[CANCER
RESEARCH
27 Part 1, 2121-2233, Xnvemlwr
1067]
Comparative Studies of the Nucleoli of Morris Hepatomas,
Embryonic Liver, and Aflatoxin B,-treated Liver of Rats'
TADAO
UNUMA,
HAROLD
P. MORRIS- AND HARRIS BUSCH
Department of Pharmacology, Haijlor University, College of Medicine, Houston, Texas 17025
SUMMARY
The nucleoli of the adult and embryonic liver, Morris 9121,
8995, 7787, and R-7 hepatomas, and the liver after prolonged
aflatoxin BI treatment were compared with respect to size and
ultrastructure. Embryonic liver cells had enlarged nucleoli with
well-demarcated fibrillar and granular components of the nucle
oli. Cells of Morris Hepatoma 7787, 9121, R-7, and 8995 had
enlarged nucleoli. The demarcation of fibrillar and granular
components in the nucleolonemas was similar to that of normal
liver in the Morris Hepatoma 7787 and was progressively less in
Hepatomas 9121, R-7, and 8995.
The ratios of the area containing fibrillar components to the
total nucleolar area of normal liver, embryonic liver, and Morris
hepatomas were within the same range despite differences in the
sizes of the nucleoli.
The morphologic resemblance of the nucleoli of the 7787
hepatoma to that of the normal liver may be related to the
greater similarity of the base composition of rapidly labeled
nucleolar RNA of this tumor to that of normal livor than that
found for other tumors. In liver cells of aflatoxin Bi-treated
rats, the enlarged nucleoli resembled those of regenerating liver.
From these studies, it appears that the Morris hepatomas
contain a spectrum of variation of nucleolar morphology rang
ing from those of the rapidly growing transplantable tumors to
that of normal liver. These data support the view (3) that
studies on ultrastructure do not provide diagnostic criteria for
tumors, at least with the method of fixation and staining em
ployed in this study.
INTRODUCTION
Morphologic features specific to nucleoli of cancer cells have
not been found in spite of the many endeavors in the field of
electron microscopy (1, 3, 4, 37). However, biochemical analyses
of the nucleolar RXA have shown a number of common charac
teristics of newly synthesized nucleolar RXA in experimental
tumors (34, 35, 47) which differentiate the nucleolar RXA of
these tissues from that of the normal liver. There is evidence (5)
that the newly synthesized nucleolar RNA of some Morris
1These studies were supported in part by grants from the
National Science Foundation, the American Cancer Society, the
Jane Coffin Childs Fund, and the USPHS Grant CA 08182.
2 Head, Nutrition and Carcinogenesis Section, Laboratory of
Biochemistry, National Cancer Institute, Bethesda, Md.
Received March 15, 1967; accepted July 13, 19(17.
hepatomas has a base composition like that of other tumors.
This study on the ultrastructure of the nucleoli of the Morris
hepatomas was designed to determine whether the ribonucleoprotein elements of the nucleoli (15, 16, 20, 28, 29, 38, 44-46,
56) of these slowly growing, more differentiated tumors were
quantitatively or qualitatively different from those of normal
liver. Although many biochemical and morphologic studies made
on the Morris hepatomas (5, 9, 13, 30-33, 36, 39) revealed some
similarities to normal liver, detailed studies have not been
reported previously on the ultrastructure of the nucleoli. The
present study showed that there are similarities between the
nucleoli of some Morris hepatomas and adult and embryonic
liver (12), such that some nucleoli of Morris hepatomas could
not be differentiated from normal liver nucleoli on the basis of
ultrastructure. In the present study, comparisons were also
made of the enlarged nucleoli of liver obtained after prolonged
treatment with aflatoxin B! and the large nucleoli of tumor cells.
Aflatoxin R¡is known to have a powerful hepatocarcinogenic
effect on the liver and its biochemical effects (7, 10, 11, 14, 22,
41-43, 48, 60) and morphologic effects (2, 6, 19, 55) have been
studied extensively. Although aflatoxin BI produced marked
increases in nucleolar size after prolonged treatment, little
change was observed in the ultrastructure of the hypertrophie
nucleoli produced after prolonged periods of administration of
this carcinogen.
MATERIALS AND METHODS
The Morris hepatomas used in this study were as follows:
Hepatoma 9121, 10th generation, 81 days after inoculation to
male ACI rats; Hepatoma 8995, llth generation, 70 days after
transplantation to male Buffalo rats; Hepatoma 7787, 8th
generation, 191 days after inoculation to male Buffalo rats;
Hepatoma R-7, 6th generation, 130 days after transplantation
to male ACI rats. .Samples were obtained from several parts of
the tumor tissues of each of two rats from areas which did not
exhibit apparent necrosis. Tissues of the control adult rat liver
were obtained from three male albino rats, weighing 175-190
gm, fed ad libitum on Purina laboratory chow (protein content
23%. Three rat embryos, 18 to 20 days old, were used for the
study on the embryonic rat liver. Both adult and embryonic
rats were obtained from Cheek-Jones Company (Houston,
Texas). The effects of aflatoxin BI (generously supplied by Dr.
G. N. Wogan, Massachusetts Institute of Technology, Cam
bridge) were studied after intraperitoneal administration of the
drug to the 24 male albino rats in doses of 750 /ig/kg, body
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2221
Tadao Unuma, Harold P. Morris, and Harris Busch
weight, for |x;riods of cither 30 minutes or 60 minutes or in daily
doses of 750 MK/kg,body weight, for 1, 5, 15, and 30 days.
For electron microscopic studies, the tissues were fixed in 3%
glutaraldehyde in 0.1 M phosphate buffer at pH 7.4 for 2 hours
at 4°C;postfixation was carried out in 2% osmium tetroxide
buffered with 0.14 M Veronal acetate at pH 7.4 for 1 hour. The
fixed tissues were dehydrated with graded concentrations of
ethanol containing uranyl acetate (46). They were embedded in
Epon-Araldite and stained with 2.5/¿ uranyl acetate in 50%
ethanol for 20 minutes and with 0.2% lead citrate for 2 minutes.
Thin sections were obtained with an LKB Ultrotome and studied
with a Philips EM 200 electron microscope.
For measurement of the size of the nucleoli, relatively large
blocks, 1.0-1.5 cu mm, of the tissues which were fixed and em
bedded for electron microscopy were cut in sections about 2-4
microns thick. The sections were stained with 0.1% toluidine
blue at pH 11.1 for 1 hour (57) and examined with a light micro
scope. To determine the number of the very small nucleoli in
normal liver, light microscopy was employed for studies on
isolated nuclei stained with Azure C. To observe all the nucleoli
in these nuclei, which range from 1 to 7 in number, it was neces
sary to focus from the bottom to the top of the nuclei. The
number of nucleoli per nucleus, the mean total nucleolar volume
per nucleus (cu ¿i)and the ratio of total nucleolar volume per
nucleus to nuclear volume were determined on the printed
pictures with a magnification of X 2000, twice the original
magnification. The volumes of round nucleoli and nuclei were
measured from a diameter by the formula for the sphere. The
volumes of elliptic nucleoli and nuclei were determined from the
largest and the smallest diameter by the formula of the ellipsoid
with the largest diameter as an axis of rotation (50). Ninety to
500 nuclei were selected at random on the sections obtained from
the three different blocks.3
The ratio of the area containing fibrillar components of the
nucleolus to the total nucleolar area (46) was determined on
electron micrographs ( X 30,000-40,000) with a planimeter (OTT,
Burrel Corporation, Pennsylvania).
The area containing fibrillar components of the nucleolus is
defined as an area in which the density is greater than that of
the intranucleolar light areas, and it contains fibrils with an
average width of 50 A but no defined granular components which
are about 150 A in diameter. RNA is present both in fibrillar
components and in granular components (28, 29, 58). In some
3The measured mean value of the diameters of the nucleoli
may be underestimated
depending on the localization of the
nucleoli within the sections. If the thickness of the sections is 2
to 4 it and measured mean values of the diameters of the nucleoli
are 0.5 to 1 p, the errors of the determination of the diameter of the
nucleoli become 4 to 10% of the obtained values according to the
following formula:
dr
(d, thickness of a section, r, real mean value of the radii of the
nucleoli). Then
_
= 2m - d ±\/(d
- 2m)1
where m is the measured mean of the radii of the nucleoli.
2222
areas in the nucleoli, in tumor cells, the fibrillar components were
not clearly demarcated from the granular components. These
fibrillogranular areas (46), although included in the granular
area, were not included in the area containing fibrillar com
ponents in the present determination.
RESULTS
Electron
Microscopy
of Nucleoli
and Nuclei
Adult Liver. The nucleoli of adult rat liver cells were small by
comparison with the nucleoli of the other tumors studied (Fig. 1).
Their nucleolonemas were well separated by relatively large light
spaces. In the nucleolonemas, areas containing only fibrillar
components were distinguished from areas containing granular
components (Fig. 2). Chromatin was distributed around the
nucleolus, along the nuclear membrane, and in a few clumps in
the nucleoplasm (Fig. 1). Interehromatinic granules and perichromatinic granules were also observed (Fig. 1).
Embryonic Liver. The nucleoli of embryonic rat liver cells
differed from those of the normal adult rat liver cells in size and
compactness. The embryonic nucleoli were large and contained
the granular components and fibrillar components which ap
peared to be clearly demarcated from the granular components
(Figs. 3, 4). On the other hand, the granular components were
distributed relatively diffusely in the nucleolus (Fig. 4). Chromatin was located along the nuclear membrane and around the
nucleolus.
Morris 9121, 8995, 7787, and R-7 Ilepatomas. As noted
previously for other transplanted tumors (46), the nucleoli of
these Morris hepatomas were large but the nucleolonemas were
not well defined (Figs. 5-12), except in the Morris 7787 hepatoma (Figs. 5, 6). In this tumor, the nucleolonemas were sepa
rated by light spaces as in the normal liver. The granular and
fibrillar components of the nucleolus were clearly demarcated
from one another in this tumor (Figs. 5, 6), but in the others the
degree of demarcation of the regions diminished in progressive
order in the hepatoma series 9121 (Figs. 7, 8), R-7 (Figs. 9, 10),
and 8995 (Figs. 11, 12) that were studied. Some fibrillogranular
areas were seen in these nucleoli (Figs. 8, 12). In the Hepatoma
7787, the fibrillar areas of the nucleoli were located mainly around
the intranucleolar light area (Fig. 6). The nucleoli of this series of
tumors were compact, and only small intranucleolar light areas
(Figs. 8, 10, 12) were found. Chromatin was distributed irregu
larly in the nucleoplasm, mainly around the nuclear membrane
and the nucleolus. Interehromatinic granules and perichromatinic
granules were also noted (Figs. 5, 7, 9, 11).
Aflatoxin B,-treated Liver. Compact nucleoli with clearly
segregated fibrillar and granular components were obtained 30 to
60 minutes after intraperitoneal administration of aflatoxin BI
in doses of 750 Mg/kg body weight. These nucleoli were often
associated with dark caps (Fig. 14) referred to as "substance
contrastée"by Bernard et al. (2). On Day 1 and Day 5 of con
tinued administration of aflatoxin BI , compactness and segrega
tion of the components of the nucleoli were observed. The dark
cap was not found in the nucleoli at this stage. Large nucleoli
were present after treatment with the drug daily for 30 days in a
dose of 750 Mg/kg body weight (Fig. 13). In these enlarged
nucleoli, the nucleolonemas were loosely arranged and there was
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Nucleolar
Ultrastructure
of Rat Liver and Hepatomas
TABLE l
Size of Nucleoli
TissueAdult
of
of nucleoli
nucleoli
nucleus2.7
per
examined5001059392969291Number
(1-7)«1.3
(1-3)1.1
(1-3)1.2
(1-3)1.0
(1-2)1.3
(1-2)1.5
(1-3)Mean
liverEmbryonic
liverMorris
9121Morris
8995Morris
7787Morris
R-7Aflatoxin
1i¡30 daysNumber
total nucleolar
volume per nucleus
<CU,i)3.9
of total
nucleolar volume |ter
nucleus to
volume0.028
nuclear
1.4*6.0
±
0.010*0.053
±
3.48.4
±
0.0220.067
±
5.310.5
±
0.0080.057
±
5.813.9
±
0.0210.057
±
7.17.4
±
0.0200.041
±
4.35.5
±
0.0140.037
±
±1.6Ratio
± 0.017
0 Range of the number of nucleoli per nucleus.
6 Standard deviation.
TABLE 2
Ratio of the Area Containing fibrillar Components to the Total
Nucleolar Area
Ratio
[15]"Morris
Embryonic liver
[201Morris
9121
[24]Morris
8995
[19]Morris
7787
[23[Aflatoxin
R-7
[16]Novikoff
B,—30 days
[21]Adult hepatoma
[10]Walker
liver«
tumor«[15]19.7
3*20.5± 3.
4.615.5
±
4.417.1
±
4.918.3
±
2.429.2±
7.013.4
±
3.019
±
43 ±
±2
" Number of nucleoli studied is in brackets.
6 Standard deviation.
c These values were obtained by Smetana et al. (46).
a clear demarcation
of the granular components from fibrillar
components. The overall appearance of these nucleoli was more
similar to that of adult or embryonic liver nucleoli rather than
that of the nucleoli of Morris hepatomas, despite the large size
of the nucleoli of livens of aflatoxin-treated
rats.
Size of Nucleoli
Table 1 presents the ratios of total nucleolar volume per
nucleus to the nuclear volume. In embryonic liver cells, this ratio
was significantly higher than that of adult liver cells (P < 0.05).
These ratios in the Morris 9121 (P < 0.01), 8995 (P < 0.05), 7787
(P < 0.05) hepatoma cells were also greater than that of the adult
liver cells. However, the ratios of the R-7 hepatoma and the
aflatoxin Bi-treated liver were not statistically different from that
of adult liver. The ratios for embryonic rat liver and Morris
hepatomas were not significantly different. The mean total
nucleolar volume per nucleus varied in the series of tumors
studied. There was no significant difference in the values for adult
liver, embryonic liver, Morris hepatomas, and aflatoxin Hr
treated liver; the exception was the 7787 hepatoma, in which the
mean total nucleolar volume was statistically greater (P < 0.05)
than that of normal liver.
The Ratio of the Area Containing
to the Total Nucleolar Area
Fibrillar
Components
As shown in Table 2, the fibrillar components of the embryonic
liver nucleoli occupied 20% of the total nucleolar area, which
was almost the same as the ratio in the adult liver nucleoli; the
great difference in the sizes of the nucleoli did not influence these
ratios. The ratios of fibrillar to total nucleolar areas of Morris
hepatoma nucleoli were also within the same range as that of the
normal liver nucleoli. The ratio of the fibrillar components to
the total nucleolar area of the nucleoli of Walker 250 carcinosarcoma (46) was strikingly less than those of the Morris hepa
tomas which are much more differentiated and slowly growing
tumors. The ratio of these components of Novikoff hepatoma
nucleoli was significantly lower than that of embryonic liver
nucleoli (P < 0.05) but was not statistically different from those
of the Morris hepatomas. The fibrillar components of the nucleoli
of rat liver cells increased significantly (P < 0.05) after continual
administration
of aflatoxin BI .
Electron Microscopy of the Cytoplasm
The endoplasmic reticulum in 18- to 20-day-old embryonic rat
liver cells was poorly developed. The rough -surfaced endoplas
mic reticulum was found almost exclusively around mitochondria
(Fig. 15) and the nuclear envelope, from which it extended into
the cytoplasm. By comparison with normal liver, the roughsurfaced endoplasmic reticulum was decreased in the series of
Morris hepatomas and was also found mainly around the mito
chondria
(Figs. 16-19). The smooth-surfaced
endoplasmic
reticulum was vesicular in these tumors and was found in large
amounts in the Morris hepatomas (Fig. 16). A few clusters of
ribosomes were found, presumably as parts of polysomes. The
mitochondria in the embryonic rat liver were large and numerous
cristae were present (Fig. 15). The mitochondria of the Morris
hepatomas were not markedly different from tumor to tumor and
from that of adult liver cells (Figs. 16-19). Long and slender
mitochondria were found in Morris 8995 hepatoma cells (Fig.
19). Glycogen-containing
areas were present in large amounts in
embryonic liver cells (Fig. 15), but not in Morris hepatoma cells
(Figs. 16-19).
DISCUSSION
The common and predominant finding of these series of Morris
hepatomas was enlargement of the nucleoli, as previously ob-
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2223
Tartan Unuma, Harold P. Morris, and Harris Busch
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tumors (24-27, 59, 61). Enlargement of nucleoli is not a diag
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aflatoxin BI , but not in Walker tumor (46). In the Morris
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the series Hepatoma 7787, 9121, R-7, and 8995. Distortion of the
nucleolar structures was also observed in the other tumors, i.e.,
fibrosarcoma of rats (3) and in Hodgkins cells (23).
The base compositions of the rapidly labeled nucleolar RXA
of Morris hepatomas (5) showed that content of adenylic acid
was 13-15 percent of the total bases by comparison with 19 per
cent in normal livei and 12 percent in the Novikoff hepatoma
(58). In Hepatoma 7787, which had the best organized nucleonemas which were separated by light areas like those of normal
liver, the content of adenylic acid in the rapidly labeled nucleolar
RNA was the highest of the tumors studied, i.e., 15.7 percent.
The ratios of the areas containing fibrillar components to the
total nucleolar area were very similar in the Morris hepatomas
to those of normal liver, and were markedly different from the
ratios for Walker tumor (46). The corresponding ratio for the
Novikoff hepatoma was intermediate (58). No significant correla
tions were obtained between the ratios of the area containing
fibrillar components to the total nucleolar area and the values of
the base compositions of the rapidly labeled nucleolar RNA of the
series of Morris hepatomas. These data support the view (3) that
studies on nucleolar ultrastructure probably will not provide
diagnostic criteria for tumors.
Although aflatoxin produces segregation of the components of
the nucleolus and appearance of substance contrastée
(2) as an
acute effect, no significant nucleolar abnormalities, except
nucleolar enlargement, were observed in chronic treatment with
1 ppm to 2 ppm of aflatoxin HI in a synthetic diet, prior to
appearance of tumors after up to 52 weeks (55).
In the present studies, acute treatment with this hepatocarcinogen produced marked changes in the nucleolar structures,
but little change except for nucleolar enlargement was found
after continued treatment with the same dose of the drug.
2224
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50. Stenram, U. Nucleolar Size in the Liver of Rats Fed on High
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51. Stenram, U. Nucleolar Size in the Liver of Rats Fed Diets
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On High and Non Protein Diet. Acta Pathol. Microbiol.
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Scand., 44: 239-246, 1958.
36. Novikoff, A. B., and Biempica, L. Cytochemical and Electron
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and Ultrastruc
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Kennzeichen der Krebszel
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len. Acta Med. Scand., 80: 78-92, 1933.
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NOVEMBER 1967
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FIGS. 1-19. The marker lines in the figures denote one micron. /, fibrillar components of the nucléolos;
g, granular components of the
nucleolus; fg, fibrillogranular area; c, nucleolus-associated chromatin; I, intranucleolar light area; i, interchromatinic granules; p,
perichromatinic granules; gl, glycogen granules. Specimens were fixed with glutaraldehyde and osmium tetroxide, embedded in Epon
Araldite, and stained with uranyl acetate and lead citrate.
FIG. 1. Nucleus of an adult rat liver cell. X 20,000.
FIG. 2. Nucleolus of an adult rat liver cell. The nucleolonemas are well separated by relatively large light areas (I). X45,000.
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1967 American Association for Cancer Research.
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FIG. 3. Nucleus of an embryonic rat liver cell. X 20,000.
FIG.
embryonic rat liver
liver cell.
and contain granular components and fibrillar
FIG. 4. Nucleolus
Nucleolus of an embryonic
cell. The
The embryonic
emlii^um*; nucleoli
»iu,ic»n.are
no large
i.
components which are clearly demarcated from granular components.
X 36,000.
NOVEMBER
1967
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1967 American Association for Cancer Research.
2227
Tadao Unuma, Harold P. Morris, and Harris Busch
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FIG. 5. Nucleus of a Morris 7787 hepatoma cell. X 15,000.
FIG. 6. Nucleolus of a Morris 7787 hepatoma cell. The nucleolonemas
2228
are separated
by light areas (1).
X 30,000.
CANCER
RESEARCH
VOL. 27
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FIG. 7. Nucleus of a Morris 9121 hepatoma cell. X 25,000.
FIG. 8. Nucleolus of a Morris 9121 hepatoma celi. The nucleoli are large, and their nucleolonemas
components are not clearly demarcated from the granular components.
X30.000.
NOVEMBER
are not well separated.
The fibrillar
1967
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1967 American Association for Cancer Research.
2229
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FIG. 9. Nucleus of a. Morris R-7 hepatoma cell.X 16,030.
FIG. 10. Nucleolus of a Morris R-7 hepatoma coll. The nucleolonemas
'
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_
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CANCER
RESEARCH
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1967 American Association for Cancer Research.
VOL. 27
Nudcolar Ultrastrudurc of Rat Liver and Hepatomas
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FIG. 11. Nucleus of a Morris 8995 hepatoma cell. X 18,000.
FIG. 12. Nucleolus of a Morris 8995 hepatoma cell. The nucleoli are large and contain few intranucleolar
NOVEMBER
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FIG. 13. Xucleolus of a liver cell from a rat treated with aflatoxin Bi in daily doses of 750 Mg/kg for 30 days. The nucleoli are large
and t hoir micleoloiic-mas are well defined. X 32,000.
FIG. 14. Nucleolns of a rat liver cell treated with aflatoxiu Bi in doses of 750 /¿g/kgfor 30 minutes. The nucleoli are compact and
their components are segregated. Dark caps (</) are associated with the nucleoli. X 37,000.
FIG. 15. Cytoplasm of an embryonic rat liver cell. Endoplasmic reticulnm is poorly developed, except around mitochondria and
around nuclear membrane. Abundant deposit of glycogen granules (gl) is found in the cytoplasm.
X 24,000.
CANCER RESEARCH VOL. 27
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1967 American Association for Cancer Research.
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FIG.
FIG.
16. Cytoplasm of a 7787 hepatoma cell. Abundant smooth-surfaced endoplasmic reticulum
17. Cytoplasm of a 9121 hepatoma cell. X 20,000.
18 Cytoplasm of an H-7 hepatoma cell. X 20,000.
19. Cytoplasm of an 8995 hepatoma cell. Long and slender mitochondria are conspicuous.
is in the cytopl (asm.
I
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X 17,000.
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1967 American Association for Cancer Research.
Comparative Studies of the Nucleoli of Morris Hepatomas,
Embryonic Liver, and Aflatoxin B 1-treated Liver of Rats
Tadao Unuma, Harold P. Morris and Harris Busch
Cancer Res 1967;27:2221-2233.
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