Primary Liver Tumors in Beagle Dogs Exposed by Inhalation to

American Journal of Pathology, Vol. 133, No. 2, November 1988
Copyright © American Association of Pathologists
Primary Liver Tumors in Beagle Dogs Exposed by
Inhalation to Aerosols ofPlutonium-238 Dioxide
NANCY A. GILLETT, DVM, PhD,
BRUCE A. MUGGENBURG, DVM, PhD,
JAMES A. MEWHINNEY, PhD,
FLETCHER F. HAHN, DVM, PhD,
FRITZ A. SEILER, BRUCE B. BOECKER, PhD,
and ROGER 0. McCLELLAN, DVM
From the Inhalation Toxicology Research Institute, Lovelace
Biomedical and Environmental Research Institute, Albuquerque,
New Mexico
Primary liver tumors developed in Beagle dogs exposed by inhalation to aerosols of 238PuO2. Initial deposition of 238PuO2 in the respiratory tract was followed by translocation of a portion of the 238Pu to the
liver and skeleton, which resulted in a large dose committment and tumor risk to all three tissues. In a population of 144 dogs exposed to 238PuO2, 112 dogs died or
were killed 4000 days after 238Pu exposure, 100 dogs
had osteosarcoma, and 28 dogs had lung cancers. At increasing times after exposure, however, liver lesions
have become more pronounced. Ten primary liver tumors in nine animals were diagnosed in the dogs dying
before 4000 days after exposure. An additional five pri-
mary liver tumors in three dogs occurred in 9 animals
killed after 4000 days after exposure. The majority of
these tumors have been fibrosarcomas. The liver tumors were usually not the cause of death, and rarely
metastasized. The occurrence of liver tumors in this
study indicates that 238Pu is an effective hepatic carcinogen. Liver carcinogenesis is assuming an increasing
importance in this study at late times after inhalation
exposure. These results suggest that the liver may be an
important organ at risk for the development of neoplasia in humans at time periods long after inhalation of
238Pu. (AmJ Pathol 1988, 133:265-276)
THE BIOLOGIC EFFECTS of two of the major isotopes of plutonium (Pu) present in the nuclear fuel
cycle, 238Pu and 239Pu, have been studied extensively
because of the potential risk of human exposure in the
unlikely event of accidental releases. Plutonium-238,
because of its abundance, high specific activity, and
relatively short half-life (86 years), produces about
80% of the alpha activity in Pu recoverable from the
spent fuel of a light water reactor. Because of the rapid
oxidation of plutonium metal, plutonium dioxide
(PuO2) is the compound most likely to be encountered after accidental release.
Previous studies have demonstrated that Pu is an
effective skeletal, pulmonary, and hepatic carcinogen,
depending on its chemical form and route of exposure.`5 Injected 239Pu is rapidly deposited in the bone
and liver, resulting in bone and liver tumors.3 6'7 Inhaled 239PuO2 is insoluble and is retained in the lung
and lung-associated lymph nodes, with the primary
effect being lung cancer.",4,5 Because of its high specific
activity, however, inhaled 238Pu becomes solubilized
and is subsequently translocated from lung to bone
and liver.8
Life-span studies in beagle dogs after single inhalation exposure to monodisperse aerosols of 238PuO2
were initiated at the Inhalation Toxicology Research
Institute in the mid- 1 970s to study the biologic effects
and relative importance of nonuniform distribution
of alpha radiation dose in the lung. Previous reports
from these studies have documented the initial effects,
predominantly bone cancer.2 This study has been in
progress for more than 10 years, and will continue for
the remaining life span of the dogs. With increasing
time after exposure, the occurrence of liver lesions
and primary liver tumors has assumed increasing
Supported by the Office of Health and Environmental
Research, U.S. Department of Energy, under Contract No.
DE-ACO4-76EV01013 in facilities fully accredited by the
American Association for Accreditation of Laboratory Animal Care.
Accepted for publication June 14, 1988.
Address reprint requests to Nancy A. Gillett, Inhalation
Toxicology Research Institute, Lovelace Biomedical and
Environmental Research Institute, P.O. Box 5890, Albuquerque, NM 87185.
265
266
GILLETT ET AL
AJP * November 1988
Table 1 -Experimental Design for Study of Beagle Dogs
Exposed by Inhalation to Aerosols of MPuO2
Number
of dogs
24
12
12
12
12
12
12
12
12
12
12
12
12
Particle
size
Projected initial
pulmonary burden
(AMAD)
(kBq/kg)
-
-
1.5
1.5
1.5
1.5
1.5
1.5
3.0
3.0
3.0
3.0
3.0
3.0
0.4
1.1
2.6
5.2
10.4
20.7
0.4
1.1
2.6
5.2
10.4
20.7
Range of initial
pulmonary burden
obtained
(kBq/kg)
0.1-0.7
0.7-2.6
1.9-5.6
3.3-10.7
4.1-16.3
9.3-37.0
0.4-1.1
0.7-3.3
1.5-6.3
2.6-14.4
5.9-34.4
14.1-55.5
prominence in this study. The range of biologic effects
seen in this study and the dose-response relationships
will be described in subsequent manuscripts. This report documents the nature of the liver lesions and tumors occurring within this study, focusing primarily
on liver pathology induced within 4000 days after inhalation exposure.
Materials and Methods
Previous reports have detailed the experimental design, exposure methods, and husbandry for this
study.2'8'9 In brief, 168 purebred beagle dogs, born and
raised in the Inhalation Toxicology Research Institute
colony, were exposed at 12-14 months of age to
monodisperse aerosols of either 238PuO2 particles or
the aerosol diluent. An equal number of male and female dogs were used in this study.
Seventy-two dogs were exposed once by inhalation
to monodisperse aerosols of 238PuO2, with an activity
median aerodynamic diameter (AMAD) of 1.5 A (cg
= 1.2) and an additional 72 dogs were exposed singly
to 238PuO2 particles with an AMAD of 3.0 q (ag = 1.1).
Twenty-four control dogs inhaled an aerosol of only
the diluent (0.01% dipalmitoyl lecithin in distilled water) used in the preparation of 238PuO2 particle suspensions. The aerosol concentration and duration of
exposure were adjusted for each animal such that 12
dogs each were exposed to achieve one of six normalized initial lung burdens (0.4, 1.1, 2.6, 5.2, 10.4, and
20.7 kBq/kg body mass). Because of the nature of inhalation exposure, a continuum of initial lung burdens was achieved among the dogs. Table 1 summarizes the experimental design of the study.
A detailed description ofthe methods used to deter-
mine the disposition of the 238Pu and the associated
dosimetry are reported elsewhere.8 In the present report, the biologic effects observed are related to the
initial pulmonary burden (IPB) of 238Pu and the cumulative average absorbed alpha dose to liver calculated to 4000 days after exposure (dpe).
Dogs were housed individually in metabolism cages
for the initial 21 days after inhalation and then transferred to kennel buildings for the remainder of their
lives. Animals with similar body burdens of 238Pu and
of the same sex were housed in pairs in dog runs in
the kennel buildings. Dogs were fed 350 g of dry kibble
(Wayne Dog Food, Continental Grain Company,
Chicago, IL) daily and water was provided ad libitum.
After exposure, dogs were observed daily, and complete physical examinations, hematology, clinical
chemistry, and radiographic surveys were performed
annually on all dogs. Individual animals received clinical care and treatment as needed as per accepted veterinary procedures, but treatments that would alter
the clinical course of neoplasms induced by 238Pu
were not performed."'
A detailed post-mortem examination was performed at the time of death, or when euthanasia was
performed based on humane considerations. Tissue
sections from all organ systems and all lesions were
formalin-fixed, paraffin-embedded, cut, and stained
with hematoxylin and eosin (H & E) using standard
histologic techniques. To minimize the variability inherent in a study of this length involving several clinicians and pathologists, clinical records from all dogs
were reviewed by one veterinarian (BAM) in conjunction with a review of pathology reports and histopathology by one pathologist (NAG). Neoplasms were
classified according to the International Histological
Classification of Tumors in Domestic Animals."
Results
Because aerosol particle size (1.5 and 3.0 ,u) and sex
did not appear to have an overall effect on the retention, distribution, and excretion patterns of the Pu or
the biologic effects observed in the liver over the first
4000 days after exposure (dpe), results of the two studies are considered together.8
After the initial pulmonary deposition of 238PU02,
an average pulmonary retention half-time of approximately 500 days was observed during a period of approximately 100 days after exposure. Thereafter, the
relatively high specific activity of the 238Pu led to the
fragmentation of the particles deposited in the lung,
followed by greatly increased dissolution and translo-
Vol. 133-oNo. 2
cation ofsolubilized 238Pu to the liver and skeleton.8"12
The pulmonary retention half-time during this time
period was approximately 100 days. The cumulative
radiation doses to the lung, liver, and skeleton
through 4000 days after inhalation exposure are
shown in Figure 1. After about 500 days, there was
little further increase in the cumulative dose to the
lung because of the depletion of the pulmonary deposit of Pu by translocation. In contrast, doses to the
liver and skeleton continued to increase throughout
the dog's lifetime because of the much longer residence time of Pu in these tissues. The liver is predicted
to attain a larger cumulative radiation dose than the
lung in dogs dying at very late times after exposure
(approximately 5000 days after exposure).
At this writing, all of the dogs had reached at least
4000 days after the initial exposure. During this interval, 112 exposed dogs and 6 control dogs died. The
principal cause of death for exposed dogs was osteosarcoma. Analysis of exposed dogs dying through
4000 dpe has shown that 11 1 osteosarcomas occurred
in 100 dogs. Of these, 72% have been the primary
cause of death. The other major cause of death in this
study has been primary lung cancer. A total of 51 primary lung tumors have been identified in 28 dogs; of
these, 8% have been the primary cause of death. Other
radiation-related effects include atrophy and fibrosis
of the tracheobronchial lymph nodes, osseous atrophy
of the maxilloturbinates, radiation osteodystrophy,
and radiation pneumonitis and pulmonary fibrosis.
With increasing time after inhalation exposure to
238PuO2, liver degeneration and primary liver tumors
have been more frequently noted clinically and
through gross and histopathologic examination. A serologic indicator of liver status is serum alanine aminotransferase (ALT), which is a hepatocytic cytoplasmic enzyme that increases in serum during periods of
altered plasma membrane permeability, and thus is
indicative of hepatocyte degeneration or necrosis.
Dogs were grouped by initial pulmonary burden (IPB)
into seven groups, and the mean group value of the
ALT derived from annual clinical chemistry determinations were plotted over time (Figures 2A and B).
Exposed dogs exhibited a significant increase in ALT
values as compared with control dogs. The groups
having higher lung burdens tended to have elevated
ALT values at earlier times after exposure. This time
course relationship showed a clear dose effect, with the
highest IPB group (25.9 kBq/kg) peaking at 3 years
after exposure, and the lowest IPB group (0.6 kBq/kg)
showing a gradual increase beginning at 6 years after
LIVER TUMORS FROM INHALED 238PU IN DOGS
267
m
0)
a
cr
CD
uii
0
0
a
z
0
-
a:
-J
0
2000
4000
DAYS AFTER INHALATION EXPOSURE
Figure 1-Radiation dose to lung, liver, and skeleton from dogs exposed
by inhalation to aerosols of 238PuO2 through 4000 days after inhalation exposure.
exposure, and continuing to increase at the last measurement. The magnitude of the increase in ALT values was not clearly dose related, but the duration of
time required to reach elevated ALT values showed a
clear stratification with dose.
A similar grouping of dogs by IPB was done to compare liver to body mass ratios obtained at the time of
necropsy among groups. The mean mass ratio from
each group was plotted against the mean activity level
as determined by IPB. Linear regression yielded a fitted line having a slope that was not statistically different from zero. Thus, the liver mass was not demonstrated to have been altered by the 238Pu burden as
measured by IPB.
A total often primary liver tumors occurred in nine
exposed dogs dying before 4000 dpe. At the time of
this report, an additional five liver tumors were identified in three of the nine exposed dogs dying at greater
than 4000 dpe. Table 2 summarizes the phenotype,
role in death, and other related information on the
tumors in these 12 dogs. None of the control dogs for
this study developed a primary liver tumor during this
time interval. Only 2 primary liver tumors have occurred in 157 control dogs to date for all of this Institute's life-span studies combined; both of these occurred after 4000 dpe. The final incidence of liver tumors in the present study may be higher as additional
dogs live longer times after 238Pu exposure.
The primary liver tumor phenotype observed was
fibrosarcoma, comprising 5 of the 15 liver tumors
present (Figure 3). In addition, one fibroma was
268
AJP * November 1988
GILLETT ET AL
250 r
250 r
-J
200
w
C')
cc
200
cc
w
LL
cr
CD)
z
0
1i00
z
100
IF
w
z
z
z
-J
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0
2
I
I
I
I
,
0
4
2
I
I
I
nXI
4~~~~~~~~~~~~~~~~~~
6
8
10
12
A
6
8
10
B
YEARS AFTER INHALATION EXPOSURE
YEARS AFTER INHALATION EXPOSURE
Figure 2A,B-Mean of serum alanine aminotransferase (ALT) values as a function of time after exposure. Dogs are grouped by lung burden with a mean
lung burden of 0, 0.6, 1.7, 3.0, 5.6, 11.1, and 25.9 kBq/kg. Note that groups with higher lung burdens tend to have peak ALT values at earlier times after
exposure.
noted. One of these fibrosarcomas was the cause of
the animal's death. Bile duct tumors, including three
benign intrahepatic bile duct cystadenomas, and one
fatal cholangiocarcinoma were the second most common liver tumor observed (Figure 4). Two microscopic carcinoids were identified, and their histogenesis was confirmed through immunocytochemical
staining for neuron-specific enolase (Figure 5).
Clinical signs related to liver dysfunction were absent in the majority of the dogs with liver tumors.
Only three of the ten liver tumors occurring before
4000 dpe were suspected clinically at the time of gross
necropsy. Exposed dogs without liver tumors also did
not show clinical evidence of liver disease despite the
of hepatic degenerative changes as discussed
subsequently. A severe granulomatous hepatitis was
presence
Table 2-Primary Liver Tumors in Beagle Dogs that Inhaled Graded Activity Levels of 238PuO2 at Approximately 13 Months
of Age and Were Maintained for Life-Span Observation
Dose to
Particle
liver to
Days
Dog
size
IPB
4000 dpe
after
number (AMAD) (kBq/kg)
(Gy)
exposure
Role of
Morphologic
diagnosis
tumor in
Metastasis
Clinical
signs
death
Cause of death
No
No
No
No
No
Yes
No
No
Incidental
Cause of death
Incidental
Incidental
Osteosarcoma
Fibrosarcoma, liver
Osteosarcoma
Osteosarcoma
No
Yes
Contributory
Squamous cell
carcinoma,
gingiva
Carcinoma, primary
site unknown
Osteosarcoma
731 S
692S
872V
705A
3.0
1.5
1.5
1.5
22.2
6.7
0.7
1.8
25.9
7.8
0.9
2.0
1161
2416
3119
3176
860B
1.5
2.2
2.5
3178
Fibroma
Fibrosarcoma
Fibrosarcoma
Intrahepatic biliary
cystadenoma
Fibrosarcoma
704S
3.0
1.5
1.5
3566
Neurofibrosarcoma
No
No
Incidental
856T
3.0
2.6
3.0
3804
No
No
Incidental
746S
1.5
1.5
1.7
3805
No
No
No
No
Incidental
Incidental
854B
735C
3.0
3.0
2.6
3831
4234
No
2.2
2.9
2.6
Intrahepatic biliary
cystadenoma
1. Fibrosarcoma
2. Intrahepatic biliary
cystadenoma
Carcinoid
No
No
Incidental
Incidental
697A
3.0
1.8
2.2
4386
No
No
Incidental
Contributory
724S
1.5
0.7
0.8
4415
No
No
Contributory
Carcinoid
Yes-hepatic
lymph node
1. Sarcoma,
No
undifferentiated
Yes-lung, TBLN,
2. Cholangiocarcinoma
hepatic lymph
node
1. Hepatocellular
No
carcinoma
2. Intrahepatic biliary
cystadenoma
No
Incidental
Osteosarcoma
Osteosarcoma
Adenocarcinoma,
lung
Osteosarcoma
Sarcoma, lung
112
LIVER TUMORS FROM INHALED 2mPU IN DOGS
Vol. 133 * No. 2
269
_>j~~~~~~~~~~~~~~~
00
A#---~~~~~~~~~~~~~~~~~~
AML ~
~
~
h
Figure 3-Liver fibrosarcoma, characterized by interwoven bundles of spindloid cells and scant matrix production. X420
an incidental finding in an exposed dog dying from an
osteosarcoma. The cumulative absorbed alpha dose
to the liver to 4000 dpe in this animal was 22.5 Gy.
The relationship of the granulomatous hepatitis and
the radiation exposure is not known.
One control dog was killed as a result of severe
diffuse hepatic cirrhosis. This was the only evidence
of hepatic disease in any of the control dogs in this
study. The cause of the cirrhosis is not known.
The cause of death in three 238Pu-exposed dogs was
a mast cell tumor, which in two dogs was confined to
the liver and spleen. Both animals died approximately
3100 days after exposure. One animal had an accumulated absorbed alpha dose to liver calculated to
4000 dpe of 4.0 Gy, while the second had a dose of
1.9 Gy. Visceral mast cell tumors in the absence of
cutaneous involvement are rare in the dog. " The similar presentation of the mast cell involvement in these
two dogs raises the possibility that the neoplasms were
radiation induced.
Although clinical signs of liver dysfunction were absent in the majority of the exposed animals, review of
the gross pathology and histopathology of these dogs
showed an increase in nonspecific hepatic degenerative changes with increasing time after exposure. A
variable degree of nodular hyperplasia was frequently
present. Vacuolar degeneration ofhepatocytes was inconsistent; large hyperplastic nodules usually exhibited diffuse vacuolar degeneration (Figure 6). It was
not uncommon to note generalized vacuolar degeneration of hepatocytes, however, which probably primarily reflected the systemic state of the animal at the
time of death. Mild periportal fibrosis and biliary hyperplasia were also noted in some animals. A common finding in the exposed dogs was the presence of
multiple small cellular aggregates randomly disseminated throughout the hepatic parenchyma. These cellular aggregates were composed of macrophages containing lipid and brown granular pigment, and were
histologically consistent with the lipogranulomata described by Bergman.13 Although these cellular aggregates were seen in control dogs, their occurrence was
much less common. Review of liver sections from all
of the dead exposed and control dogs did not reveal
any liver lesion that could be interpreted as being specific for the radiation exposure. Rather, there ap-
270
GILLETT ET AL
,0
O
AJP * November 1988
oAS
P
Figure 4-Intrahepatic biliary cystadenoma. The benign neoplasm is a well delineated, multi-loculated cystic mass, containing papillary projections lined by
low cuboidal epithelial cells. x131
peared to be an increase in incidence and severity of
nonspecific degenerative changes in the exposed dogs
as compared with control animals.
Discussion
Although primary liver tumors have been observed
after other routes of exposure to plutonium, the occurrence of primary liver tumors after inhalation exposure to 238PuO2 has not been reported previously in
the open scientific literature. Because of the specific
activity of 238Pu, inhaled particles tend to fragment in
the lung, enhancing dissolution of the 238PuO2 and
translocation to the liver and bone.8 With increasing
time after exposure, the accumulated liver dose approaches the average organ dose received by the lung
and exceeds that of bone. The predominant incidence
of respiratory and skeletal tumors suggests that the
spatial distribution of dose within these organs results
in a higher tumor incidence or that these tissues are
more sensitive to neoplastic induction after radiation
exposure than is the liver. The increasing incidence of
liver tumors in the long-term surviving dogs in this
study, which is still in progress, however, supports
speculation that the liver may have been neglected as
an organ at risk for plutonium-induced neoplasia in
humans, particularly at late times after exposure.'4
In all but two of the 12 animals with liver tumors,
the neoplasm occurred later than 3000 days after initial exposure. This long latent period has been observed by Taylor et a13 for liver tumors occurring in
beagle dogs after a single intravenous injection of
239Pu and by Brooks et al6 in Chinese hamsters injected with 239PuO2 or 237Pu citrate. In the Taylor
study at the University of Utah, all of the liver tumors
occurred in animals living longer than 3000 dpe; the
average age at recognition was 4240 dpe. The long latent period observed in both studies probably reflects
the long life span of the hepatic cell and its low mitotic
activity because cell division is required for the expression of radiation-induced tumors. In addition, the life
shortening induced from a Pu-related osteosarcoma
LIVER TUMORS FROM INHALED
Vol. 133 * No. 2
Figure 5-Liver carcinoid, diagnosed based on
morphologic appearance and positive immunocytochemical staining for neuron-specific enolase. Border of normal hepatocytes present in
upper left comer. X262.5
2mPU
IN DOGS
271
_
%%~~~1
A*1
I~~~~~~~*.~~~~~1
3%,5
or lung tumor in many of the dogs exposed to higher
levels of plutonium probably interposed before the expression of liver tumors.
In both the Utah and ITRI studies, liver tumors predominantly occurred at lower burdens of Pu. This
may be related to both the long latent period required
for liver tumor expression and competing risk from
pulmonary and skeletal cancer in animals with higher
burdens. Both of these factors are likely to lead to an
underestimate of the risk for plutonium-induced liver
cancer for small intakes of 238Pu02. Microdistribution
of the Pu may also play a contributing role in the development of hepatic tumors in animals receiving
lower doses. At higher dose levels, cell death and necrosis of hepatocytes, where deposition initially oc-
curs, results in a localized deposition in reticuloendothelial cells. This pattern is less remarkable at the
lower dose levels.35 Other investigators have demonstrated that the increased uniformity of dose to liver
occurring at low liver burdens of Pu may play a role
in the occurrence of liver tumors in animals. 15,16
The phenotypic distribution of the liver tumors in
the present study was unusual compared with the distribution from anticipated, spontaneously occurring
liver tumors in the dog. Fibrosarcomas were the most
common histologic type. Another unusual finding
was the presence of two liver carcinoids. Spontaneous
liver tumors are not common in the dog, and typically
appear to be hepatocellular adenoma, hepatocellular
carcinoma, and cholangiocarcinoma.'in7 A recently
272
GILLETT ET AL
AJP * November 1988
Figure 6-Nodular hyperplasia (large arrow) and secondary vacuolar degeneration (small arrow) were common in the livers of beagles exposed to 238Pu02.
x86.4
reported review of spontaneous liver tumors in 1867
canine necropsies showed a primary liver tumor incidence of less than 3%. '7 Of these, hepatocellular carcinomas and cholangiocarcinomas represented 63% of
the cases. Only one fibrosarcoma was diagnosed. Carcinoid of the liver also is a rare neoplasm that appears
to originate from cells with argentaffin properties in
the bile duct epithelium.18 Because of the low rate of
spontaneous liver tumors in the Beagle and the unusual histologic types observed, all of the liver tumors
in this series were judged to be radiation induced.
In a report from the University of Utah, 47 primary
liver tumors were noted to have occurred in beagle
dogs following a single intravenous injection of 239Pu
in a soluble form.'9 Liver tumors occurred only in
239Pu-exposed dogs that lived at least 7.6 years. In contrast to the present study, the most frequently occurring tumor was the bile duct adenoma (62%), followed
by the bile duct carcinoma (21%). Only two (4%) fibrosarcomas were observed. As in the present study,
clinical illness that could be related to the induced
liver tumors was infrequent. The liver malignancy
was the cause of death in only eight or 17% of the beagles that were diagnosed as having liver tumors.
A review of the incidence of internal mast cell tumors in beagles receiving intravenous doses of 239Pu
or 241Am'9 supports the hypothesis that the two visceral mast cell tumors occurring in the present study
are related to the radionuclide exposure. Within the
two intravenous injection studies discussed previously,'9 five mast cell tumors occurred that were
considered to be hepatic in origin. It was speculated
that the moderate frequency of hepatic mast cell tumors observed in the dogs injected with actinide radionuclides may be related to the relatively high number of mast cells lining the sinusoids in the canine
liver.
The cells of origin for the liver neoplasms seen in
both studies are uncertain. Comparison of the intrahepatic cellular distribution of plutonium in beagles
after inhalation exposure or intravenous injection
showed that the hepatic distribution was not dependent on the route of administration, but was dependent on total activity in the liver.15 The difference in
Vol. 133
*
No. 2
tumor phenotypes between the present study and
those occurring in beagles injected with 239Pu therefore does not appear to be dependent on intrahepatic
cellular distribution of dose. Soluble Pu is preferentially deposited in the hepatocyte, where it is associated with lysosomes.20-23 Investigators have shown
that after high levels of exposure, Pu was initially deposited in hepatocytes; radiation-induced death of
these cells resulted in the loss ofthe Pu to the reticuloendothelial cells by phagocytosis.3 Because of the high
cellular concentration ofPu in the hepatocyte and low
concentration in bile duct epithilium, investigators
have postulated that the bile duct tumors occurring in
dogs receiving intravenous Pu arose from hepatocytes.3 The embryologic origin of bile ducts, which
originate from hepatic cords that are adjacent to connective tissue surrounding branches ofthe portal vein,
is consistent with this hypothesis. The absence of hepatocellular dysplasia argues against the postulate that
the hepatocyte is the primary cell of origin, however.
The rare occurrence of hepatocellular carcinomas is
striking in this study (1 out of 15), particularly in view
of the fact that the hepatocyte receives the highest radiation dose from the deposited plutonium. Studies in
other species have demonstrated that alpha irradiation of the liver can induce a high incidence of hepatocellular adenomas and carcinomas.6 It is possible that
the relative resistance of the canine hepatocyte to
transformation by ionizing radiation may represent a
species-specific phenomena. The wide distribution of
tumor phenotypes seen in the present study suggests
multiple cells of origin for the different tumor types.
The fibrosarcomas, which developed in the absence
ofmarked fibroplasia within the liver, are of particular
interest. One can speculate that the Ito cell may be the
cell of origin for the fibrosarcomas seen in this study.
Focal aggregates of lipid, pigment, and macrophages,
termed lipogranulomata, have been correlated with
changes in the perisinusoidal Ito cell population in the
liver.'3 The function of Ito cells has been related to
vitamin A storage and fibrogenesis.24 Fibroplasia, a
potential source of cells of origin for fibrosarcomas,
was not a striking feature of exposed dogs; however,
the formation of lipogranulomata was often marked.
This may represent injury to a cell population capable
of fibrogenesis, and hence may be capable of "neoplastic expression as a fibrosarcoma.
Studies investigating the biologic effects of inhaled
238PuO2 in rats and Syrian hamsters have not shown
the significant increase in bone and liver tumors that
has been demonstrated in the Beagle dog.25'26 In these
studies, the in vivo solubility of 238PU02 particles was
not significantly different from the in vivo solubility
LIVER TUMORS FROM INHALED
2-mPU IN DOGS
273
of 239PuO2 particles. This species difference probably
reflects the more rapid mechanical clearance of particles from the lung by rodents.27'28 The 238PuO2 particles do not substantially fragment, allowing increased
dissolution ofthe Pu, for approximately 100 days after
exposure. A much larger percentage of the Pu particles have been cleared by this time in rodents than in
dogs. Thus, the majority of the Pu dose is delivered to
the lung in rodents. In addition, the rat liver clears
soluble Pu rapidly whereas the dog retains Pu in the
liver.29 Likewise, a prolonged retention half-time for
Pu in the liver is observed in Chinese hamsters.30 The
retention pattern of Pu in the liver of man is not
known. The clearance of particles from the lung in
man more closely resembles dogs than rodents, however. Thus, it is probable that a similar solubilization
of the Pu and subsequent translocation to liver and
skeleton would occur in man after inhalation exposure to 238PuO2, as has been shown by this study in
the dog.
Although epidemiologic information is not available for the effects of inhaled plutonium in humans,
the occurrence ofliver tumors in man after Thorotrast
injection has provided a model for liver tumors resulting from alpha irradiation. Thorotrast, a colloidal
mixture containing thorium dioxide, was used as a diagnostic radiologic contrast medium in the 1930s and
1940s.3'-33 The latent period for these tumors, similar
to that observed in the present study, was long, with
few tumors observed before 20 years after injection.
The cumulative tumor incidence increased rapidly
between 20 and 40 years after the injection.3' The risk
for liver cancer induced by alpha particle irradiation
has been calculated from Thorotrast data to be high if
the latent period and competing risk factors are considered.34 Risk factors derived from life-span studies
of beagle dogs exposed to soluble beta emitters that
translocate to the liver in significant amounts are similar to those derived for humans from the Thorotrast
studies.35
Thorotrast was deposited in the reticuloendothelial
cells throughout the body; in the liver, the colloidal
material was primarily deposited in Kupffer cells.33 A
variety of liver tumors that have been classified predominantly as hemangioendotheliomas and cholangiocarcinomas have been described in those individuals receiving Thorotrast.32'33'36 Several experimental
studies have shown that the neoplastic effect results
from the alpha particle irradiation rather than from a
foreign body effect or chemical toxicity.37-39 The intrahepatic cellular distribution of the Thorotrast
differs from soluble Pu, which is preferentially deposited in the hepatocyte. This difference in cellular dis-
274
AIP * November 1988
GILLETT ET AL
tribution of dose may account for the difference in
phenotype noted in the Thorotrast series and the present studies.
A study of the biologic effects of Thorotrast in deer
mice resulted in a high incidence (32%) of liver fibrosarcomas,38 equivalent to that reported in the present
study. In addition, liver carcinoids occurred in the
study in deer mice. Hence the distribution of tumor
phenotype may be a species dependent phenomena.
Correlation ofthe liver:body mass ratios at the time
of death with activity level (IPB) indicated a slight decrease in liver mass with increasing amount of 238Pu.
This decrease was not statistically significant; however, many of the dogs in the control and low-dose
group are still alive at this time. As these animals die,
the statistical error for the ratios at these levels should
decrease greatly, which would then increase the
chance for a statement of statistical significance. Dramatic changes in liver weights were previously reported in beagle dogs receiving 107 kBq 239Pu/kg intravenously.3 Severe centrolobular degeneration and
necrosis resulted in marked liver atrophy in approximately 25% of the animals at this activity level. At
lower activity levels of Pu (1.7 kBq/kg), the weight of
the liver appeared to increase gradually with increasing age, although this trend was not established definitively.40
As shown in Figures 2A and B, the mean ALT values of the exposed dogs, grouped by IPB levels, are
significantly different from control dog levels, particularly at increasing time after exposure. In a similar
study conducted at Pacific Northwest Laboratories
(PNL) investigating the long-term effects ofinhalation
of 238PuO2 in beagles, ALT values were elevated only
after 48 months after exposure and only in the high
dose level dogs (initial alveolar deposition of 140 Bq).'
At later times in the PNL study, significant elevations
in ALT may be evident as the lower dose dogs accumulate a significant liver radiation dose. Significant
elevations in mean ALT values were observed in beagle dogs after intravenous injection of 239Pu at doses
of 0.016 ,uCi/kg (0.6 kBq/kg) or greater when measurements were made at 5 years after exposure.4'42 At
increasing times after exposure, ALT elevation was
progressive and evident in lower dose levels.'9
Histologic examination of all the livers from both
control and exposed dogs in the present study did not
show distinct lesions that were specific for the plutonium exposure. Investigators have reported significant mortality from primary plutonium-induced liver
disease at approximately 400 days after injection in
beagle dogs receiving 3 1iCi/kg (1 11 kBq/kg) of 239Pu
intravenously.43 Lesions consisted of marked hepatic
atrophy and severe secondary portal hypertension.
Similar lesions were not observed in the ITRI study;
however, the doses were significantly smaller and the
dose rate to the liver was markedly reduced. The degenerative changes present in the ITRI study, particularly nodular hyperplasia and lipogranulomata, are
seen frequently in older dogs as part of the aging process. 3 Periportal fibrosis and biliary hyperplasia were
also evident in some exposed dogs. Similar degenerative lesions have been observed in beagle dogs receiving 239Pu intravenously.'9 Nodular hyperplasia of the
liver has also been reported in beagle dogs that inhaled
238Pu02 and 239Pu(NO3)4 in ongoing studies at the Pacific Northwest Laboratory." Dogs exposed by inhalation to 238Pu exhibit these degenerative liver changes
at an age when comparable control animals have no
evidence of liver disease. The lesions observed appear
to be a nonspecific reaction to liver injury. Previous
investigators have suggested that irradiation of the
liver by 239Pu acts through the aging mechanism by
accelerating the rate of induction of small critical
changes in genetic material.39 The lesions observed in
this study are compatible with that hypothesis.
This study demonstrates that inhaled 238Pu02 is an
effective hepatic carcinogen. Other investigators have
emphasized that liver neoplasia may be an important
organ at risk in humans after intake of low levels of
Pu. Some investigators have speculated that the risk
from liver tumors might exceed the risk from bone
sarcomas for low level intakes of Pu in humans. 14 The
long life span of humans and additional carcinogens
to which the human liver can be subjected, such as
alcohol, might enhance liver tumor expression above
that seen in studies involving laboratory animals. The
final phase of this life-span study will provide critical
information regarding the risk of liver tumors from
238PuO2 inhalation exposure, including the development of a quantitative estimate of risk.
References
1. Park JF: Late effects of inhaled plutonium in dogs, Radiation Research; Biomedical, Chemical, and Physical
Perspectives. Edited by OF Nygaard, HI Adler, WK
Sinclair. New York, Academic Press, 1975, pp 12331247
2. Hahn FF, Mewhinney JA, Merickel BS, Guilmette RA,
Boecker BB, McClellan RO: Primary bone neoplasms
in Beagle dogs exposed by inhalation to aerosols of plutonium-238 dioxide. JNCI 1981, 67:917-927
3. Taylor GN, Jee WS, Williams J, Shabestari L: Hepatic
changes induced by 239Pu, Radiobiology of Plutonium.
Edited by BJ Stover, WS Jee. Salt Lake City, J. W. Press,
1972, pp 105-127
4. Lundgren DL, Gillett NA, Hahn FF, Griffith WC, Mc-
Vol. 133 *
No.2
Clellan RO: Effects of protraction of the alpha dose to
the lungs of mice by repeated inhalation exposure to
aerosols of 239PuO2. Radiat Res, 1987, 111:201-244
5. Dagle GE, Sanders CL, Park JF, Mahaffey JA: Pulmonary carcinogenesis with inhaled plutonium in rats and
dogs, Pulmonary Toxicology of Respirable Particles.
Edited by CL Sanders, FT Cross, GE Dagle, JA Mahaffey. Hanford Life Sciences Symposium, Technical
Information Center, U.S. Department of Energy, 1980,
pp 601-615
6. Brooks AL, Benjamin SA, Hahn FF, Brownstein DG,
Griffith WC, McClellan RO: The induction of liver tumors by 239Pu citrate or 239PuO2 particles in the Chinese
hamster. Radiat Res 1983, 96:135-151
7. Benjamin SA, Brooks AL, McClellan RO: Biological
effectiveness of 239Pu, '44Ce and 90Sr citrate in producing chromosome damage, bone-related tumours, liver
tumours, and life shortening in the Chinese hamster,
Biological and Environmental Effects of Low Level Radiation. Vol II. Vienna, International Atomic Energy
Agency, 1976, pp 143-152
8. Mewhinney JA, Diel JH: Retention of inhaled 238PuO2
in Beagles: A mechanistic approach to description.
Health Phys 1983,45:39-60
9. Bielfelt SW, Wilson AJ, Redman HC, McClellan RO,
Rosenblatt LS: A breeding program for the establishment and maintenance of a stable gene pool in a Beagle
dog colony to be utilized for long-term experiments.
Am J of Vet Res 1969, 30:2221-2229
10. Kirk RW: Current Veterinary Therapy. VIII. Small Animal Practice. Philadelphia, W. B. Saunders Company,
1983
11. International Histological Classification of Tumors of
Domestic Animals: Bulletin of the World Health Organization, 1974, 50:1-142 and 1976; 53:137-304
12. Diel JH, Mewhinney JA: Fragmentation of inhaled
238PuO2 particles in the lung. Health Phys 1983, 44:
135-143
13. Bergman JR: Nodular hyperplasia in the liver of the
dog: An association with changes in the ito cell population. Vet Pathol 1985, 22:427-438
14. Mays CW, Taylor GN, Jee WSS, Dougherty TS: Speculated risk to bone and liver from 239Pu. Health Phys
1970, 19:601-610
15. Gearhart JM, Diel JH, McClellan RO. Intrahepatic distribution of plutonium in Beagles. Radiat Res 1980, 84:
343-352
16. Brooks AL, Guilmette RA, Evans MJ, Diel JH: The induction ofchromosome aberrations in the livers of Chinese hamsters by injected Thorotrast, The Radiobiology of Radium and Thorotrast. Edited by W Gossner,
GE Gerber, U Hagen, A Luz. Baltimore, Urban &
Schwarzenberg, Muncheu-Wieu, 1986, 197-200
17. Trigo FJ, Thompson H, Breeze RG, Nash AS: The pathology of liver tumours in the dog. J Comp Pathol
1982, 92:21-39
18. Patnaik AK, Lieberman PH, Hurvitz Al, Johnson GF:
Canine hepatic carcinoids. Vet Pathol 1981, 18:445453
19. Taylor GN, Mays CW, Wrenn ME, Shabestari L, Lloyd
R: Incidence of liver tumors in Beagles with body burdens of 239Pu or 24'Am, Life-span Radiation Effects
Studies in Animals: What Can They Tell Us? Edited by
LIVER TUMORS FROM INHALED 2PU IN DOGS
275
RC Thompson, JA Mahaffey. USDOE Conf. 830951,
1986,268-285
20. Bair WJ, Thompson RC: Plutonium: Biomedical research. Science 1974, 183:715-722
21. Jung W, Thies WG, Seidel A: The role of lysosomes in
239Pu binding in rat liver: Comparison of sucrose and
metrizamide density gradient studies with whole liver
and purified hepatocytes. Int J Radiat Biol 1985, 48:
807-810
22. Danpure CJ, Taylor DM. The effect of internally deposited plutonium-239 on the lysosomes of rat liver. Radiat Res 1974, 59:679-692
23. Lindenbaum A, Rosenthal MW: Deposition patterns
and toxicity of plutonium and americium in liver.
Health Phys 1972, 22:597-605
24. McGee JO'D, Patrick RS: The role of perisinusoidal
cells in hepatic fibrogenesis. Lab Invest 1972, 26:429440
25. Sanders CL, Dagle GE, Cannon WC, Powers GJ, Meier
PM: Inhalation carcinogenesis of high fired 238PuO2 in
rats. Radiat Res 1977, 71:528-546
26. Sanders CL: Inhalation toxicology of 238PuO2 and
239PuO2 in Syrian golden hamsters. Radiat Res 1977,
70:334-344
27. Snipes MB: Retention of relatively insoluble particles
inhaled by dogs, rats, and mice. Current Concepts in
Lung Dosimetry. Technical Information Center, U.S.
Department of Energy, 1983
28. Snipes MB, Boecker BB, McClellan RO: Respiratory
tract clearance of inhaled particles in laboratory animals, Lung Modelling for Inhalation of Radioactive
Materials. Edited by H Smith, G Gerber. Commission
of the European Communities, 1984, pp 63-71
29. Brooks AL, Guilmette RA, Hahn FF, Jirtle RL: Uptake
and clearance of plutonium-238 from liver cells transplanted into fat pads of F344 rats. Int J Radiat Biol
1986, 50:631-639
30. Brooks AL, Diel JH, McClellan RO: The influence of
testicular microanatomy on the potential genetic dose
from internally deposited plutonium-239 citrate in Chinese hamsters, mouse and man. Radiat Res 1979, 77:
292-302
31. van Kaick G, Mirth H, Kaul H, Wesch H, Immich H,
Liebermann D, Lorenz D, Lorenz WJ, Luhrs H, Scheer
KE, Wagner G, Wegener K: Report on the German
Thorotrast study, The Radiobiology of Radium and
Thorotrast. Edited by W Gossner, G Gerber, U Hagen,
A Luz. Baltimore, Urban & Schwarzenberg, Munchen,
1986, pp 114-118
32. da Silva Horta J: Late effects of Thorotrast on the liver
and spleen and their efferent lymph nodes. Ann NY
Acad Sci 1967, 145:676-699
33. Grampa G: Radiation injury with particular reference
to Thorotrast. Pathology Annual 1971, 6:147-169
34. Mays CW: Risk estimates for liver, Critical Issues in
Setting Radiation Dose Limits. Bethesda, MD, NCRP,
April, 1982, 182-196
35. Muggenburg BA, Boecker BB, Hahn FF, Griffith WC,
McClellan RO: The risk of liver tumors in dogs and
man from radioactive aerosols. Life-span Radiation
Effects Studies in Animals: What Can They Tell Us?
Edited by RC Thompson, JA Mahaffey. USDOE Conf.
830951,1986,556-563
276
GILLETT ET AL
36. Kojiro M, Nakashima T, Iti Y, Ikezaki H: Pathomorphological study on Thorotrast-induced hepatic malignancies, The Radiobiology of Radium and Thorotrast.
Edited by W Gossner, G Gerber, U Hagen, A Luz. Baltimore, Urban & Schwarzenberg, Munchen, 1986, pp
119-122
37. Brooks AL, Guilmette RA, Evans MJ, Diel JH: The induction of chromosome aberrations in the livers of Chinese hamsters by injected Thorotrast, The Radiobiology of Radium and Thorotrast. Edited by W Gossner,
G Gerber, U Hagen, A Luz. Baltimore, Urban &
Schwarzenberg, Munchen, 1986, pp 197-201
38. Taylor GN, Mays CW, Lloyd RD, Jones CW, Rojas J,
Wrenn ME, Ayoroa G, Kaul A, Riedel W: Liver cancer
induction by 241'Am and Thorotrast in deer mice and
grasshopper mice, The Radiobiology of Radium and
Thorotrast. Edited by W Gossner, G Gerber, U Hagen,
A Luz. Baltimore, Urban & Schwarzenberg, Munchen,
1986
39. Wesch H, Riedel W, Hasenohrl K, Wegener K, Kaul
A, Muth H, van Kaick G: German Thorotrast study:
Results of the long-term animals studies on the effect of
incorporated radioactive and nonradioactive particles,
The Radiobiology of Radium and Thorotrast. Edited
by W Gossner, G Gerber, U Hagen, A Luz. Baltimore,
Urban & Schwarzenberg, Munchen, 1986, pp 186-188
40. Stover BJ, Atherton DR, Buster DS: Protracted hepatic,
splenic, and renal retention of 239Pu in the Beagle.
Health Phys 1971, 20:369-374
AJP * November 1988
41. Stevens W, Berliner D: Serum transaminase levels in
Beagle dogs burdened with plutonium-239. Radiat Res
1964, 23:420-429
42. Stevens W, Nabors CJ, Berliner D: A comparison of
serum transaminase levels and other serum constituents in dogs burdened with 239Pu, 228Th, 228Ra and
226Ra. Annals NY Acad Sci 1967, 145(Art. 3):817-828
43. Taylor GN, Dougherty TF, Christensen WR: Some toxicity aspects of internally deposited 239Pu, Pathology of
Irradiation. Edited by CC Berdjis. Baltimore, Williams
and Wilkins, 1971, 110-119
44. Dagle GE, Park JF, Weller RE, Ragan HA, Stevens DL:
Pathology associated with inhaled plutonium in Beagles, Life-span Radiation Effects Studies in Animals:
What Can They Tell Us? Edited by RC Thompson, JA
Mahaffey. USDOE Conf. 830951, 1986,471-476
Acknowledgment
Conduction of this life-span study has involved the active
participation of a broad range of individuals from the ITRI
scientific, technical, and support staffs. Although it is not
possible to list all of these participants, it is important to
recognize that a large, long-term study such as this could not
be undertaken without the participants functioning
smoothly as an integrated team. The authors thank all of
them for their participation, particularly members of the
Aerosol Science, Animal Care, Radiobiology, and Pathology Groups.

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