(CANCER RESEARCH 26, 834-843,May 1966]
Nodules in the Livers of C3H Mice after Long-Term Carbon Tetrachloride Administration:
A Light and Electron Microscopic Study1
DAVID
B. CONFER2 AND
RICHARD J. STENGER3
The Department of Pathology, College of Medicine, University of Cincinnati and Cincinnati General Hospital, Cincinnati, Ohio, and the
Department of Pathology, Cleveland Metropolitan General Hospital and It'estern Reserve University School of Medicine, Cleveland, Ohio
Summary
Parenchymal nodules developed in the livers of C3H mice
given carbon tetrachloride by rectal administration. These nonencapsulated nodules varied from 4 to 14 mm in diameter and
were sharply demarcated from the adjacent parenchyma. Despite
a lack of organized lobular structure, the nodules contained
occasional biliary ductules and scattered blood vessels resem
bling capillaries. Histologically, the cells composing the nodules
were less bizarre than those of the contiguous parenchyma and
were, in fact, comparable, in many ways, to the livers of untreated
animals.
Ultrastructurally, when contrasted with control parenchyma,
the cells of the nodules exhibited enlarged mitochondria, hypertrophied microbodies, prominent Golgi complexes, and abundant
dilated rough-surfaced endoplasmic reticulum with numerous
attached ribosomes. Nuclei were frequently invaginated and
glycogen was abundant. Lysosomes and autophagic vacuoles
were few in number.
The morphologic evidence suggests that the nodules represent
areas of hyperplasia rather than neoplasia, and this possibility
is discussed. Moreover, it would appear that the dilated roughsurfaced endoplasmic reticulum of the nodule cells reflects
metabolic activity rather than cellular injury.
Introduction
Carbon tetrachloride has been shown to produce tumor-like
nodules in the livers of several strains of mice (10, 11, 13-15).
Special attention has been paid to the C3H mouse which has a
1This investigation was supported in part by institutional
grants to the University of Cincinnati (IN-79 and IN-79A) from
the American Cancer Society and in part by a Public Health Serv
ice Research Grant (AM-08416) from the National Institute of
Arthritis and Metabolic Diseases and by a Public Health Service
Graduate Training Grant (2G-T1-GM-1122) and a Research Career
Program Award (1-K3-GM-22.575) from the Institute of General
Medical Sciences. Presented in part at the Sixty-second Annual
Meeting of the American Association of Pathologists and Bac
teriologists, Philadelphia, Pa., March 6, 1965.
2Present address: Department of Pathology, University of
Cincinnati College of Medicine, Cincinnati General Hospital,
Cincinnati, Ohio.
8Present address: Department of Pathology, Cleveland Metro
politan General Hospital, 3395Scranton Road, Cleveland, Ohio.
Received for publication August 23, 1965; revised November 18,
1965.
834
high incidence of both CCl4-induced (10, 11, 14) and spon
taneous nodules (2,7, 12). Whether the nodules induced by CCU
are truly neoplastic has been previously considered (11, 12), al
though they usually have been interpreted as hepatomas (10, 11,
14).
The purpose of the present experiment was to study the his
tologie and ultrastructural characteristics of such CCU-induced
nodules in the livers of C3H mice.
Material
and Methods
Twenty-five male C3H mice, from the Roscoe B. Jackson
Memorial Laboratory (Bar Harbor, Me.) were given twice
weekly rectal instillations of 0.1 ml of 40% solution of CC14dis
solved in olive oil. Ten control mice were given pure olive oil in
a similar manner. All animals were 5 weeks old at the beginning
of the experiment. After 40-52 administrations (20-26 weeks),
the experimental animals were divided into 2 groups; a group of
14 was sacrificed 9 days after their last treatment, while the re
maining 11 experimental animals were sacrificed at periods from
3 to 37 weeks after their last treatment. At least 9 days were al
lowed to elaspse between the last dose and the sacrifice of the
animals in order that the liver would have sufficient time to re
cover from the acute effects of CC14.
Portions of the nodules and of the adjacent parenchyma were
fixed, for light microscopy, in Zenker's solution or neutral forma
lin. Hematoxylin and eosin, periodic acid-Schiff, and reticulum
stains were done on this material.
For electron microscopy, slices of the nodules and of adjacent
parenchyma were trimmed into small blocks (1-2 cu mm) in 1%
Os04 buffered to pH 7.25-7.35 with phosphate (17). After being
trimmed the blocks were immediately transferred into larger
quantities of the fixative, and fixation was continued for 2 hr at
4°C.Subsequently, the tissue was dehydrated in graded acetones
and embedded in Araldite epoxy resin (26). Thin sections were
prepared with a Porter-Blum MT-1 microtome, stained with
lead hydroxide (18), and examined with an RCA EMU-Õ5For
-3G electron microscope.
Results
Of the 2 groups of experimental animals, the group of 14, sacri
ficed 9 days after their last dose of CC14, yielded 5 nodules in 5
livers. The group of 11, killed 3-37 weeks after their last dose,
yielded 11 nodules in 8 livers. The nodules from the 2 groups of
experimental animals showed identical gross, histologie, and
CANCER RESEARCH VOL. 26
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CCh-induced Hepatic Nodules
ultrastructural
gether.
features and, therefore, will be considered to
Gross Features
Grossly, the nodules (Fig. 1) were pale pink or white, and they
varied from 4 to 14 mm in diameter. The nodules were not en
capsulated but were sharply demarcated from the contiguous
parenchyma. There was no extension, either by direct invasion
into adjacent structures or by distant metastasis, in any of the
animals studied. Cirrhosis failed to develop in any of the experi
mental livers.
Light Microscopy
In hematoxylin and eosin-stained sections the nodules were
seen to consist of uniform parenchymal cells with relatively small
nuclei (Fig. 2). Although no organized lobular structure was
recognizable (Figs. 2^), scattered, well-formed bile ductules ap
peared within the substance of the nodules, as well as at their
periphery (Fig. 3).
The nodules abutted directly upon the adjacent parenchyma
(Fig. 2), and the contrast between the cells of the nodules and
those comprising the remainder of the parenchyma was striking,
especially in respect to nuclear features. Nuclei in the nodules
were small and uniform, with 1-2 nucleoli; those in the surround
ing parenchyma were large and irregular, and often contained
multiple nucleoli.
Interposed between groups of parenchymal cells were occa
sional regular, cylindrical blood vessels, resembling capillaries
(Fig. 4).
Electron Microscopy
CONTROLS.
The normal ultrastructure of the mouse liver has
been amply discussed elsewhere (29) ; the controls of the present
experiment differed in no respect from this. Parenchymal cell
nuclei (Fig. 5) were regular in shape, with evenly dispersed chromatin and 1-2 nucleoli, often partially applied to the nuclear
membrane (Fig. 5). Mitochondria were small or medium-sized
and dispersed at random through the cytoplasm. Several cristae
could be seen in each mitochondrion, usually near the periphery
of the organelle (Fig. 5). The rough-surfaced endoplasmic reticulum was rather sparse (Fig. 5) and cisternae were surrounded by
numerous apparently free ribosomes; the latter, however, might
represent attached ribosomes where the cisternae were tangentially cut. Microbodies were present as small, dense, singlemembrane-limited particles with denser, laminated or serpentine
nucleoids in their centers (Fig. 5). The Golgi complexes were
small, with many dilated vesicles (Fig. 5). Occasional lysosomes
and autophagic vacuoles were seen, most often near Golgi zones
(Fig. 5). Glycogen was plentiful and an occasional lipid droplet
was encountered (Fig. 5).
NODULES.Many nuclei of the nodule cells exhibited strikingly
irregular profiles (Figs. 6, 8) with tongues of cytoplasm filling
clefts formed by invaginations of the nuclear membrane. Such
invaginations, in cross sections, appeared as nuclear inclusions
filled with cytoplasmic components (Fig. 7).
The mitochondria in many cells were enlarged and contained
increased numbers of cristae which, on occasion, seemed to form
MAY 1966
a central sheaf (Figs. 9, 10). The rough-surfaced endoplasmic
retieulum in many instances was moderately dilated. The vesicles
and cisternae were usually filled with a fine, flocculent substance
(Figs. 9, 11) but rarely with lipid-like granules. Numerous ribo
somes were attached to the dilated cisternae (Figs. 9, 11). Polyribosomes were occasionally prominent (Figs. 9, 11).
One of the most unusual features was an enlargement of microbodies in many of the cells. In addition, microbody nucleoids
underwent marked elaboration and appeared as greatly enlarged,
laminated profiles or as a jumble of threadlike densities (Figs.
12, 13).
The Golgi zones were often quite increased in size and con
tained many layers of flattened sacs and vesicles, frequently filled
with electron-dense material (Figs. 13, 14). Glycogen was abun
dant (Figs. 9, 10, 13, 14, 15); lipid droplets appeared in small
numbers (Figs. 7, 9, 11-14). Both autophagic vacuoles and lyso
somes were infrequently seen.
Within the substance of the nodules occasional bile ductules
were encountered (Fig. 15). These were lined by cuboidal cells
with relatively large nuclei. The cell membranes of adjacent cells
interdigitated. Desmosomes were evident on the lumen side of
the cells at their junctions with one another (Fig. 15). The entire
ductule was separated from surrounding parenchymal cells and
adjacent sinusoids by an almost continuous, amorphous base
ment membrane (Fig. 15).
Now and then blood vessels intervened between adjacent
groups of hepatic cells (Fig. 16). The peri vascular space con
tained a basement membrane, interposed between the endothelium and the parenchyma. The endothelial cells were mod
erately thick. Occasional pseudopodia projected from their
external surfaces, and tight intercellular junctions were manifest
between neighboring cells. Such structures were thought to be
the ultrastructural counterparts of the capillary-like vascular
channels viewed by conventional microscopy (Fig. 4).
The ultrastructural features of the nodule cells also differed
from those of the adjacent parenchyma. The latter, however,
were quite extensive and will constitute the basis of a separate
report.
Discussion
The hepatic parenchymal nodules reported here, despite their
size and multiplicity, appear to represent foci of regenerative
hyperplasia rather than true neoplasia. There was no frank in
vasion of the adjacent parenchyma or metastasis beyond the
confines of the liver. Moreover, the cells of the nodules had histo
logie features which were less atypical than those of the con
tiguous parenchyma. This was especially true of nuclear char
acteristics. Nuclei of the nodule cells were small and uniform in
size with only 1-2 nucleoli, whereas many of those in the adjacent
parenchyma were large and hyperchromatic, with multiple
nucleoli. There was no consistent increase in mitotic activity in
the nodules.
These benign cytologie characteristics were also manifest at the
ultrastructural level. Thus, it has been reported that neoplastic
cells often show marked degrees of cytoplasmic dedifferentiation,
with a decrease in the size and number of mitochondria as well as
an increase in the amount of free ribosomes (5, 9, 20). These
features were coupled with a sparsity of other cytoplasmic comS35
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David B. Confer and Richard J. Stetiger
ponents (5, 9, 20). In contrast, the cells of the nodules reported
here contained many mitochondria (Figs. 9, 10, 13, 14) and ex
hibited hypertrophied Golgi complexes (Figs. 13 14), prominent
rough-surfaced endoplasmic reticulum (Figs. 9, 11, 14), and rich
glycogen stores (Figs. 9, 10, 13-15). The generalized increase of
interchromatin granules, reported in the nuclei of many neoplastic cells (6), was also lacking in the CCl4-induced nodules re
ported here. Finally, as has also been suggested in previous re
ports (23, 30), the numerous enlarged microbodies (Figs. 12, 13)
might constitute an added indication of regenerative activity.
The cytologie characteristics of the nodules reported here stand
in contrast to those of hepatomas studied by electron micros
copy. Thus, the ultrastructural features of hepatomas induced by
p-dimethylaminoazobenzene (1, 27), as well as those described
in several transplantable hepatomas of various origins (28),
differed significantly from the lesions described here. In a spon
taneous transplantable hepatoma from a substrain of C3H mice
(9), for example, the neoplastic cells exhibited small mito
chondria, many free ribosomes, and Golgi areas filled with empty
vesicles, whereas the nodules induced in the present experiment
showed increased numbers of large mitochondria with abundant
cristae (Figs. 9, 10), few free ribosomes (Figs. 9, 11), and Golgi
areas containing vesicles filled with an electron-dense material
(Figs. 13, 14).
CCU-induced hepatic nodules have often been interpreted as
hepatomas (10, 11, 14); however, such tumors have proved to be
notably resistant to transplantation (3, 11). Successful trans
plantation, reported in only one series of experiments, has been
accomplished with the line of C3H mice utilized by Andervont
(4). Such tumors appeared as cords of cells, 2 cells thick, flanked
on either side by sinusoids. Bile ductules were encountered, but
were most often at the periphery of the lesions (10, 11). The nod
ules in the present experiment were produced in the substrain of
C3H mice from the Roscoe B. Jackson Memorial Laboratory.
Andervont (2) has stated that these 2 substrains apparently had
significantly different characteristics, so that the results ob
tained in one could not be equated with results in the other. In
actuality, the nodules reported here were not characterized by
obvious cell cords (Figs. 2-4), and bile ductules were found with
regularity within their substance (Fig. 3). This position of the
ductules would seem to eliminate the possibility that they were
simply preexisting structures incorporated within an expanding
neoplasm.
Studies of acute CC14poisoning have shown that dilated roughsurfaced endoplasmic reticulum might reflect cell injury (22, 24,
25). In contrast, this did not seem to be the case with the dilated
endoplasmic reticulum of the nodules shown here (Fig. 11). This
dilation could not reasonably have been a direct toxic effect,
since it was present as long as 37 weeks after the last administra
tion of CCU. It also could not have been related to the structural
disarray of cirrhosis, since none of the livers were cirrhotic. More
over, the hyperplastic nodule cells exhibited none of the stigmata
of injury, such as increased numbers of autophagic vacuoles (8)
or dissociation of ribosomes from the endoplasmic reticulum (25).
Instead, the cells of the nodules exhibited areas replete with
glycogen (Figs. 9, 10, 13-15), many mitochondria with increased
numbers of cristae (Figs. 9, 10), and abundant cisternae of
rough-surfaced endoplasmic reticulum with numerous, closely
applied ribosomes (Figs. 9, 11, 14). In consideration of all these
points, it seems likely that the dilated rough-surfaced endo
plasmic reticulum reflected metabolic activity rather than cellu
lar injury, and might in this respect be analogous to the dilated
rough-surfaced endoplasmic reticulum of active fibroblasts (19,
21) or plasma cells (16).
Acknowledgments
The authors wish to express their thanks to Dr. Edward A. Gall
and to Dr. Winfield S. Morgan for their valuable support and
advice. We would also like to acknowledge the excellent technical
assistance of Miss Elizabeth A. Johnson and Mr. John N. William
son.
References
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FIGS. 1-16.—Sections for electron microscopy were stained with lead hydroxide.
FIG. 1.—Nodules in the liver of a CCU-treated C3H mouse. The liver contains discrete, sharply demarcated, white nodules.
X 2.7.
FIG. 2.—Junction between a nodule and the adjacent parenchyma. The cells of the nodule, on the upper right, are seen,
abutting directly upon those of the contiguous parenchyma. Note the difference in size and appearance of the nuclei in the 2 areas.
X 144.
FIG. 3.—Portion of a nodule. The cuboidal epithelium of the dilated bile ductules contrasts with the flattened epithelium of the
large blood vessels. X 240.
FIG. 4.—Portion of a nodule. A capillary-like blood vessel, cut in longitudinal section, is shown. X 240.
FIG. 5.—Control liver. This portion of a control liver cell exhibits many normal features including a nucleus (.V) with a nucleoli»
(A'«)partially applied to the nuclear membrane, many mitochondria (M), small microbodies (Mb), sparse rough-surfaced endoplasmic
reticulum (HER), areas replete with glycogen (Gly) and a few small lipid bodies (L). R, Free ribosome; G, Golgi zone; Ly, lysosorne.
X 24,400.
FIG. 0.—Portion of a nodule. The nucleus (Ar) presents a very irregular profile. A myelin figure (arrow) is seen in the cytoplasm.
X 16,800.
FIG. 7.—Portion of a nodule: The nucleus (A') contains a large inclusion filled primarily by lipid droplets (L). The double nuclear
membrane (arrows) can be seen surrounding the inclusion.
X 23,100.
FIG. 8.—Portion of a nodule. An irregular, horseshoe-shaped nucleus is shown here. X 23,500.
FIG. 9.—Portion of a nodule. The mitochondria (M) in this picture are enlarged and one shows a central sheaf of cristae. Small
segments of dilated rough-surfaced endoplasmic reticulum (RER) are also present.
X 24,400.
FIG. 10.—Portion of a nodule. Hypertrophied mitochondria (M) are shown with increased numbers of cristae.
X 28,600.
FIG. 11.—Portion of a nodule. Many dilated cisternae of rough-surfaced endoplasmic reticulum (RER), filled with a flocculent mate
rial, may be seen here. Note small ribosomes studding the membranes. L, Lipid. X 24,400.
FIG. 12.—Portion of a nodule. This picture contains 3 hypertrophied microbodies (Mb) with elaborate, dense nucleoids. L, Lipid.
X 44,700.
FIG. 13.—Portion of a nodule. Several large microbodies (Mb) can be seen in this picture. A prominent Golgi zone (G) is also shown.
X 23,500.
FIG. 14.—Portion of a nodule. An enlarged Golgi zone (G) fills a large portion of this picture. Note the electron-dense material in
many of the Golgi vesicles. L, Lipid. X 22,700.
FIG. 15.—Portion of a nodule containing a bile dilettile : The cuboidal cells of a bile duct ule (BD) are illustrated here. The cells may
be seen to interdigitale along their borders, and desmosomes (D) are prominent. A thin, amorphous basement membrane (arrows)
separates the bile ductule from adjacent cells. X 11,500.
FIG. 16.—Portion of a nodule. This micrograph shows a cross-sectioned blood vessel whose lining cells exhibit tight, intercellular
junctions (/), as well as an occasional pseudopodium (P). Notice the duplication of the basement membrane (arrows) in the perivascular space. X 23,500.
MAY I960
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David B. Confer and Richard J. Stenger
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VOL. 26
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VOL. 26
CClt-induced Hepatic Xcdules
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Nodules in the Livers of C3H Mice after Long-Term Carbon
Tetrachloride Administration: A Light and Electron Microscopic
Study
David B. Confer and Richard J. Stenger
Cancer Res 1966;26:834-843.
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