The Numerical Proportions of Cell Types in Rat Liver during
Carcinogenesis by 4-Dimethylaminoazobenzene (DAB)*
R.
D A O U S T t AND A . C A N T E R O
(Research Laboratories, Montreal Cancer Institute, Notre Dame Hospital and University of
Montreal, Montreal, Canada)
For differential counts of cell types in the tissue sections,
a reticle delimiting a square field was placed in one of the
oculars of a binocular microscope. The field was placed at
random on a liver section, and all nuclei included in the field
were examined under oil immersion. Differential counts of
the following cell types were made: parenchymal cells,~littoral
cells, s bile duct cells, cells of connective tissue, and cells of
the blood vessel walls. Once all the nuclei in a field had been
examined and classified, the counts were pursued in similar
fields taken at random on different parts of the liver sections
until at least 6,000 nuclei had been examined per animal.
The results of these counts represent the apparent distribution
of the cell types. Such data must be corrected for the relative
nuclear sizes, since bigger nuclei have greater chances to be
included in any section (1, 4).
To correct the results of the differential counts, the mean
nuclear length of each cell type was calculated from measurements made by ocular micrometry on 200 parenchymal nuclei
and 50 nuclei of each other type per animal. 4The measurements
were made under oil immersion by a microscope which had
been adjusted so that one division of the eye-piece micrometer
corresponded to 1 micron on the stage micrometer. The objective
was placed at random on a liver section, and the latter was
displaced perpendicularly to the micrometer scale. The first 25
nuclei of each type, the centers of which fell within the length
of the scale (50 #), were measured. All measurements were
T h e l i v e r is a n o r g a n w h i c h is f r e q u e n t l y u t i l i z e d
for b i o c h e m i c a l i n v e s t i g a t i o n s b e c a u s e of its l a r g e
v o l u m e a n d its a p p a r e n t h o m o g e n e i t y . A c t u a l l y ,
t h e l i v e r is n o t a h o m o g e n e o u s o r g a n , since i t
c o n t a i n s , in a d d i t i o n to t h e h e p a t i c cells, a n app r e c i a b l e n u m b e r of o t h e r cells t h e p r e s e n c e of
w h i c h m u s t be t a k e n i n t o c o n s i d e r a t i o n w h e n
interpreting the biochemical data. Moreover, the
h i s t o l o g i c a l a r c h i t e c t u r e of t h i s o r g a n is k n o w n
t o be m a r k e d l y a l t e r e d in d i f f e r e n t p a t h o l o g i c a l
conditions. During carcinogenesis by 4-dimethyla m i n o a z o b e n z e n e ( D A B ) , for i n s t a n c e , i m p o r t a n t
c h a n g e s are o b s e r v e d in t h e r e l a t i v e p r o p o r t i o n s
of t h e cell t y p e s as well as in t h e n a t u r e a n d t h e
v o l u m e of t h e e x t r a c e l l u l a r spaces (11, 15-17).
T h e l i v e r b e c o m e s h i g h l y h e t e r o g e n e o u s in s u c h
a condition. I n order to evaluate q u a n t i t a t i v e l y
t h e c h a n g e s t a k i n g place in t h e cellular p o p u l a t i o n
of t h e l i v e r d u r i n g carcinogenesis, we h a v e u n d e r t a k e n to d e t e r m i n e t h e n u m e r i c a l p r o p o r t i o n s of
t h e v a r i o u s cell t y p e s p r e s e n t in t h e l i v e r of n o r m a l
r a t s a n d r a t s fed D A B for d i f f e r e n t t i m e i n t e r v a l s .
MATERIALS
AND METHODS
Male, adult, albino rats (Wistar strain, 175-225 gm.) fed
Purina Fox Chow and water ad libitum were separated into
three groups. The animals of the first group were continued
on Purina Fox Chow and water. Those in the second group
were transferred to a semi-synthetic, low-protein diet (basal
diet, Ref. 14, diet 8). Animals in the third group were transferred to the semi-synthetic diet containing the azo-dye DAB
at a concentration of 0.06 per cent. Normal rats were sacrificed
in groups of six at 60-day intervals during a 180-day period.
Rats fed the basal or DAB-containing diets were sacrificed
in groups of six at $0-day intervals during the same period. 1
Under ether anesthesia, the animals were exsanguinated from
the abdominal aorta, livers were excised with scissors, weighed,
and fixed in Susa fluid. Histological sections of the median
and lateral left lobes (5 # thick) were prepared by the usual
procedure and stained with eosin and hematoxylin.
* This investigation was supported by the National Cancer
Institute of Canada. Preliminary reports on the present work
have been published (7, 8).
t Research Associate of the National Cancer Institute
of Canada.
Received for publication March 23, 1959.
1 The animals were given injections subcutaneously with
0.1 ml/100 gm body weight of a 0.1 per cent solution of colchicine, 4 hours before sacrifice, for measuring the incidences
of mitoses in the same sections as those used in the present
study. Colchicine injection had no effect on the cytological
composition of the liver, however, since differential counts
of cell types in intact and colchicine-injected animals showed
no appreciable difference between the two groups.
2 The term parenchymal cells instead of parenchymal nuclei
is used throughout the present article, but these terms are
not actually equivalent, since many parenchymal cells are
binucleated (3, 5, 20, 24).
3 The term littoral cell used here after Abercrombie and
Harkness (2) refers to the cells lining the blood slnusoids. They
include the Kupffer cells and the so-called undifferentiated
cells which cannot be easily distinguished one from the other.
4 The measurements on parenchymal nuclei and those on
nuclei of the other types were actually made in two different
series. The mean diameters of the parenchymal nuclei were
computed from a series of measurements on 200 nuclei per
animal carried out for studying the distribution of nuclear
sizes in liver parenchyma during DAB carcinogenesis (unpublished results). The nuclear lengths of the four other cell
types, i.e., means of 50 nuclei of each type per animal, were
determined in a second series of measurements.
757
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Cancer Research
758
made in the center of the field after the initial position of the
nuclei on the micrometer had been noted. For each nucleus,
the lengths of the long and short axes were determined (evaluated to 89
micron), and the nuclear length was taken as the mean
of the two values. When $5 nuclei of each type had been
measured, the slide was displaced at random, and the measurements were pursued in another part of the section. Hence,
the measurements on parenchymal nuclei were made in eight
different portions of the sections for a total of $00 nuclei
per animal, while those on the nuclei of the other types
were carried out in two different portions for a total of 50
nuclei of each type. Mter the mean nuclear length of each
cell type had been calculated, the data on the apparent distribution of cell types were corrected according to Abercrombie
(1), and the true distribution values were obtained. The results
were finally expressed as the per cent distributions of cell types.
RESULTS
Normal rat liver.--The per cent distributions
of cell types in normal rat livers are shown in
Table 1. In 200-gm. rats (0 days), the parenchymal
Vol. 19, A u g u s t , 1959
The values obtained for these two groups were
intermediate between those obtained at 0 and
180 days. This confirms t h a t a quantitative change
takes place in the cellular composition of the adult
r a t liver as the organ increases in weight.
Liver of rats fed the basal dict.--In rats fed
the basal diet (Table ~), the body weights and
liver weights were observed to decrease between
0 and 60 days and to increase afterward. These
changes were accompanied by an initial (statistically significant) decrease in the percentage of
littoral cells followed b y a slight increase after
the 90-day interval. The percentages of parenchymal cells and other cell types showed reciprocal
variations. Feeding the basal diet thus altered
the cytological composition of the liver in a direction opposite to t h a t observed in animals fed
the normal diet. At the 60-, 1~0-, and 180-day
TABLE 1
NUMERICAL PROPORTIONS OF CELL TYPES IN NORMAL ADULT RAT LIVER
PER CENT DISTRIBUTION OF CELL TYPES ( •
TIME
(days)
O*
60
180
BODY WEIGHT
(gm.+S.D.)
?.0O+6
356 + 14
380 + 35
445 • 55
LIVER WEIGHT
(gin. • S.D.)
6.7+0.7
11.4+1.6
10.0+1.4
15.7_+$.4
Parenchymal
cells
Littoral
cells
Cells of
bile ducts
Cells of
conn. tissue
60.7+1.7
56.1+1.5
58.7+!$.$
55.7_+~.4
30.$+~.$
35.6_+1.$
34.3+1.9
37.7+$.1
4.1+0.9
3.5+0.9
3.5+1.0
2.6+1.0
~.$__0.4
!~.9_+0.6
1.9-4-0.4
1.7_+0.3
Cells of blood
vessel walls
2.8_1.0
1.9_+0.5
1.6_+0.4
$.3_0.6
* Time at which the animals were separated into three groups for studying the effect of (a) normal diet, (b) the
basal diet, and (c) the carcinogenic diet.
cells composed 61 per cent of the total number
of cells and the littoral cells 30 per cent, the
remaining 9 per cent being accounted for by the
bile duct cells, the connective tissue cells, and the
cells of the blood vessel walls. These proportions
were observed to v a r y with age in the normal
animals. The percentages of littoral cells were
statistically higher in the rats sacrificed at later
time intervals and the percentages of parenchymal
cells and other cell types correspondingly lower.
In the last group of normal rats investigated
(180 days, 442 gm. body weight on the average),
the parenchymal cells were found to make up
56 per cent of the total number of liver cells, the
littoral cells 88 per cent, and the other cell types
6 per cent.
I t m a y be noted that, although the rats of the
l~0-day group had a higher mean body weight
than those of the 60-day group, the mean liver
weight in t h a t group (for some unknown reason)
was lower than in the latter group, and the percentage of littoral cells was accordingly lower.
intervals, times at which both normal rats and
rats fed the basal diet were investigated, the
percentages of parenchymal cells were all statistically higher and the littoral cell values statistically
lower in rats fed the basal diet than in the normal
animals. By the end of the experiment (180 days),
the values in rats fed the basal diet were close
to those obtained at 0 day, b u t much different
from the results obtained in the rats fed the normal
diet for a corresponding time interval.
Liver of rats fed the carcinogenic dict.--The rats
fed the DAB-containing diet (Table 3) showed
a progressive decrease in body weight and liver
weight up to 60 days, as was observed in rats
fed the basal diet. These initial decreases were
followed by progressive increases. After 180 days,
the body weights of the DAB-fed animals were
similar to those of animals fed the basal diet,
b u t the liver weights were much higher in the
former group than in the latter owing to the
development of tumor masses, between 90 days
and 180 days, in the liver of rats fed DAB.
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DAOUST AND
CANTERO---Proportiortsof Cell Types in Rat Liver
The most striking histological change observed
in the liver of DAB-fed animals is the formation
of bands of bile duct and connective tissue cells
as a result of an extensive proliferation of these
tissue elements. The trabeculae surround nodules
of parenchymal tissue (cirrhotic liver), and tumor
masses develop at later stages from either the
759
from 3 per cent to 17 per cent during the same
time interval. These changes were accompanied
by striking decreases in the proportions of parenchymal and littoral cells. The livers of rats fed
DAB for 90 days were composed, on the average,
of ~4 per cent of parenchymal cells, 15 per cent
of littoral cells, 43 per cent of bile duct cells,
TABLE 2
N U M E R I C A L PROPORTIONS OF CELL TYPES IN T H E LIVER OF RATS F E D BASAL DIET
PEa
T I M E ON DIET
BODY WEIGHT
(gin. + S.D.)
211 • 15
180 • 23
155•
177 • 22
201 • 36
173 • 36
190 •
(+S.D.)
LIVER WEIGHT
Parenchymal
cells
(days)
O
3O
60
90*
1~0
150
180t
CENT DISTRIBUTION OF CELL TYPES
(gm.+S.D.)
8.9•
7.4•
5.2•
7.2+1.7
7.0•
7.8•
7.7•
Littoral
cells
33.0•
31.5•
30.3•
28.2•
29.6•
30.7•
30.8•
60.5•
61.8•
62.4•
62.6•
64.2•
63.5 •
63.0•
Cells of
bile ducts
Cells of
conn. tissue
2.3•
2.5•
2.8•
3.8_+1.3
2.6•
2.4_+1.1
2.7• .8
2.5•
2.2•
2.1•
2.4•
1.6•
1.3•
1.5•
.5
Cells of blood
vessel walls
1.7+0.4
2.0•
.6
2.4•
3.0_+0.6
2.0•
2.1•
2.0•
* One animal of that group showed an exceptionally high percentage of bile duct and connective tissue cells, and
the results were excluded from the means. The per cent distribution of cell types in that liver was the following:
parenchymal, 54.9; littoral, 29.8; bile duct, 7.0; connective tissue, 5.3; blood vessel walls, 3.0.
t One animal of that group showed an exceptionally high percentage of littoral cells, and the results were excluded from the means. The per cent distribution of cell types in that liver was the following: parenchymal, 50.8;
littoral, 41.4; bile duct, 4.3; connective tissue, 1.9; blood vessel walls, 1.6.
TABLE S
NUMERICAL PROPORTIONS OF CELL TYPES IN THE LIVER OF RATS FED DAB
P E a C E N T DISTRIBUTION O F CELL TYPES
TI~
ON
DIET
BODY WEIGHT
LIVER WEIGHT
Parenehymalcells I
(days) (gm.+S.D.)
0
211 • 15
30
165•
6O
118 • 17
9O
144 • 15
120
163 __+26
150
181•
180
200 • 63
Hepa toma*
Cholan giomat
(+S.D.)
(gin. + S.D.)
8.9•
6.3•
5.2_+1.3
6.8•
9.7•
11.2•
15.7•
Littoral
cells
Cells of
bile ducts
i
Cells of
: conn. tissue
Cells of blood
vessel walls
60.5+2.0
42.3•
35.6•
~.6•
33.6•
28.4•
80.4•
33.0___0.6
34.2•
21.6•
14.8•
?,0.9•
17.3•
21.2•
2.3•
12.7•
28.2•
43.6•
32.1•
38.4•
34.2•
2.5• .5
9.3•
! 13.3•
17.0_+2.0
12.9•
15.3•
13.0_+4.6
1.7+0.4
1.5+0.2
1.3+0.8
1.0•
0.5_+0.8
0.6+0.7
1.2+0.5
57.7•
16.1_+5.6
15.1•
9.9+7.2
1.2+0.7
66.5_+4.9
33.5_+4.9
* Means of counts on seven different hepatomas which developed in animals between 90 and 180 days.
'f Means of counts on two cholangiomas which arose after 120 and 150 days of DAB feeding.
hyperplastic parenchyma (hepatomas) or the hyperplastic bile ducts (cholangiomas). The present
quantiative analyses revealed that the proportion
of bile duct cells increased progressively from an
original value of ~ per cent to one of 43 per cent
during the first 90 days of DAB feeding, and the
percentage of connective tissue cells increased
17 per cent of connective tissue cells, and 1 per
cent of cells in the blood vessel walls (Table
5). Such a composition is entirely different from
those observed in rats fed normal or basal diets.
The variations in the distribution of cell types
were maximal after 90 days of DAB feeding,
and the proportions observed in cirrhotic liver
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760
Cancer Research
Vol. 19, August, 1959
at later time intervals were about equivalent to clusively of parenchymal cells. In fact, such data
those obtained at the 60-day period.
cannot be translated in terms of any structural
The tumor masses which developed from 90 unit unless it is determined by histochemical methdays on, in the liver of the DAB-fed animals, ods in which tissue element the analyzed subpresented particular distributions of cell types. stances are located (9, 10).
The numerical proportions of cell types in the
In hepatomas, the parenchymal cells made up
58 per cent of the total number of cells, a value liver were found to be greatly altered during
close to the normal one; the littoral cells 16 per carcinogenesis by DAB. The percentage of parencent, a value lower than normal; the bile duct chymal cells in cirrhotic liver was as low as 24
cells 15 per cent and the connective tissue cells per cent on the average, after 90 days of DAB
10 per cent, values higher than normal; and the feeding, and the nonparenchymal elements were
cells of the blood vessel walls 1 per cent. The present in a proportion of 76 per cent. The liver
cholangiomas, on the other hand, were composed tumors, for their part, showed particular cell disalmost exclusively of bile duct and connective tributions differing from those observed in both
tissue cells in proportions of 67 and 88 per cent, the normal and the cirrhotic livers. Grant and
respectively.
Rees (12) have recently confirmed the preliminary
DISCUSSION
results obtained in the present study (7) and found
The number of parenchymal cells in the liver that the changes taking place in the cell population
of young adult rats accounted for only 61 per of the liver during carcinogenesis by thioacetamide
cent of the total number of cells (although they were of the same order as those described for
represent 85 per cent of the total volume [~2]) DAB-fed animals. From a quantitative point of
and the nonparenchymal cells were present in view, therefore, the normal, the cirrhotic, and the
a proportion of 39 per cent. Similar percentages neoplastic livers show entirely different cytological
of parenchymal cells in normal rat liver were compositions and cannot be considered as "homolreported by Abercrombie and Harkness, 59 per ogous" tissues. Since cells of different types usually
cent (~); Stowell, 60 per cent (21); Sibatani show quantitative differences in their chemical
and Fukuda, 65 per cent (19); and Grant and composition, changes in the proportions of cell
Rees, 67 per cent (12). Figures of the same order types probably alter the average chemical comwere also reported for mouse liver, 56 per cent position of a tissue. It follows that variations
(~5), and beef liver, 70 per cent (18). The results in the composition of the "average liver cell" during
obtained in different laboratories vary whether: DAB carcinogenesis may be due, in part at least,
(a) young or older animals are used, (b) the nuclear to changes in the cellular composition of the tissue
counts are carried out exclusively on the cells and that they do not necessarily reflect actual
composing the liver lobules or on both the lobular variations in the cells.
The present analyses have dealt exclusively
and interlobular cells, (e) blood elements are included in the counts or excluded, and (d) the with the cell types which are permanent structural
results of the crude counts on tissue sections are units of liver tissue. The blood cells were not
corrected or not for the relative nuclear sizes. included in the counts, since the percentage of
Each of the first three factors usually affects the these cells varies depending on whether the aniresults to only a small extent, but correcting the mals are exsanguinated or the livers perfused
data for relative nuclear sizes decreases appreci- before carrying on analyses. Other cell types which
ably the percentage of parenchymal cells. In the have been excluded from the counts are the necrotpresent work, for instance, the crude counts gave ic cells and the inflammatory cells which may be
an apparent percentage of parenchymal cells of found in the necrotic areas of cirrhotic and neo69 per cent (7), in agreement with the high (un- plastic livers. The number of these cells was judged
corrected) figures reported by some authors, while to be negligible in cirrhotic liver but appreciable
a true percentage of 61 per cent was obtained in some tumors. Necrotic and inflammatory cells
after correction. In any case, the different in- are probably included in varying numbers in the
vestigations lead to the same conclusion, namely, samples of neoplastic livers taken for biochemical
that the normal liver contains a high number assays, since it is impossible to dissect liver tumors
of nonparenchymal cells and that, for an organ so as to exclude all the necrotic regions.
In addition to the important variations taking
which is often considered as homogeneous, the
liver shows an appreciable degree of cellular het- place in the cell population of the liver during
erogeneity. The biochemical data obtained with carcinogenesis, changes are also observed in the
liver homogenates cannot, therefore, be inter- amount of fibrous elements as well as in the nature
preted as though the organ were composed ex- and the relative volume of extracellular fluids
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Research.
DAOUST A N D
CANwERo--Proportionsof Cell
Types in Rat Liver
761
(11, 15-17). The elastic and collagen fibers are in liver sections of rats fed (a) a normal diet,
present in amounts higher than normal in cirrhotic (b) a low-protein, basal diet, or (c) a diet conliver and the fibrous tissue becomes especially raining the carcinogen 4-dimethylaminoazobenabundant in some tumors. The bile ducts enlarge zene (DAB).
progressively during DAB feeding and sometimes
1. The liver of young adult (~00 gm.) normal
form huge cysts lined by a single layer of epithelial rats contained 61 per cent parenchymal cells,
cells and filled with serous fluid. Areas of necrosis 30 per cent littoral ceils, and 9 per cent of other
containing disintegrated cells and abundant fluid cell types. The relative number of littoral cells
are observed in other regions and may occupy was observed to increase with age.
relatively large volumes in liver tumors. The ratio
2. In rats fed the low-protein, basal diet, the
of extracellular fluid to cellular material thus losses of body weights and liver weights were
increases during the carcinogenic process and may accompanied by slight increases in the proportion
reach high values in neoplasia. It is likely that of parenchymal cells and decreases in the perthe samples of normal, cirrhotic, and neoplastic centage of littoral cells.
livers used for assays on homogenates contain
3. The cytological composition of the liver
different quantities of extracellular fluid.
showed striking changes in rats fed DAB, due
It may be concluded from the histological anal- mainly to an extensive proliferation of the bile
yses that the biochemical data obtained with ducts and connective tissue dements. In c/rhomogenates of liver tissue represent the mean rhotic liver, after 90 days of DAB feeding, the
composition or activity of several cell types and parenchymal ceils accounted for only 24 per cent
different extracellular fluids. The biochemical anal- of the total number of ceils and the littoral ceils
yses do bring valuable information on the chemical for 15 per cent, while the other cell types were
properties of organs and tissues, and there is no present in a proportion of 61 per cent. Liver
need to discuss here the importance of the bio- tumors (hepatomas and cholangiomas) which arose
chemical contributions. It should be realized, how- between 90 and 180 days of DAB feeding showed
ever, that assays on homogenates do not give cellular distributions different from those of both
any insight into the properties of the various the normal and the cirrhotic livers.
tissue elements and that the results, therefore,
The results were discussed in relation to the
cannot be directly translated in terms of the interpretation of the biochemical data derived
structural units. Attempts to deduce the chemical from assays on homogenates of normal, cirrhotic,
properties of tissue elements from analyses on and neoplastic livers.
homogenates are generally hazardous and can be
ACKNOWLEDGMENTS
grossly misleading when normal and pathological
tissues are compared, even if precautions are taken
The authors wish to thank Miss Yolande Poirier for valuto use the best reference bases available for ex- able technical assistance.
pressing the biochemical data (8, 13, 23). The
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Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1959 American Association for Cancer
Research.
The Numerical Proportions of Cell Types in Rat Liver during
Carcinogenesis by 4-Dimethylaminoazobenzene (DAB)
R. Daoust and A. Cantero
Cancer Res 1959;19:757-762.
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