission.
467
f o r the U. S . Atomic
468
C. T. RAYLOR A X D C . 1). V A N CLEAVE
radiography, although used in industrial metals testing (Harriiigton, Johns, Wiles and Garrett, '48), has not apparently
been applied to biological material. Gamma rays could bc
expected to be inefficient as compared to electrons or alpha
particles in producing darkening of a photographic eniulsion,
and the resolution of the resulting image might he expected
to be poor. ITseful autographs were, ncverthcless, obtained.
MATI.;RTAT>S -4ND MHTIIODS
The Be7 was obtained from the Oak Ridge National Laboratory. The injected solution was carrier-free, isotonic, and in
most instances a t a pH of 4.5-5.0. Decay curves were obtained
on all material received from Oak Ridge. These curves corresponded to the 53-day period expected. Three compounds of
beryllium were employed : sulfate, chloride, and citrate, i.e.,
the Be7 was injected as a weak solution of the respective acid.
All animals employed were female albino rats, weighing
approximately 200 grams a t time of injection, and were obttiiiied f r o m Carworth Farms.
In order to obtain radioautographs from aiiinials injected
with Re7, it was necessary to administer the substance in
millicurie levels of activity ; the individual doses ranged from
1.5 t o 11.2mc. All injections except the largest were given
via the internal jugular vein, in most instances as a single
injection. The largest dose was administered intraperitoneally
over a period of oiie week. On the basis of the formula for
internal irradiation dosage, given by Maririelli, Quimby and
Hirie ('4S), the maximum gamma, hard and soft x-ray irradiation from a single intravenous dose of 10 mc of Be7/mt
was computed to be approximately 240r.e.p. per rat a t the
eiid of the first day. This figure takes into account the excretion rate and decay curve of the injected beryllium. The
LD50/30 days for u7hole body x-irradiation in the rat has
been stated to be 600r (lle Bruyn, '48). Nothing suggesting
i*adiatioiidamage in any organ 01' tissue has heen seen in our
aiiiirials.
DEPOSITION O F R A D I O B E R P L L I U M
469
In terms of actual Re molecules present in the animal a t
any time, the dosages of I)eryllium employed in these experirnents were far below the T,D50 figure of 0.5mg Be/kg body
weight reported by Aldridge e t al. ('49), our largest intravenous dose being only 0.0030/0 of the reported TlD50.
12oclio~rutograplaictcchniqucs
The procedure requires the use of a thin, intensifyind0 niptallic screen between the histologic section on a glass slide
arid the sheet of photographic emulsion, the whole maintained
in firm contact during the exposure. The screen consisted of
gold or platinum foil 0.5 mil (12.5 p ) in thickness. Lead foil
in our experience produced pseudophotographic artifacts. The
purpose of the thin intensifying screen of metals of high Z
number was to increase the number of secondary electrons
which a r e produced hy the gamma rays ejected upwards from
the tissue section. F o r the lead screens used in industrial
gamma radiography, IIarrington et al. ( '48) found that nearly
52% of the blacliening of the film was due to electrons ejected
from the screen in front of the film and nearly 48% from
electrons ejected from a similar screen placed in back of the
film. The blackening due to absorption of ganima rays by the
film itself was small. I n the present work, a back screen gave
images of such poor resolution that this screen was not used.
The film used routinely was Ansco Process film which has a
fine-grained, single coated, slow emulsion with a low background density.
To prevent leaching and displacement of beryllium, the
organs and tissues to be autographed were fixed in 80% alcohol and imbedded in celloidin or paraffin. Sections 5 p thick,
and roughly 1em2 in area, were attached to slides in the usual
manner. The celloidin or paraffin was removed, the slides
dipped in 0.5% celloidin and then dried. The activity of a
single 5 p section was determined by the usual counting procedures immediately after slides were dipped in thin celloidin
and dried. It was found that a suitable autograph could hc
obtained in 4 days' exposure time from a section with appvosi-
470
C. T. I i AYL OR A N D C. D. V A N CLEAVE
mately 125 counts per minute. Sections with 8-2Oc.p.m. required about 6 w e e k s ’ exposure to obtain a n autograph.
Counts Tvere made with a bismuth-wall gamma counter having
a counting efficiency of approsimately 1%. During the e s posure period, the slide-foil-film unit was kept in a d r y refrigerator at 5°C.
The distal two-thirds of the femur was selected in a11
instances for representative bone sections. The femur w a s
sectioned longitudinnlly in the undecalcified state at 5 p by
the method of Axelrod (’47).
In order to compare areas of blackening in the autograph
with the corresponding histological section, the latter w a s
eiilargcd about 5 diameters, and a tracing was made of the
magnified image. The autograph, carefully oriented to COF
respond with landmarks or the outline of the histological section, mas then projected on the tracing and the lnlsckened
areas added to the tracing. Further magnification of the
autograph resulted in loss of definition.
R 128UL‘l’S
Autographs of sufficient contrast t o be reproduced photographically were obtained from 9 animals. Five had received
beryllium as the sulfate, three a s the chloride, and one as
the citrate. Many autographs that did not permit reproduction were useful with the naked eye.
The highest concentrations of radioactive beryllium sulfate and chloride, after inti*avenous injection, were in liver,
spleen, slieleton, and kidney, in that order. These were the
only organs from which autographs could be obtained following sulfate or chloride administration ; other organs contained
Ixrylliurri i n amounts too small for autographic purposes.
Radiographs of liver and spleen were relatively easily obtained from sections ttilicn within the first three days after
injection of the sulfate 01’ chloride. Bone autographs were
best in the sulfate group n.hen sections were obtained a t
approximately 8 days, and with chloride a t 8 to 30 days, after
injection. Kidney autographs were difficult to obtain.
DEPOSITION O F RAUIORERYLLIUM
47 1
Beryllium citrate behaved strikingly different in its concentration in the animal body. As Schubert and White ('50)
showed, skeletal deposition was mainly involved ; liver, spleen,
and kidney were variable and erratic sites of concentration.
Only one animal in our group offered reproducible autographs
following the citrate injection, and these autographs were
from femur.
Liver. A characteristic pattern of deposition of Be7 was
shown in all radioautographs of the liver (figs. 1, 3, 5,6, 7, 8).
It appeared within two hours after injection (fig. 1) and was
still present 49 days later (fig. 6). By tracing the autograph
onto sections (figs. 2 and 4 ) , it was found that most of the
heavily darkened areas corresponded with the periphery of
hepatic lobules. The central vein of the lobule was usually
easily located even at low magnification. Where several complete lobules adjoined, it was especially plain that the area
of activity corresponded to the periphery, and, as a concentrated blackening, did not progress inward toward the central
vein. Nothing in 01- around the portal canals was correlated
with the film blackening. Collecting veins of the afferent and
efferent systems and the bile capillaries showed no blackening.
However, it was apparent that a homogeneous darkening pel*vaded the entire area of the section and can be supposed to
represent a distribution of beryllium. It seems unlikely that
this uniform darkening represented a fogging of the emulsion
from the scattered intense areas of activity, because in spleen
autographs small areas of white pulp remained clear in the
midst of intense surrounding activity. From the evidence offered by the autographs alone, it would be useless to attempt
to localize the cellular elements responsible f o r concentrating
beryllium within the periphery of the liver lobule. Judging
from the variation in intensity of blackening, however, it is
tempting to suppose that the concentrations represent discrete entities.
Comparison of figures 3, 6, and 8 shows that the chemical
form and the amount in which Be7 was administered had no
effect on the pattern of deposition in the liver.
472
C. T. I<AYLO€i A N D C. I). V A S CLEAVIS
S'pZeo~z. There was a coiistant pattern of deposition in the
spleen (figs. 9, 11, 12, 13). Activity was sharply confined to
the red pulp areas, a s secn in figure 10 where a typical autograph is projected upon its stained section. This pattern
was unaltered by time, conccntratioii of beryllium, or the
compound injected. The pattern of beryllium concentration
resembles, in most respects, that shown by Murray ('48) for
plutonium and b a i - i ~ m ~ The
~ " . spleen radiographs ( l I u r r a y ,
'45) of phosphorous:i2,or stontiurn"', or yttriumQ1and radium,
are, however, quite the reverse picture in that deposition of
these compounds was confined to white pulp, mostly around
the arteriole.
Kidney. Kidney sections with enough activity to give an
autograph were rarely obtained. The only reproducible autograph is shown in figure 14, obtained after Be7C1, injection.
The pale central arca of the autograph can be identified in
the histological section a s medulla and the inner edge of
darkening corresponds to the cortico-medullary junction. The
cortical area, then, contained all the darkening. The fairly
uniform darkening in the cortical area does not suggest
glomerular concentration of activity a s is indicated after injection of plutonium (Bloom, '48). The inner zone of cortical
darltening corresponded in position with both proximal and
distal convoluted tubules, and neither can be identified from
a n autograph.
Boize. A selection of autographs of uiidecalcified femur
sections iiidicatirig the pattern of distribution of beryllium
in grou7ing bone is shown in figures 15 through 29.
Beryllium given as the citrate over a period of one week
accumulated mainly in the newly formed spongiosa adjacent
to the epiphyseal plate (figs. 15 and 16). It was clear that
epiphyseal plate did not contain any activity (fig. 16). Instead, a more diffuse distribution is indicated in the entire
epiphysis with extensions into the metaphysis. Faint lines
of activity marked the cut edges of thc compact bone in the
shaft and may indicate periosteal and endostcal deposition.
LeBlond et al. ('SO), working with radiophosphorous in the
DEPOSITION O F RADIOBERYLLIUM
473
rat, obtained autographs that a t first glance are not unlike
this one, but in their material the greatest concentration of
activity was in the epiphyseal plate material. There was
nothing to suggest incorporation of beryllium into preexisting
bone, a condition similar to what Comar, Lotz, and Eoyd ('5'2)
have shown f o r Ca45in the pig. None of the blackening corresponded with hone marrow in the histologic section,
One day after intravenous injection of Ke7S0, (figs. 1 7
and IS), beryllium had begun to deposit in the osteoid tissue
in discrete loci, in addition to accumulating diffusely throughout the distal end of the hone. A few intensely darkened spots
indicate deposition in the metaphysis and one spot in the
marrow of the shaft. These sharply defined spots of intense
activity in both primary and secondary spongiosa and presumably in the marrow cavity were frequent occurrences ill
bone autographs. We were unable to correlate them with
histologically recognizable structures in the stained sections.
Eight days after administration of Be7S0, (figs. 19-22),
the autographs indicated that the process of deposition in
spongiosa, begun during the first 24 hours, had continued.
The marrow cavity was free of activity.
Later stages have been followed in autographs of animals
sacrificed a t 28 and 62 days after sulfate injection which were
too faint to photograph. I n the older animal, deposition in
the primary spongiosa was decreased, although localized spots
of activity in the older spongy bone and adjacent marrow were
now more prominent. There was no clear evidence of beryllium
retention in compact bone.
Two days after Re7Cl, injection, autographs of femur (figs.
27-29) showed heavy deposition of beryllium in a narrow
zone of osteoid proximal to the epiphyseal plate. I n autographs of the femur of a 49-day animal (figs. 23-26), the
major activity appeared in discrete spots in the spongy bone
of both epiphysis and metaphysis. Some of the metaphyseal
spots may well have been in the marrow cavity. Compact
bone remained free of activity.
474
C. T. K A P L O R AN11 C. 1). V A N CLEAVE
I>ISCUSS1O N
It is believed that the radioautographs shown here represent a definite histologic extension of the niicrocheniical and
pathologic methods used by previous workers in tracing the
localization of the extremely toxic beryllium niolecule in the
living body. These autographs represent, furthermore, the
picture of deposition of beryllium in essentially normal animals since extremely minute quantities of Re7 were employed.
The resolution obtained with the Be7 autographs is decidedly
less than has been obtained from radioisotopes which emit
electrons, and leaves something to be desired.
The autographic patterns obtained from liver and spleen
were constant and the autographs themselves could be obtained with ease when niillicurie amounts of Be7 were employed. The indicatioiis from the liver pictures a s to the
areas of concentration of Be in the liver may possibly he a t
variance with the pathological findings of Scott ('48) and
Aldridge ('49) when toxic doses of Be were used. However,
both of these workers were using doses of beryllium far in
excess of any used in the present experiments. This may account for such discrepancies. Furthermore, pathologically
speaking, the concentration of beryllium at the periphery of
the lobule, as we invariably found, does not necessarily preclude the possibility of a mid-zonal necrosis which the other
workers found.
The spleen autographs confirm Aldridge 's description of
the complete lack of hei-yllium concentration in the white
pulp in any of the animals he used.
The almost complete failure to obtain autographs from
the kidney sections of these animals was surprising in view
of the assay data ( V a n Cleave and Icaylor, ' 5 3 ) on cxcrction of Re7, particularly with the citrate, where a s much
as 65-75cJo of the initial intravenous dose was excreted in
24 hours. With the passage of such large amounts of radioactivity through the kidney, one would not have predicted
such a dearth of autographs, especially if the kidney sections
were obtained in the first 24-hour period after the animals
DEPOSITION O F RAUlOBERYLLIUM
475
were injected. The one autograph illustrated showed a concentration of Be7 principally in renal cortex. This might in
a general way support Scott's ('48) findings that histological
changes in the kidney following toxic amounts of Be were
not nearly as extensive as in liver and spleen, and in general
such changes as were seen were confined to parts of the convoluted tubules.
The bone autographs, showing beryllium deposit in osteoid
tissue rather than in the mineral structure, have fairly well
substantiated the predictions of Crowley, Hamilton and Scott
('49) in their work with wet-assay methods using carrier-free
radioberyllium in the rat. Such autographs have added to
the picture of beryllium concentration so f a r a s skeleton is
concerned.
SUMMARY
1. Three different chemical forms of carrier-free radioberyllium (Be7 as the chloride, sulfate, and citrate) were
administered to r a t s by intravenous and intraperitoneal injection. The animals were sacrificed at various time intervals
following injection. Radioautographs with sufficient contrast
for photographic reproduction were obtained from 9 animals.
2. The only organs from which autographs could be obtained with any degree of regularity were liver, spleen, and
skeleton. Kidney autographs were rarely obtained.
3. Conclusions drawn from comparison of such autographs
and their respective histologic sections a r e as follows :
a. Re7 accumulated in discrete loci at the periphery of
the liver lobule, in addition to distrilnuting uniformly in lesser amounts throughout the lobule.
There was no indication of its presence in efferent
or afferent veins, lnranches of the hepatic artery, or
in bile capillaries.
b. I n spleen, beryllium was concentrated only in red pulp ;
none in the white pulp.
c. I n kidney, beryllium was found in cortex only, chiefly
in a broad zone adjacent to the cortico-medullary
junction. There mas no evidence of glomerular concentration.
476
C. T. l i A Y L O R A N D C. U. V A N CLEAVE
d. Autographs of liver and spleen suggested no aiiatonlical diff ereiice between the three chemical forms
in which Be7 was administered.
e. In the distal end of thc f emur, beryllium 1 x 7 ~ deposited
s
first in osteoid tissue adjacent to the cpiphyseal
plate. After intervals u p to 49 days, it was found
in discrete loci in oldcr spongiosa; some loci were
in mcdullary cavity. KO clear evidence of its presence in compact bone was found, although traccs
appeared in periosteum.
LITERATURE CITED
ALDRIDGE,
W. N., J. 31. RARNESAND F. A . DEN2 1949 Espcrimeiital lxrylliuin
poisoning. Brit. J. Exp. Path., SO: 375-388.
AXELROD,
I). J. 1947 An improved nicthod f o r cutting nm1eealcified bonr sections
and its application t o 1:idio-autograpli~. Anat. Rcc., 9 X : 19-24.
RLoonr, W. 19-18 Histopathology of i~radiation. N.N.E.S. McGraw Hill (20.
Chap. XIV.
COnIAK, C. L., W. E. LOTZAND G. A. B O Y D 1952 Autoradiogral)liic studies of
c:rlcinm, phosplioms, :ind strontiuni distribution i n the lioncs of the
growing pig. Anat. Rec., 90: 113-129.
CKOWLEY,J. F., J. G. HAnrmox A N D I<. G. SCOTT1949 The metabolism of
carrier-free radioberyllium i n tlic rat. J. Biol. Chcm., 177 : 975-984.
DE BRUY'S, P. P. 11. 1948 Tlic cffcct of s-r:iys on the lymphatic nodule, with
wferences t o the dose and relative sensitivities of diff'crent sl'ccies.
Anat. nee,., 10.1: 373-404.
HARRINGTOX,
. L., 1.1. E. JOIINS,
:
3
A. 1'. \vII.ES AND c. GARRETT 1048 Tlic
fundamental action of iiitcnsifying serccns in ganniia mdiography. Can.
J. RCS. (F) 26: 5'40-551.
LIWLOND,
C. P., G. W. WILKINSON,
L. F. RELANGRR
AND J. ROBICHON 1950 R.a(lioautogixpliic visualizatioii of bone forinntion in the rat. Arn. J. a n a t . ,
86: 289-341.
MARINELIJ,L. I)., E. H. Q U I h I n Y A N D G . J . HINE 1948 Dosage determillatioll
with radioactive isotopes. 11. Pmcticnl eoiisiderations in tlicrapy a n d
protection. Am. J. Roent. and Rad. Tlier., 59: 260-281.
MURRAY,
K. G. 1918 Histopathology of irradiation. Ed. by W. Bloom. N.N.E.s.
McGraw Hill Go. Chap. V I I : 2,43447.
SCIIUBERT,
J., A K D M. R.. WHITE 1950 Effect of citrate salts and other e1icinic:il
factors on the distribution and exwetion of beryllium J. Lab. and Clia.
Med., 35: 854-864.
SCOTT,J. K. 1948 The pathnlogic anatomy of aente cxpcriniental bcrylliuin
poisoning. Arch. Path., 4 5 : 354-359.
VAN CLEAVE:,C. D., A N D C. T. KAYLOR1953 The distribution and retention of
carrier-free rndioberglliimi in tlrc rat. Arch. lndust. Hyg. and Occup.
Med., 7 : 367-375.
PLATE
ESPL.4NhTION
OF FIGUKES
1 hutogr:lpli of 5 p section of liver of r a t ii1,jectt.d tivo hours prcrioosly ivitli
6 mc of Be'SO,.
x
approx. 2.5.
2
Tracing of Iiistological section of liver ivliose autograph is sliown i n figiire 1.
Autograph mas superimposed on tlie tracing and some of the areas of hl:ickcliing mere stippled on. Tliese darkened areas vere invariably around ccntr:il
veins, wliich are shown as small, irregular liolcs i n this drawing. X 2.6.
3
hutograpli of 5 p section of liver of rat injected tliree (lays previousl!. vitli
6 nic Bc'80,. X approx. 3.
4
Tracing of histological eectioii whose autograph is shown i n figure 3. Areas
of blackening are stiplilcd. Central reins irregnlar Iioles. X 3.
5
Autograph of 5 p section of liver of r a t injected two days prerious1-j with
7 nic of Rc'CI,. The black streaks are artifacts. The mottled pattern of darkening scen i n figures 1 and 3 is :ipp:went in this and in the n e s t tlirec figures.
x approx. 2.5.
6
Autograph of 5 ,u section of liver of r a t injected 40 days prcriously with 7 nic
EeY;12. x 3.
7
Autogr:il)h of 5 p section of liver of r a t injected 24 lionrs previously witli
G nic Re'SO,. X 2.
8
Autograpli of 5 r section of liver of r a t injcetcd tlirec days Iireriously wit11
1.5 nic Bc'SO, plus 0.5 nig stable BeSO,. x 2.
9
Autograph of 5 r section of spleen of rat. injected two IIOWS ~ i ~ e v i o i i swith
l~
Gme 13e7S0,. X 2.
10
Tracing of histological scctioii mliose autograph is shown in figure 9. T l ~ c
unstippled areas, corrrslionsl,onding t o the clear areas of the : ~ u t o g r a p l ~
a r, e tile
\vhite pulp with central arteries indicated in a few instances. x appros. 2 .
11 Autograph of 5 EL section of spleen of rat injected two days previously 6 t h
7 me Bc'CI,. X approx. 3.
1 2 Autograpli of 5 r section of spleen of rat injected 24 hours previously with
6 iiic Bc'SO,. The dark edge is a n artifact. X approx. 2.
13 Autograph of 5 P section of spleen of r a t injected three days previously
G iiic &'SO,.
X approx. 2.5.
TTTitll
14 Autograph of 5 P scctioii of kidney of r a t injected with a total of 5 IIIC of
Be'CI, over the preceding 8 days and sacrificed 24 hours after the last injection. X iipprox. 2.
478
PLATE 1
479
PLATE 2
EXPLANATION O F FIGURES
13
Autogr:rpli of 5 p undecalcified longituclinal scction of distal end of f e m u r of
a rat injected intraperitoneally over a period of one week with 11.21~lc Be'
citrate, and sacrificed 24 hours :ifter last injection. X 1.7.
1G
Tracing of liist,ologic:il section whose autograph is slromn in figure 15. The
position of tlie cpipliysral ]iI:ite is indicated aiid the extent of compact bone.
Thc corresponcliiig area of iiitcnsc hlackening in autograph is stippled. X 2.
17
Autograph of 5 p section of fcwiur of
prcriously with G nic Bc'SO,. X 1.5.
78
Tracing of Iiisto1ogic:il section whosc autograph is shown ill figure 17. l<]iipliyscal plate, com~iactbone :tnd niedullnry carit?. are shown and tlie area of
darkeiiiug in thc autogr:ipli is stippled. X 1.3.
19
Autograph of 3 p section of feinur of a r a t injected 8 days previously with
9.6 nic Be'SO,. X 1.5.
20
Tracing of histoloyicd section whose xntograpli is shomn i n figure 19. Arcas
of d:irlieuing in :rutogr:rph are stippled. X 1.5.
ii
w t injectcd i1itr:trenously 21 hours
21-23 Autographs of t w o other 5 p scctious of femur of s:iiiic r a t :is clcscriliecl in
figure 19. Each X 1.5.
23
Autograph of 5 p scctioii of fciuur of :I r a t injected 49 days p?cviou;ly wit11
7 l l l C Bc'CI,. x 1.5.
24
1'r:icing of histological section wliose autoyr:ilih is shown in figure 23. Areas
of darkening on autograph :ire stilipled. X 1.5.
25 Autogr:iph of another 5 p section of femur of same rat as described i n figure
23. x 1.5.
26
Tracing of histological seclioii whose autograph is shown in figure 24. Darkened areas of autogmpli arc stipp1t.d. X 1.5.
27-28 Autograplis of 5 p scctioiis of femur of r a t iiijwtcd two days previously
with i iiic Ee'Cl,. X 1.5.
29
Tracing of histological section whose autograph is shown in figure 28. Darkeiicd arras of nutogrnlili are stipliletl. X 1.5.
480
481
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