Download Histochemical Profile of Mouse Hepatocellular

[CANCER RESEARCH 51. 1952-1958. April 1. 1991)
Histochemical Profile of Mouse Hepatocellular Adenomas and Carcinomas Induced
by a Single Dose of Diethylnitrosamine1
Hans JörgHacker,2 Hussein Mtiro,1 Peter Bannasch, and Stan D. Vesselinovitch
Division of Cytopathology [H. J. //., H. M., P. B./, German Cancer Research Center, Im ,\euenheimer Feld 280, D-6900 Heidelberg, Federal Republic of Germany, and
Departments of Radiology and Pathology ¡S.D. V.], University of Chicago, The Pritzker School of Medicine, Chicago, Illinois
ABSTRACT
In continuation of earlier studies on murine neoplastic liver lesions, we
characterized by histochemical methods the phenotype of hepatocellular
adenomas and carcinomas induced by single injections of diethylnitrosamine (1.25, 2.5, or 5.0 /jg/g of body weight) in 15-day-old C57BL/6 x
male C3H FI mice.
The hepatocellular adenomas were composed predominantly of basophilic cells but stored excessive amounts of fat and glycogen in large
portions of the tumors. Irrespective of the carcinogenic dose, the adeno
mas showed a consistent histochemical pattern. Glycogen synthase and
phosphorylase »erehighly active in the hepatocytes that stored glycogen.
In cells poor in, or free of, this polysaccharide, these enzymes were only
moderately active or even inactive. In glycogen-storing parts of the
adenomas, the activity of adenylate cyclase was reduced compared with
normal liver parenchyma, but in fat-storing portions it was elevated. In a
few adenomas, uniform increase in adenylate cyclase activity could be
encountered. The levels of ATPase, acid phosphatase, and glucose-6phosphatase were either increased or decreased. Glucose-6-phosphate
dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase showed
an increased activity in all adenomas compared with preneoplastic foci,
which in turn exhibited a higher glucose-6-phosphate dehydrogenase and
glyceraldehyde-3-phosphate dehydrogenase activity than the surrounding
parenchyma or the liver of untreated controls.
The hepatocellular carcinomas showed remarkable histochemical
changes compared with adenomas. The levels of fat and glycogen and the
activities of glycogen synthase, phosphorylase, and in most cases also
that of glucose-6-phosphate dehydrogenase, were reduced significantly.
In contrast, adenylate cyclase, glucose-6-phosphatase, glyceraldehyde-3phosphate dehydrogenase, and also alkaline phosphatase showed a strik
ing elevation in developing carcinomas. Similar, although more pro
nounced, histochemical changes were seen in the advanced hepatocellular
carcinomas. These observations indicated that progression from adeno
mas to hepatocellular carcinomas was associated with a change in the
activity of several enzymes involved in cell membrane function, glycogen
metabolism, the oxidative pentose phosphate pathway, and glycolysis.
INTRODUCTION
Several studies (1,2) showed that low, single doses of DEN4
their number decreased concurrently with the emergence of the
hepatocellular adenomas. The hepatocellular carcinomas began
to develop either within existing adenomas or within the hep
atocellular parenchyma. Hepatocellular carcinomas grew rap
idly, involved surrounding tissue, and metastasized to lungs (1).
The temporal relationship between foci, adenomas, and carci
nomas clearly pointed to time-dependent tumor progression, a
phenomenon well established in several experimental systems
and observed in human carcinogenesis.
In a previous paper (3), the early focal lesions induced in
mice by a single dose of DEN were characterized histochemically. The main feature of these lesions was a decrease in
GóPase and an increase in both G6PDH and GAPDH, indi
cating a shift from the gluconeogenic situation predominating
in normal liver to a glucose consumption by pentose phosphate
pathway and glycolysis. The focal hepatic lesions also showed
increased labeling indices as well as an increase in their diam
eters and volumes with time. Thus, it was concluded that the
changes mentioned above acted in concert, manifesting the
acquired functional and replicating potential of focal lesions.
The present article reports on the histochemical characteristics
of the subsequently emerging hepatocellular adenomas and
carcinomas.
MATERIALS
AND METHODS
Tumors were induced in 15-day-old male C57BL/6 x C3H F, mice
by single injections of DEN (1.25, 2.5, or 5.0 ng/g of body weight) as
described earlier (4). The DEN-treated and nontreated control animals
were killed 44 and 68 weeks following the administration of the
carcinogen. The objective was to sample primarily hepatocellular ade
nomas and carcinomas for histochemical characterization.
Histology. Preneoplastic lesions in DEN-treated mouse livers were
classified as intermediate cell foci as defined earlier (3). Neoplastic
lesions were classified as hepatocellular adenomas and frank hepato
cellular carcinomas (1). Hepatocellular carcinomas that were observed
as emerging within the hepatocellular adenomas were referred to as
early carcinomas. No such distinction was observed in the "late"
administered to infant mice resulted in a sequential emergence
of preneoplastic hepatic foci, heptocellular adenomas, and hep
atocellular carcinomas. The frequency of developing lesions, as
well as the time of their emergence, were dose-dependent. The
preneoplastic foci increased progressively until their number
reached the maximal response to a given dose. Subsequently,
carcinomas.
Histochemistry of Enzymes and Metabolic Products. Pretreatment of
tissues and histochemical procedures used were identical to those in
the preceding study (3). Briefly, pieces from shock frozen liver tissue of
normal (one specimen) and carcinogen-treated (3 specimens, 1 for each
dose) mice were frozen onto the same tissue holder, and serial sections
of all 3 pieces (0.15 cnr each) were cut simultaneously in a Cryostat
(Jung, Nussloch, Federal Republic of Germany). The sections were
Received 7/6/90; accepted 1/17/91.
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the respective histochemical reaction. With this technique, it was pos
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
sible not only to produce sections of the same thickness but also to
' This work was supported in part by the National Cancer Institute Grant CAtreat them simultaneously under identical conditions for the specific
25522. H. Mtiro was a guest research scientist of the German Cancer Research
Center.
histochemical assays. The thickness of the sections was varied (6 to 14
: To whom requests for reprints should be addressed.
urn) in accordance with the reaction intensity of the enzyme to be
3 Present address: Tanzania Tumor Center. Dar-es-Salaam, Tanzania.
demonstrated. The activities of the following enzymes were demon
'The abbreviations used are: DEN. diethylnitrosamine; SYN. glycogen syn
thase, EC 2.4.1.11: PHO. glycogen phosphorylase. EC 2.4.1.1; GóPase. glucosestrated: SYN, PHO, GóPase, GAPDH, ATPase, 7GT, and ACPase.
6-phosphatase, EC 3.1.3.9; G6PDH. glucose-6-phosphate dehydrogenase. EC
In addition, 2 other enzymes, ADC and ALKPase, were studied in
1.1.149; GAPDH. glyceraldehyde-3-phosphate
dehydrogenase. EC 1.2.1.12;
preneoplastic and neoplastic lesions since they play a pivotal role in the
ATPase. adenosine triphosphatase. EC 3.6.3.1: ADC. adenylate cyclase, EC
regulation of cell metabolism (5) and transport processes (6, 7), respec
4.6.1.1; ALKPase, alkaline phosphatase. EC 3.1.3.1: iGT, -y-glutamyl transpeptively. Details of the histochemical assays for ADC are given by Mayer
tidase, EC 2.3.2.2; ACPase, acid phosphatase, EC 3.1.3.2.
1952
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HISTOCHEMICAL
Table 1 Number of preneoplastic
fociDEN(ng/g
PATTERN OF MOUSE LIVER TUMORS
and neoplastic
lesions studied histochemically
Intermediate
cell
at different stages of the experiment
hepatocellular
hepatocellular
hepatocellular
carcinomasWk4400013WkcarcinomasWk440004Wk68404All
carcinomasWk4400017Wk689817
wt)1.252.55.0Irrespective
body
685813Late
doseIrrespective
of
of timeAnimalsinvestigated6121230Wk4430407072Wk68022AdenomasWk4435165191Wk68202040Early
et al. (8) and those for ALKPase are given by Lojda et al. (9).
Furthermore, sections were stained for basophilia with toluidine blue
and for the presence of neutral lipids with Fettrot B. Others were
treated with either the periodic acid-Schiff reaction or Lugol's iodine
to demonstrate glycogen.
Evaluation of Histochemical Reactions. The histochemical reactions
were evaluated under a comparison microscope (Leitz, Wetzlar, Federal
Republic of Germany), which allowed exact correlation of the various
histochemical parameters with specified focal lesions in the serial
sections. The intensity of the histochemical reactions was estimated
semiquantitatively using 5 grades (no change, increase, strong increase,
decrease, strong decrease) as compared with the reaction in the concur
rently processed tissue of the untreated control animals.
RESULTS
At 44 weeks, a large number of hepatocellular adenomas were
observed in addition to the intermediate cell foci described
earlier. At 68 weeks, the samples studied contained both hepa
tocellular adenomas and carcinomas (Table 1).
The histological appearance and the histochemical pattern of
the intermediate cell foci that were observed at 44 weeks did
not differ from foci detected at earlier time points (3). The foci
were active in SYN and PHO. In comparison with normal liver
parenchyma, ADC was reduced, and ALKPase andiGT did not
show a change. GoPase and ACPase activities were decreased.
However, G6PDH and GAPDH were increased in comparison
with the surrounding liver tissue and the parenchyma of the
untreated animals.
The histochemical patterns of the adenomas, early carcino
mas, and the late carcinomas (fully developed carcinomas) are
summarized in Table 2. For comparison, the histochemical
patterns of intermediate cell foci are presented in Table 2,
Table 2 Increase, decrease, or no change in the level of investigated
histochemical markers in later stages of mouse hepatocarcinogenesis
lesionHistochemical
markersFat
Type of
carcinoma*
carcinoma'i
Column 2. Adenomas were predominantly composed of basophilic cells in addition to cells that stored excessive amounts of
glycogen (Fig. 2a) and fat (Fig. Ib), respectively. The basophilic
cells often formed a ring along the periphery of the adenomas.
This cell population frequently showed an increase in the num
ber of mitotic figures. SYN and PHO were highly active in the
cells storing glycogen in excess but were only moderately active
or even inactive in the basophilic cells poor in, or free of, this
polysaccharide (Fig. le). The ADC was generally reduced in
glycogen storage portions of adenomas (Fig. \d) but regularly
elevated in fat-storing areas. In some adenomas, ADC was
uniformly elevated despite a conspicuous glycogen deposition.
GoPase (Fig. le), ATPase, and ACPase were increased in some
but were decreased in other areas of adenomas. ALKPase was
increased around the capillaries but showed a normal or de
creased activity in the adenoma cells (Fig. I/). The 7GT could
not be detected in any of the hepatocellular adenomas. The
activity of G6PDH and GAPDH was strongly increased in all
adenomas (Fig. l, g and h), exceeding that observed in the
intermediate cell foci. With the exception of decreased SYN
and PHO, no significant differences in the histochemical pat
terns were found between the ring of basophilic cells and the
rest of adenomas.
The appearance of hepatocellular carcinomas was accom
panied by additional histochemical changes (Fig. 2, a-h); fat,
glycogen (Fig. 2a), as well as the activities of SYN, PHO (Fig.
2e), ATPase, and in many cases also that of G6PDH (Fig. 2/),
were strongly reduced in comparison with adenomas. In con
trast, ADC (Fig. 2c), ACPase (Fig. 2e\ and GAPDH (Fig. 2h)
showed an increase in their activities. GoPase (Fig. 2c) and
ALKPase (Fig. 2g) were more elevated in the early carcinomas
than in the surrounding adenomas. Similarly to the preceding
lesions, -yGT was lacking. The fully developed hepatocellular
carcinomas showed essentially the same histochemical pattern
as the early carcinomas, although most of the changes were
more manifest (Fig. 3, a-h).
Late
DISCUSSION
Glycogen
Tori
i
SYN
NCNC
tori
li
PHO
Toriî-'ftorjÎÎÎÎtori
ilÃŽ
ADCG
iiTTNCNC]Adenoma"î" 1ÎÎ1îli
PaseG6PDHGAPDHATPase
6
JÃŽÃŽ
ALKPaseTGTACPaseFociNCiNCNCToriEarly1lî}NC
NCî
î
" Compared with intermediate foci.
* Focal carcinomas observed within or outside of adenomas.
c Fully developed hepatocellular carcinomas.
d}. increase; J, decrease; NC. no change; |j, strong increase; ({. strong
decrease.
' In fat-storing portions and cells around veins, uniformly in new adenomas.
^In glycogen storage portions.
Sequential histochemical changes occurring during hepato
carcinogenesis from preneoplastic lesions to the hepatocellular
adenomas were studied extensively to date in rats (10, 11). In
mice, however, histochemical studies were of more limited
scope. The present study was directed to fill this void by
investigating a large number of enzymes, particularly those of
alternative pathways of carbohydrate metabolism.
The histochemical pattern of preneoplastic hepatic lesions
induced in the infant mouse model by DEN has been described
previously (3). In the present investigation, the spectrum of
enzymes studied in the early lesions was complemented by
demonstration of the activities of ADC and ALKPase, which
are involved in signal transduction and transport processes of
1953
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MIMIK
III MIC M
I'M
I I KN NI
MOI SI
I l\ I K II \1NKS
f
m •
Fig. 1. Serial sections through .1hepiitocellular adenoma. Remnant liver parenclnma is visible at the left margin of the micrographs. Bar. 100 firn. a. glycogen:
reduced deposition in large areas of the adenoma; A, neutral lipids: pronounced storage in some portions of the adenoma (niroir.v). Insci, higher magnification. Note
intensely stained lipid droplets in contrast to nuclei of the tumor cells, c. glycogen synthase: decreased activity in large areas of the adenoma. ¡I.adenylate cyclase:
increased en/yme activitv in the majority of adenoma cells, e. glucose 6-phosphatase: activity is decreased in some hut slightly increased in other parts of the adenoma.
/. alkaline phosphatase: weak activity in adenoma cells but strong in capillaries (arrows). Inset, higher magnification. Note, enzyme activity is confined to the cell
membrane of adenoma cells, g. glucose-6-phospliate dehydrogenase: strongly increased in adenoma. A, glyceraldehyde-3-phosphate dehydrogenase: strongly increased
in adenoma.
1954
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HISTOCHEMICAL
PATTERN OF MOUSE LIVER TUMORS
Fig. 2. Serial sections through an area showing transition from adenoma (upper, lower, and left margin) to carcinoma. Bar. 100 i/m. a, glycogen: gradient leading
from marked storage in the adenoma to almost complete reduction in the center of the carcinoma, h. glycogen phosphorylase: decrease activity in subpopulations of
the adenoma and in all cells of the carcinoma, r. adenylate cyclase: strongly increased enzyme activity in carcinoma in contrast to the surrounding adenoma tissue, d,
glucose-6-phosphatase: variable activity in adenoma but strong increase in the majority of carcinoma cells. £'.
acid phosphatase: activity of the enzyme is increased at
the transition /one between adenoma and carcinoma and in the central part of the carcinoma (righi upper corner), f. glucose-6-phosphate dehydrogenase: variable
activity in adenoma but decreased in carcinoma, g. alkaline phosphatase: low activity in adenoma but strong increase in carcinoma, h. glyceraldehyde-3-phosphate
dehydrogenase: increased activity in adenoma but even higher in carcinoma.
1955
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HISTOCHEMICAL
ff
,••
<».•/
PATTERN OF MOl'SE
LIVER Tl'MORS
a
*•*
-. - - *r
M .
—¿^C^Sf
:. -. .S-¿?i.&.
j*%v¡
">
'£j&\:Î
^¿.V-.
*•*•;:•
r^.
H
•••jPSWfiT
^?
îsln
'
^?
Fig. Ì.
Serial sections through a frank hepatoccllular carcinoma. Kur. 100 firn, a, glycogen: s(rongl\ reduced in majority of tumor cells, h. glycogcn phosphorylase:
decrease of enzyme activity throughout the tumor, r. adenylate cyclase: activity is found in plasma membrane as well as in cytoplasm of carcinoma cells, d. glucose-6phosphatase: all tumor cells show a strong increase in activity, e. acid phosphatase: strongly increased en/yme activity in all tumor cells. / glucose-6-phosphate
dehydrogenase: strongly increased activity in some tumor areas but decrease in most of the carcinomas cells. #. alkaline phosphatase: strong increase in activity
throughout the carcinoma, h. glyceraldehyde-3-phosphate dehydrogenase: en/yme activity is strongly increased.
1956
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HISTOCHEMICAl.
PATTERN OF MOUSE LIVER TUMORS
the plasma membrane. Whereas ADC activity was reduced, that
of ALKPase remained unchanged in preneoplastic lesions.
The adenomas shared a number of features with the preneo
plastic foci. Thus, in addition to the pronounced basophilia,
which has been known for some time, the adenomas were
particularly characterized by strongly increased activities of
G6PDH as detected in a number of tumor types, including
hepatocellular tumors of the rat, by biochemical (12) and histochemical methods (13). In contrast to early lesions, the mu
rine adenomas usually stored fat and especially glycogen in
excess in large tumor areas with SYN and PHO being active at
these sites. GoPase-activity, which was regularly reduced in
preneoplastic foci, was frequently increased in adenomas. It is
difficult to understand the significance of this histochemical
pattern since in addition to the anabolic and catabolic enzymes
of glycogen metabolism, key enzymes of the oxidative pentose
phosphate pathway (G6PDH) and glycolysis (GAPDH) were
strongly increased in their activities at the same time. In line
with earlier interpretations of findings in rat liver (10, 13), it
may be speculated that an increased intracellular level of the
central metabolite G6P is responsible for the simultaneous
activation of these alternative pathways of carbohydrate
metabolism.
Additional metabolic changes accompanied the transition
from adenomas to carcinomas. Thus, glycogen, SYN, and PHO
were reduced. Surprisingly, there was also a decrease in G6PDH
activity in most carcinomas. This is at variance with the pre
vious findings in rat liver (10) and does not support the concept
that the increase in G6PDH activity in preneoplastic lesions
merely reflects the proliferative activity of the respective tu
mors. Our studies were focused in the oxidative part of the
pentose phosphate pathway. There is, however, also the nonoxidative pathway, which provides precursors for ribonucleotide synthesis and glycolysis without production of NADPH.
Whether both parts are acting or either of them is preferentially
stimulated under normal conditions is dependent on the relative
requirements for NADPH and ribose-5-phosphate (14). Thus,
in the fast-growing hepatocellular carcinomas with low G6PDH
activity, most of the ribonucleotide precursors might be sup
plied by an elevated nonoxidative pentose phosphate pathway
and glycolysis as indicated by the increased GAPDH activity
observed in the present investigation. Another feature of devel
oping carcinoma cells was the striking increase in ALKPase,
an enzyme known to catalyze the hydrolysis of various phos
phate esters, in liver, especially proteins phosphorylated at
tyrosine residues ( 15). The elevation of ALKPase was correlated
with an increased ADC. The ability of cyclic AMP to induce
alkaline phosphatase has been described by Firestone and Heath
in mouse L-cells(lo). However, an isoenzyme shift of ALKPase
leading to a protein of higher catalytic activity cannot be pre
cluded (6, 7, 17).
The sequential cellular changes observed in the experimental
model used have been shown to be consistent with earlier studies
in the same infant mouse model (3), which is highly sensitive
to the hepatocarcinogen DEN (4). Following treatment of the
adult mice with the same carcinogen (DEN), even after its
repeated applications, the frequency of preneoplastic and neoplastic lesions was significantly lower (2). Tinctorial foci similar
to those observed in the infant mouse model were also induced
in rat livers by a single dose of aflatoxin B, (18) and were
referred to as "tigroid cell foci." However, although both types
of foci share similar morphology and lack of 7GT, they are
different with respect to other parameters. Thus, GoPase is
regularly reduced in mouse foci but frequently unchanged in rat
tigroid cell foci. On the other hand, 7GT-positive hepatocellular
foci were encountered after feeding mice safrole (19). Glycogen
storage foci being precursor lesions of hepatocellular carcino
mas in the rat can also be found in mouse liver after adminis
tration of ethylnitrosourea (20), DEN, and dieldrin (21). There
fore, several factors such as species, strain, age at treatment,
sex, experimental protocol, and the type of carcinogen may
influence the kinetics of emerging lesions and some aspects of
their phenotypic expression (22). Common characteristics,
however, predominate across the lesions.
The present article characterized for the first time the se
quential change in the histochemical pattern taking place from
small preneoplastic hepatocellular foci to adenomas and carci
nomas triggered by single carcinogenic treatment of infant mice.
In many respects, the observed histochemical changes during
hepatocarcinogenesis in the infant mouse model are comparable
with those seen during hepatocarcinogenesis in the rat. Despite
some conspicuous differences in the histochemical pattern that
are not fully understood as yet, a shift from glycogen metabo
lism towards the pentose phosphate pathway and particularly
to glycolysis seems to be a common denominator of hepatocar
cinogenesis. These metabolic changes are apparently closely
related to the neoplastic conversion of hepatocytes (10, 13).
ACKNOWLEDGMENTS
We thank Erika Filsinger for excellent histochemical work, Gabriele
Becker and Erwin Zang for printing the photomicrographs. Dr. D.
Mayer for critically reading the manuscript, and Brigitte Pétillonand
Myrtle Tuzar for typing the manuscript.
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1958
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Histochemical Profile of Mouse Hepatocellular Adenomas and
Carcinomas Induced by a Single Dose of Diethylnitrosamine
Hans Jörg Hacker, Hussein Mtiro, Peter Bannasch, et al.
Cancer Res 1991;51:1952-1958.
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