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Br.
J.
Cancer
(1989),
60,
827-833
©
The
Macmillan
Press
Cell
population
kinetics
and
ploidy
rate
of
early
focal
lesions
during
hepatocarcinogenesis
in
the
rat
Ph.
Castelain',
A.
Deleener',
M.
Kirsch-Volders'
&
H.
Barbason2
'Laboratory
for
Anthropogenetics,
VUB,
Pleinlaan
2,
1050
Brussels,
and
2Tour
de
Pathologie
B35,
ULG,
Sart
Tilman,
4000
Liege,
Belgium.
Summary
We
have
studied
the
changes
in
cell
population
kinetics
and
DNA-content
of
cycling
parenchymal
cells
during
the
very
early
steps
of
rat
hepatocarcinogenesis
in
Faber's
protocol.
Adult
rats
were
initiated
by
a
single
dose
of
diethylnitrosamine
(DENA,
200
mg
kg-1),
followed 2
weeks
later
by
a
2-week
diet
of
0.03%
2-acetylaminofluorene
(2-AAF)
as
selection
phase.
In
the
middle
of
selection
time,
a
single
necrogenic
dose
of
carbon
tetrachloride
(CCL4,
2
ml
kg-
)
was
administered
by
gavage.
Twenty
four
hours
thereafter,
radiolabelled
thymidine
(3H-TdR,
1.5
ftCi
g'
)
was
given
by
repeated
injections
during
24
h.
An
emergence
of
small,
pyroninophilic
('tigroid')
foci
was
observed
at
the
second,
fifth
and
eighth
days
after
the
proliferative
stimulus.
The
focal
putative
precancerous
cells
presented
a
significant
higher
labelling
index
(LI)
than
the
non-affected
parenchymal
cells
for
all
exposure
times.
However,
the
labelling
intensity
decreased
from
the
second
to
the
eighth
day
after
CC14,
suggesting
a
dilution
of
the
radiolabelled
DNA
by
repeated
divisions
within
the
foci.
The
nuclei
of
the
same
foci
were
analysed
for
DNA-content
by
feulgen
microdensitometry
on
neighbouring
sections.
A
gradual
reduction
of
nuclear
DNA-content
was
observed
in
66%
of
the
foci
at
the
fifth
day
and
in
100%
of
foci
at
the
eight
day,
as
compared
to
surrounding
tissue
and
untreated
animals,
where
labelling
and
DNA-content
remain
in
the
same
ratio.
It
is
well
established
now
that
carcinogenesis
is
a
multistep
process
(Bannasch
et
al.,
1980;
Farber
&
Cameron,
1980;
Farber
&
Sarma,
1987).
In
rat
hepatocarcinogenesis,
many
models
are
available
which
generate
preneoplastic
lesions
as
foci
and
nodules,
which
are
believed
to
be
preferential
sites
for
ultimate
cancer
development
(Farber
&
Cameron,
1980).
Some
of
these
protocols
allow
a
step-by-step
analysis
of
these
early
preneoplastic
foci
and
nodules,
because
of
their
syn-
chroneous
emergence
(Farber
et
al.,
1976;
Lans
et
al.,
1983).
A
lot
of
biochemical,
enzymatic
and/or
genetic
alterations
were
described
in
the
past,
but
the
problem
is
that
none
of
these
markers
seems
to
persist
until
the
final
cancerous
stages.
In
previous
works
(Deleener
et
al.,
1987;
Kirsch-Volders
et
al.,
1986)
it
was
shown
that
nodular
cells,
induced
by
a
triphasic
protocol
(initiation,
selection,
promotion)
were
predominantely
diploid,
in
contrast
to
the
mainly
tetraploid
cell
population
of
a
non-treated
adult
rat
liver.
These
findings
were
also
reported
with
several
other
carcinogenic
regimens
(Bassleer
et
al.,
1985;
Godoy
et
al.,
1976;
Inui
et
al.,
1971;
Pugh
&
Goldfarb,
1978;
Schwarze
et
al.,
1984;
Styles
et
al.,
1985,
1987;
Sargent
et
al.,
1989)
and
even
in
primary
liver
cancer
of
man
(Saetar
et
al.,
1987).
These
downward
shifts
in
ploidy
level
were
observed
by
classical
cytodensitometry
(Inui
et
al.,
1971;
Neal
&
Bulter,
1978),
flow-cytometry
of
inter-
phase
cells
(Schwarze
et
al.,
1984;
Styles
et
al.,
1985)
or
by
chromosome
counting
of
dividing
hepatocytes
(Becker
et
al.,
1971).
In
other
studies,
however,
preneoplastic
and
cancerous
lesions
were
not
unequivocally
diploid,
but
also
showed
a
tetraploid
pattern
(Bassleer
et
al.,
1985;
Kuo
et
al.,
1987;
Mori
et
al.,
1982;
Sarafoff
et
al.,
1986).
Besides
the
biological
meaning
of
diploidisation,
the
ques-
tion
arises
at
what
time
and
in
which
cells
this
phenomenon
develops.
Hitherto,
many
authors
described
the
growth
kinetics
of
hyperplastic
and
putative
premalignant
cell
populations.
In
these
works,
emphasis
was
laid
on
the
impor-
tance
of
cellular
proliferation
after
initiation
(Columbano
et
al.,
1981;
Ying
et
al.,
1982)
and
on
the
effect
of
several
carcinogens
upon
cell
loss,
repair
mechanisms
and
the
con-
commitant
de
novo
DNA
synthesis
(Albert
et
al.,
1972;
Bursch
et
al.,
1985;
Yager
&
Potter,
1975).
Moreover,
it
is
generally
accepted
that
there
is
a
significant
increase
of
DNA
synthesis
and
mitotic
activity
in
foci
and
nodules
induced
by
Correspondence:
Ph.
Castelain.
Received
26
September
1988;
and
in
revised
form
8
June
1989.
carcinogens.
The
latter
was
demonstrated
by
combinations
of
enzyme
histochemical
techniques
and
histoautoradiography
after
chronic
or
pulse-labelling
with
radioactive
precursors
of
DNA
(Barbason
et
al.,
1983;
Enomoto
&
Faber,
1982;
Kitagawa
&
Sugano,
1973;
Pugh
&
Goldfarb,
1978;
Rabes
&
Szymkowiak,
1979;
Rotstein
et
al.,
1984,
1986;
Schulte-
Hermann
et
al.,
1983).
None
of
these
works,
however,
reports
the
link
between
the
proliferative
activity
of
putative
preneoplastic
lesions
and
the
ploidy
of
their
nuclei.
In
this
study,
we
tried
to
follow
the
fate
of
the
cycling
cells
during
the
selection
phase
of
the
biphasic
protocol
for
hepatocarcinogenesis
(i.e.
period
of
focal
growth).
This
was
done
by
following
the
incorporation
of
a
radioactive
precur-
sor
for
DNA
on
autoradiographs.
In
this
way,
cells
were
labelled
which
resist
the
cytotoxic
effects
of
the
'selector'
2-AAF
and
of
the
necrogenic
agents
CC14.
This
period
is
interesting
in
the
analysis
because
the
resistent
cells
will
grow
out
to
possible
preneoplastic
lesions
during
this
time.
His-
tological
changes
were
detected
by
classical
Haematoxylin
and
Eosin
staining
and
methyl
green-pyronine
staining;
DNA-content
was
analysed
on
Feulgen-stained
serial
sections
by
microdensitometry.
Materials
and
methods
Experimentalprotocol
(Figure
1)
Initiation-selection
(IS-CC14)
protocol
Twenty
male
Wistar-
R
rats
(IOPS
AF)
HAN,
Iffa
Credo,
about
3
months
old,
were
injected
i.p.
with
a
necrogenic
dose
of
diethylnit-
rosamine
(DENA)
200
mg
kg-'
i.s.
0.9%
NaCl
for
initiation.
Two
weeks
later
a
selection
regimen
was
given,
as
described
by
Lans
et
al.
(1983).
A
0.03%
solution
of
2-
acetylaminofluorene
(2-AAF)
was
added
to
the
UAR
(04)
basal
diet.
This
regimen
was
given
during
2
weeks.
In
the
middle
of
this
period,
carbon
tetrachloride
(CC
14)
was
administered
by
gavage
at
a
dose
of
2
ml
kg-',
diluted
with
an
equal
volume
of
corn
oil.
This
serves
as
a
proliferative
stimulus
for
non-inhibited
hepatocytes.
CC14
protocol
As
a
comparison,
10
age-matched
animals
were
treated
only
with
CC14
at
the
same
time-point
as
the
rats
treated
in
the
IS
protocol.
No
DENA
or
2-AAF
was
given
to
them.
Untreated
controls
As
a
supplementary
control,
five
rats
receive
a
normal
regimen,
and
will
be
referred
further
on
as
the
'untreated
group'.
Br.
J.
Cancer
(1989),
60,
827-833
'."
The
Macmillan
Press
Ltd.,
1989
828
P.
CASTELAIN
et
al.
2
weeks
NN
2
weeks
2-AFF
1
week
NN
CCI4
DENA
C
T
I
I
illz!
111
xl
NN
2
days
5
days
8
days
14
days
CC14
2
days
8
days
Figure
1
Experimental
protocol
for
rat
hepatocarcinogenesis.
Wistar
R
male
rats
about
250
g.
DENA,
diethylnitrosamine,
200
mg
kg-';
2-AAF,
2-acetylaminofluorene,
0.03%;
CCI4,
car-
bon
tetrachloride,
i.g.
2
ml
kg-',
1:1
dil.
in
corn
oil;
T*,
3H-TdR,
1.5
pCig-',
6
i.p.
injections
during
24
hours;
NN,
normal
nourishment.
Incorporation
of
radioactive
thymidine
Initiation-selection
protocol
(IS-CCd4)
In
order
to
label
a
significant
proliferating
cell-fraction,
a
continuous
24-h
incor-
poration
of
6-3H-TdR
was
performed
as
follows:
the
radiolabelled
thymidine
(1.5
tLCi
g-',
specific
activity
5
Ci
mmol-'
diluted
in
a.d.,
Amersham
Int)
was
injected
i.p.
four
times
at
6-h
intervals
from
the
24th
hour
to
the
48th
hour
after
the
CC14
administration.
In
this
way,
all
cells
passing
through
DNA
synthesis
in
the
time
span
of
24
h
are
labelled
(assuming
the
mean
S-phase
duration
of
normal
liver
cells
to
be
about
7
h)
(Rabes
&
Szymkowiak,
1979;
Barbason
et
al.,
1983).
Radiotoxicity,
caused
by
the
3H-TdR
was
unlikely.
No
significant
lethalithy
is
expected,
since
each
cell
received
no
more
than
3
tLCi
g-'
body
weight
in
total
(1.5
glCi
g'
at
6-h
interval
labels
at
a
maximum
twice
in
the
same
cell,
since
the
mean
t,
is
±
40
h
and
the
total
3H-TdR-
administration
lasts
for
24
h).
All
animals
were
treated
in
the
same
way.
They
were
killed
in
groups
of
five
respectively
2
days, 5
days,
8
days
or
14
days
after
the
CC14
administration.
The
animals
of
this
experimental
group
are
referred
to
as
'IS-2,
5
or
8d'
in
the
text.
Controls
The
CC14
group
and
untreated
group
received
no
3H-TdR.
The
10
animals
of
the
CC14
group
were
killed
in
groups
of
five
respectively
2
days
and
8
days
after
the
necrogenic
stimulus.
The
livers
of
these
control
animals
were
only
used
to
analyse
the
DNA-content
of
hepatocytes.
Histology
After
excision,
pieces
of
liver
were
fixed
in
either
10%
for-
malin
or
Carnoy
solution
(6:3:1
of
absolute
ethanol:chloroform:acetic
acid)
and
embedded
in
paraffin.
Sections
of
7
,m
were
cut
serially
and
stained
with
Haematoxylin
and
Eosin
(H&E),
methyl
green-pyronine
(Unna-Brachet
stain)
(UB)
and
Feulgen.
All
stained
slices
were
dehydrated
in
changes
of
graded
ethanol
and
mounted
with
DPX
(Fluka).
At
least
one
section
was
processed
for
histoautoradio-
graphy.
For
this
purpose,
slides
were
coated
with
K5-emulsion
(Ilford)
by
dipping
and
stored
in
the
dark
at
4°C
for
1,
2,
3,
4
or
5
weeks.
After
3
weeks,
a
plateau
in
the
labelling
intensity
was
reached
(optimal
labelling
without
too
great
background
labelling).
The
slices
were
developed
and
post-stained
with
Unna-Brachet.
Cells
with
five
or
more
grains
over
their
nucleus
were
considered
as
labelled
(this
was
based
on
the
analysis
of
the
background
level
of
the
autoradiography,
which
was
less
than
five
grains
per
unit
area
(
area
of
one
nucleus).
For
the
Feulgen
reaction,
Carnoy-fixed
slices
were
first
hydrolysed
under
mild
conditions
(1
N
HCI
at
room
temperature
for
17
h).
This
hydrolysis
time
was
chosen
because
of
the
stability
of
the
Feulgen
stain
at
this
time-point
(hydrolysis
curve
not
shown).
After
rinsing,
Feulgen
stain
was
performed
during
1
h,
and
rinsed
with
saturation
buffer
during
10 min.
Morphometry
and
cytodensitometry
Early
lesions
were
visualised
with
H&E
and
UB
stain.
The
slices
were
projected
on
a
drawing
table;
the
lobes
and
early
foci
were
drawn
with
an
16.5
times
enlargement.
The
areas
of
the
lobe
and
focal
sections
were
measured
on
a
HP
9874A
digitizer.
The
labelling
index
(LI),
defined
as
the
percentage
of
labelled
nuclei
in
the
total
number
of
nuclei,
was
measured
on
a
glarex
projection
screen,
mounted
upon
a
Zeiss
micro-
scope.
Using
a
magnification
of
400
times,
constant
areas
(370
ium)
were
randomly
analysed.
The
DNA-content
of
focal
and
non-focal
nuclei
was
deter-
mined
by
Feulgen
microdensitometry.
For
this
purpose,
pyroninophilic
foci
(as
determined
by
the
UB
stain)
were
photographed
with
high
magnification.
The
same
lesions
were
relocated
on
a
Feulgen-stained
section.
Densitometric
measurements
were
performed
on
a
computer-assisted
image
analysing
device
Magiscan
2A
(Joyce-Loebl,
GB)
connected
to
a
Zeiss
photomicroscope
III
with
a
Bosch
TV
camera
(TYK
9A,
Chalnicon
tube).
Results
Morphometric
data
on
growth
of
hyperplastic
foci
Two
days
after
IS-CC14,
an
important
necrosis
was
observed,
predominantly
in
centrolobular
areas.
These
areas
were
char-
acterised
by
karyorhexic,
karyolytic
and
heteropycnotic
nuclei.
Eight
days
after
IS-CC14
these
degenerating
cells
are
no
longer
observed
and
non-parenchymal
oval
cells
appeared.
After
labelling
with
3H-TdR,
these
necrotic
cells
were
heavily
labelled
at
2
days
after
IS-CC14.
This
labelling
disappeared
after
8
days
in
these
areas.
The
H&E,
and
especially
the
UB-staining
revealed
little,
pyroninophilic
foci
('tigroid'
foci)
from
the
second
day
after
IS-CC14
on.
They
were
characterised
by
cells
with
clusters
of
RNA
in
their
cytoplasm
and
sometimes
very
prominent
nucleoli,
as
previously
reported
(Bannasch
et
al.,
1985).
The
morphomet-
ric
data,
given
in
Table
I,
show
that
there
was
an
increase
of
the
volume
and
of
the
number
of
these
foci
in
the
course
of
the
exposure
time.
From
the
second
to
the
fifth
day,
the
number
of
foci
increases
with
a
factor
2,
and
the
volume
remains
somewhat
constant.
From
the
fifth
to
the
eighth
day,
the
volume
is
doubling,
while
the
number
of
foci
increases
with
a
factor
3.
The
increase
in
the
fraction
of
focal
tissue
(the
total
area
of
the
foci
as
a
percentage
of
the
total
liver
section
area)
indicates
that
the
growth
of
the
foci
exceeds
by
far
the
reparative
growth
of
the
rest
of
the
liver
parenchyma.
The
increase
in
number
and
in
volume
of
the
tigroid
foci
reaches
a
maximum
at
the
eighth
day
after
the
IS-CC14
induction
(mean
values
per
treatment
in
Table
I).
However,
after
14
days,
it
appears
that
there
is
a
confluence
of
the
pyroninophilic
foci:
they
are
not
observable
as
single
entities
any
more.
A
similar
observation
was
made
in
livers
of
animals
which
were
treated
with
CC14
alone:
also
in
this
case,
2
days
after
CC14
small
pyroninophilic
foci
were
noticed,
which
persisted
until
the
eighth
day.
No
morphometric
data
were
collected
on
this
focal
proliferation,
since
these
foci
were
very
small
and
not
sharply
delimited
in
the
surrounding
liver
tissue.
Proliferative
activity
of
resistant
cells
In
order
to
know
which
cells
were
cycling
in
the
24
h
period
immediately
after
the
CC14
induction,
the
labelling
index
was
determined
in
necrotic
regions,
normal
cells
and
focal
cells.
The
results
are
given
in
Table
II.
Two
days
after
CC14,
there
is
a
huge
incorporation
in
the
1-1
HEPATOCARCINOGENESIS
IN
THE
RAT
829
Table
I
Data
of
relative
focal
area,
fraction
of
focal
tissue,
and
number
of
foci
per
unit
area
Treatment
No.
Relative
focal
s.d.
Fraction
of
Number
of
area
(%)
focal
tissue
foci
per
cm2
(per
mille)
2
days
1
6.32
1.28
0.40
5.70
2
8.28
1.63
0.34
5.14
3
-
4
-
_
5
16.50
4.51
2.16
10.38
Mean
9.43
4.21
0.97
7.10
5
days
1
8.31
2.85
0.67
13.43
2
11.80
0.57
1.91
17.57
3
14.70
1.00
3.17
22.13
4
-
_
_
_
5
8.68
3.59
0.31 4.81
Mean
10.87
2.59
1.52
14.50
8
days
1
19.48
3.68 5.35
26.51
2
35.10
0.18
3.50
105.44
3
26.90
3.78
5.10
59.78
4
23.70
2.80
4.54
19.33
5
16.20
1.61
3.17
20.74
Mean
24.28
6.52
4.30
46.36
aRelative
focal
area
is
the
mean
area
of
the
foci
(in
fnm2)
divided
by
the
total
area
of
the
lobe.
bFraction
of
focal
tissue
is
the
sum
of
the
areas
of
all
foci
in
a
lobe
divided
by
the
total
area
of
that
lobe.
The
treatment
is
indicated
by
the
number
of
days
after
the
proliferative
stimulus
(CC14)
in
the
IS
protocol.
Table
II
Mean
labelling
index
(Ll
in
%)
of
necrotic,
normal
and
focal
cells
for
different
periods
after
the
necrogenic
stimulus
(in
the
IS
protocol)
Labelling
index
(%)
Non-affected
Treatment
No.
Necrotic
zone parenchyma
Focal
tissue
2
days
1
30.6
4.4
-
2
43.8
9.5
76.1
3
36.6
8.4
71.9
4
49.6
10.7
-
5
47.6
12.2
77.3
Mean
37.6
7.6
75.1
5
days
1
24.1
12.6
73.1
2
20.9
4.1
56.9
3
38.7
10.1
81.6
4
45.6
35.1
-
5
24.3
9.1
-
Mean
33.7
10.3
70.5
8
days
1
-
10.7
50.3
2
-
11.7
49.9
3
-
9.4
39.6
4
-
16.8
45.6
5
-
2.7
49.9
Mean
-
10.6
47.1
14
days
1
-
4.8
2
-
3.8
-
3
-
1.6
-
4
-
2.4
-
S
-
_
Mean
-
3.6
necrotic
zones
(38%
LI).
This
LI
decreases
to
34%
until
the
fifth
day
and
decreases
further
to
nearly
0%
afterwards,
indicating
a
massive
cell
loss.
In
the
non-affected
paren-
chyma,
the
LI
increases
somewhat
from
the
second
to
the
eighth
day
to
10.6%
and
decreases
after
the
eighth
day
until
the
fourteenth
day
to
3.6%.
The
foci
show
a
very
high
LI
compared
to
the
surrounding
parenchyma.
They
are
particularly
strongly
labelled
after
the
second
day
and
after
the
fifth
day,
but
between
the
fifth
and
the
eighth
day,
the
LI
decreases
to
about
67%
(compared
to
the
value
at
5
days).
The
relationship
between
the
volume
of
a
focus
and
its
mean
LI
was
investigated
by
calculating
the
correlation
between
them
(Figure
2):
a
significant
negative
correlation
coefficient
of
0.76
was
obtained.
100
-
,
90-
x
80-
.70
c
60-
S
50-
40
-j
40-4
3U,
0
1
2
3
4
5
6
Fraction
of
focal
tissue
(%o)
Figure
2
Correlation
between
the
relative
area
of
the
foci
(frac-
tion
of
focal
area)
and
the
labelling
index.
DNA
content
The
nuclear
DNA
content
was
determined
on
serial
sections
after
Feulgen
staining
and
analysed
with
a
computer-aided
cytodensitometer.
The
histograms
of
DNA-content
are
given
on
Figure
3.
Figure
3
givens
the
distributions
of
DNA-content
from
hepatocytes
of
normal,
perifocal
and
focal
tissue,
collected
from
the
several
animals
submitted
to
the
same
treatment.
As
a
general
remark,
it
appears
that
there
is
a
broad
range
in
the
C-value
distribution,
especially
for
the
normal
tissue.
In
the
first
group
(2
days
after
IS-CCl4)
only
one
animal
shows
measurable
foci
(Figure
3).
In
this
animal,
no
significant
shift
to
lower
C-values
can
be
noted
in
the
focal
tissue,
as
com-
pared
to
the
normal
parenchyma.
Moreover,
there
is
a
significant
difference
between
the
distribution
of
this
animal
and
the
merged
data
from
the
other
animals
of
the
same
group,
indicating
a
strong
interindividual
variation.
On
the
fifth
day
after
IS-CC14,
the
shift
of
the
modal
C-value
is
more
clear.
On
the
eighth
day,
the
downward
trend
is
confirmed.
It
appears
that
about
60%
of
the
focal
cells
have
a
ploidy
rate
beneath
4C
(compared
to
36%
on
the
fifth
day
and
14%
on
the
second
day).
There
is
no
significant
difference
between
the
DNA-profiles
of
the
untreated
livers
and
these
of
the
livers
only
treated
by
the
CC14
(see
Figure
3).
If
there
is
no
initiation
or
selection,
the
mean
result
is
a
bimodal
distribution
round
the
4C-
and
8C-values
These
results
were
also
obtained
in
'focal'
cells
generated
by
the
CCI4
alone.
This
suggests
that
these
foci
are
only
cirrhotic
lesions;
it
is
known
that
a
treatment
with
CC14
alone,
even
with
this
high
dose,
is
not
sufficient
to
induce
hepatocarcinogenesis
in
rats.
In
Figure
4,
a
survey
of
the
C-values
is
given.
The
percen-
tage
C-value
is
given
per
treatment
and
per
kind
of
tissue.
The
classes
are
defined
here
as
the
integrated
part
(in
%)
of
the
histograms
in
Figure
3:
in
order
to
define
2C,
4C
and
8C
fractions,
two
boundaries
were
created
around
the
three
modal
C-values
(see
as
an
example
the
DNA
profile
of
the
untreated
liver
in
Figure
5).
For
the
data
of
the
animals
treated
with
the
IS-protocol,
a
clearcut
increase
in
the
2C
fraction
is
visible.
At
the
same
time
the
4C
fraction
and
the
8C
fraction
decrease.
A
striking
observation
is
that
the
decrease
in
the
8C
fraction
is
the
strongest,
as
compared
to
the
decrease
of
the
4C
fraction.
This
evolution
is
observed
in
the
course
of
the
treatment,
as
well
as
within
the
different
type
of
tissues
(non-focal,
perifocal
and
focal).
That
the
reduction
of
nuclear
ploidy
rate
really
corres-
ponds
to
a
reduction
of
cellular
ploidy
rate
is
proven
by
the
nuclearity
analysis
summarised
in
Table
III.
Of
the
focal
and
of
the
normal
cells
95-97%
are
mononucleated.
Discussion
Morphometric
data
on
the
growth
of
hyperplasic
foci
Between
the
second
and
the
fifth
days
after
CCI4
the
mean
focal
area
is
relatively
constant
while
the
number
of
foci
per
I
.
.-
I
r
=
0.76
a
0
0
0
830
P.
CASTELAIN
et
al.
Untreated
animals
IS
(2
days)
non-focal,
1
animal
8
2C
4C
8C
n
=
122
7
K
8
1
5
22
29
36
43
50
1
8
15
22
29
36
IS
(2
days)
perifocal,
1
43 50
animal
8-
2C
4C
8C
n
=
191
67
1i1
Only
CCI4
(2
days)
non-focal
16-
2C
4C
8C
n
=
1845
14
12
10
f
o/1,
9
Ou
6^
4.
2'
O'
1
8
1
5
22
29
36
43
50
IS
(2
days)
non-focal
16
14
12
10
8
6
4
2
0
2C
4C
8C
n
=
426
A
-
-
..4...
1
8
15
22
29
36
43
50
IS
(2
days)
focal,
1
animal
1
8
1
5
22 29
36
43
50
Only
CCI4
(2
days)
focal
16
2C
4C
14-
12
i
10.
%
8.
16
14
12
10
%
8
6
4
2
n
16
14
12
10
%
8
6
4
2
(
8C
n
=
1527
A.Q
Rl.
hn
IS
(5
days)
non-focal
n
=
516
2C
4C
8C
Lh~J
h~LA
8
1
5
22
29
36
43
50
IS
(5
days)
perifocal
n
=
108
2C
4C
8C
ii'I
1
8
15
22
29
36
43
50
IS
(5
days)
focal
161
2C
4C
BC
n
=203
14
1
211
4
2
0
M R
.
1
8
15
22
29
36 43
16
14
12
10
%
8
6
4
2
0
Only
CCI4
(8
days)
non-focal
n
=
2082
8C
2C
4C
14,
12,
1
0
6
4
2
8
15
22
29
36
43 50
16
14
12
10
%
8
6
4
2
0
16
14
12
10
%
8,
6
4
2
0
50
Only
CC14
(8
days)
focal
n
=
1470
2C
4C
8C
8
1
5
22 29
36
43
16
14
12
10
%8
6
4
2
IS
(8
days)
non-focal
n
=
292
2C
4C
8C
1
8
15
22
29
36
43
50
IS
(8
days)
perifocal
2C
4C
BC
n =
417
Lkm
1
8
15
22
29
36 43
50
IS
(8
days)
focal
2C
4C
BC
I
n
=
536
8
__
50
1
8
15
22
29
36
43 50
Figure
3
Merged
histograms of
proportion
of
cells
in
%
(ordinate)
with
a
given
integrated
optical
density
in
arbitrary
units
(abscissa).
The
data
are
given
per
treatment
(IS,
treated
wth
DENA,
2-AAF
and
CC14
controls
are
only
treated
with
CC14)
and
per
kind
of
tissue
(non-focal,
perifocal
and
focal).
The
treatments
and
the
kind
of
tissue
are
indicated
above
each
histogram.
The
number
of
cells
analysed
is
designated
by
n.
The
modal
C-values
(2C,
4C
and
8C)
are
delimited
by
the
vertical
bars
in
the
histograms.
unit
area
doubles,
in
parallel
with
the
doubling
of
the
frac-
tion
of
focal
tissue
(Table
I).
From
these
data,
it
can
be
deduced
that
the
increase
in
the
total
focal
mass
can
be
attributed
mainly
to
the
increase
in
the
number
of
foci.
At
8
days
after
CCI4,
there
is
a
maximum
in
the
fraction
of
focal
tissue.
This
fraction
has
increased
by
a
factor
of
3
(compared
to
the
value
at
5
days).
However,
the
increase
cannot
be
related
only
to
the
increase
in
number,
but
also
to
an
inc-
rease
in
volume
of
the
existing
foci.
Between
the
fifth
and
eighth
days
there
is
an
increase
in
the
mean
diameter
of
the
tigroid
foci.
The
larger
the
diameter
of
a
focus,
the
greater
is
the
probability
of
seeing
it
in
a
random
section.
oJ....
6
4
2
O
r
i
i
w
v
li
_
_~~~m
...A...A
-
_
_-
-m
-
.+4
I
I
JL-
0
;)
1-
Ui
3VU
'+13
JU
m m
HEPATOCARCINOGENESIS
IN
THE
RAT
831
CCI4
only
S
cJ
cv
a)
Cr
Li)
U-
100
90
80
70
60
50
40
30
20
10
0
C
c
4C
'
4(
A
IS,
Treatment
duration
Figure
4
Summarising
column-chart
of
ploidy
level
(abscissa)
for
the
different
treatments
(2
days,
5
days
and
8
days)
and
for
the
different
tissues.
Percentages
(ordinate)
are
calculated
by
the
integration
of
several
classes
in
the
histograms
in
Figure
3.
16
14
12
10
%
8
6
4
2
0
2C
4C
8C
8
1
5
22
29
36
43
50
.4
owl
.i14-
1-9
10-18
19-50
Figure
5
DNA
profile
of
untreated
liver,
to
illustrate
the
way
by
which
C-fractions
were
calculated.
The
grouping
of
classes
is
shown
in
abscissa.
The
modal
C-values
are
given
by
the
dotted
lines.
Table
III
Frequency
of
binucleated
cells
in
normal
and
in
focal
tissue
(in
%)
%
binucleated
cells
Treatment
No.
Normal
cells
Focal
cells
2
days
1
3.4
2
7.1
-
3
4.4
-
4
6.2
_
5
8.1
-
5
days
1
3.8
2
2.2
-
3
2.8
-
4
3.2
-
5
2.4
-
8
days
1
3.3
5.2
2
5.6
3.3
3
3.4
4.7
4
2.6
-
5
2.6
-
14
days
1
3.2
-
2
4.1
-
3
4.2
-
4
4.6
-
5
5.6
-
Autoradiographic
data
on
proliferating
cells
The
massive
incorporation
of
3H-TdR
at
2
days
after
CC14
may
be
an
indication
of
DNA
repair
synthesis
as
a
regenerative
response
against
the
xenobiotic
(Yager
et
al.,
1975).
This
is
followed
by
a
massive
cellular
loss,
for
virtually
no
labelling
can
be
detected
in
the
centrolobular
regions
from
the
eighth
day
onwards.
On
the
other
hand,
it
cannot
be
excluded
that
a
part
of
the
labelling
can
be
ascribed
to
the
proliferation
of
non-parenchymal
cells
such
as
endothelial
cells
(Tatematsu
et
al.,
1984)
or
Kupfercells
(Bouwens
et
al.,
1986).
However,
almost
no
labelled
non-parenchymal
cells
are
observed
later
on
in
the
liver.
For
that
reason,
the
non-parenchymal
cells
represent
only
a
minority
among
the
labelled
cells
from
the
centrolobular
zone.
Massive
re-
utilisation
of
the
3H-TdR
is
unlikely,
the
biological
half-life
of
the
radioactive
precursor
being
only
I
h.
The
experiment
further
indicates
that
selection
with
2-AAF
does
not
produce
a
complete
inhibition
of
the
DNA
synthetic
activity
as
depicted
by
the
hypothesis
of
Solt
et
al.
(1977),
since
3.6-10.6%
of
the
cell
population
is
cycling
in
the
non-focal
parenchyma.
When
the
data
of
LI
of
tigroid
foci
are
considered
(Table
II),
it
appears
that
there
is
a
reduction
by
a
factor
1.6
between
the
second
and
the
eighth
days
after
CC14.
This
reduction
of LI
can
be
the
consequence
of
a
cellular
loss,
or
of
cellular
division
of
at
least
a
part
of
the
focal
cell
popula-
tion.
When
this
finding
is
compared
to
the
morphometric
data
of
focal
growth
discussed
above,
it
is
clear
that
the
latter
is
more
likely
to
occur.
In
the
timespan
of
6
days
the
fraction
of
focal
tissue
increased
by
a
factor
of
4.4,
which
implies
two
rounds
of
cell
division.
Another
argument
for
their
intense
mitotic
activity
is
found
in
the
inverse
relationship
between
the
total
volume
of
the
foci
and
their
mean
LI.
Figure
2
shows
the
high
correla-
tion
between
the
volume
of
the
focus
and
the
mean
LI.
This
can
be
explained
by
the
dilution
of
the
3H-TdR
in
the
focus
by
repeated
divisions.
This
does
not
exclude
the
possibility
that
there
is
DNA
repair
in
the
foci,
but
in
this
case
we
would
see
a
strong
labelling
in
the
focal
cells,
even
14
days
after
the
CC14
induction.
This
phenomenon
has
not
been
observed
here.
Many
data
in
the
literature
evoke
the
presence
of
highly
proliferating.cells
in
preneoplastic
lesions
in
other
protocols
(Pugh
&
Goldfarb,
1978;
Enomoto
&
Farber,
1982;
Garcea
et
al.,
1987).
IS
+
CC14
100
90
80
70
60
50
40
30
20
10
0
U-
:r_
(11
*
%2C
*
%4C
M
%8C
0O>
I\6
I
m
-.
0%
---
l
832
P.
CASTELAIN
et
al.
Cytodensitometric
data
on
DNA
content
The
broad
spread
in
the
DNA
distribution
of
the
cell
popula-
tions
(Figure
3)
can
partly
be
explained
by
the
fact
that
the
analysis
of
DNA
content
was
made
on
histological
sections.
In
contrast
to
flow-cytometric
measurements,
it
is
more
difficult
to
distinguish
between
the
different
maxima
of
2C,
4C,
etc.
In
our
system,
however,
the
produced
foci
are
so
small
that
a
liver
perfusion
before
flow-cytometry
would
produce
a
mixture
of
focal
and
non-focal
cells.
Consequently,
the
DNA-profiles
of
these
entities
would
be
masked
by
those
of
the
non-affected
parenchymal
cells.
A
second
explanation
for
the
broad
histograms
is
the
presence
of
a
significant
S-phase
fraction
in
the
treated
livers.
The
reduction
in
DNA
content
of
focal
cells
is
only
clear
in
the
period
when
the
number
and
fraction
of
foci
is
at
its
highest,
i.e.
8
days
after
the
CCl4
induction.
Five
days
after
CCl4,
this
trend
is
not
so
clear.
After
14
days,
the
diploid
foci
are
not
visible
as
separate
units
any
more.
This
may
be
the
consequence
of
an
intensive
proliferation
of
these
foci
and
of
the
rest
of
the
parenchyma
(the
latter
can
occur
now
because
there
is
no
mito-inhibition
by
the
2-AAF
any
longer),
so
that
they
dissipate
in
the
surrounding
parenchyma.
It
is
striking
that
perifocal
nuclei
show
intermediate
values
between
those
of
the
normal
and
the
focal
tissue.
However,
this
may
be
the
consequence
of
a
technical
artefact.
It
cannot
be
excluded
that
the
observed
perifocal
cell
population
is
a
mixture
of
focal
and
normal
cells,
because
of
the
difficulty
in
delineating
perfectly
the
foci
on
serial
sections.
It
must
further
be
stressed
that
there
is
an
important
interindividual
variation
between
animals
receiving
the
same
treatment,
so
that
care
must
be
taken
with
the
summing
up
of
the
individual
data.
In
spite
of
this
variation
a
trend
in
the
reduction
to
lower
C-values
can
be
seen
in
the
merged
data.
This
means
that
the
differences
between
different
tissues
in
the
same
animal
are
always
significantly
greater
than
the
differences
between
different
animals.
Emphasis
must
also
be
laid
on
the
fact
that
these
C-values
do
not
correspond
to
actual
ploidy
values.
A
4C
value,
for
example,
can
be
a
2N-nucleus
in
G2
or
a
4N-nucleus
in
G,.
Without
chromosome
counting
it
is
impossible
to
discriminate
between
them
on
that
basis
alone.
The
question
remains
of
by
which
mechanism
preneoplas-
tic
focal
cells
preferentially
contain
nuclei
in
the
2C
DNA-
range.
A
theoretically
conceivable
hypothesis
resides
in
the
fact
that
there
could
exist
a
population
of
hepatocytes,
delayed
or
blocked
in
G2
phase
(Bassleer
et
al.,
1985).
This
phenomenon
was
reported
in
skin
(Gelfant,
1977)
and
recently
suggested
in
adult
liver
(Daoust,
1987).
In
this
way,
an
appropriate
stimulus
could
drive
blocked
'tetraploid'
(4C)
nuclei
into
mitosis
within
a
few
hours
and
give
rise
to
diploid
hepatocytes
in
Go.
This
mechanism
is
unlikely
to
occur
in
our
model,
because
of
the
quasi-absence
of
unlabelled
foci,
at
least
in
the
early
stages.
A
second
possibility
is
based
upon
the
fact
that
CCl4
causes
a
massive
centrolubular
necrosis
in
the
liver,
because
of
the
metabolic
zonation
in
this
organ
(Jungermann,
1986).
This
could
suggest
that
the
remaining
periportal
parenchymal
cells
would
be
the
preferential
candidates
for
liver
regenera-
tion
(Fabrikant,
1968;
Grisham,
1962).
Since
these
periportal
cells
are
mostly
diploid
(Sulkin,
1943)
it
could
be
expected
that
emerging
cycling
cells
would
have
this
ploidy
level.
However,
early
foci
5
days
after
IS-CC14
are
not
exclusively
diploid;
foci
of
higher
ploidy
rate
also
emerge
at
that
time.
Moreover,
it
was
reported
earlier
that
there
was
no
preferen-
tial
lobular
distribution
of
early
foci
(Solt
et
al.,
1977).
Thus,
selective
necrosis
of
tetraploid
cells
cannot
be
considered
as
the
only
mechanism
of
diploidisation.
Rather,
even
tetraploid
focal
cells
can
emerge,
together
with
the
diploid
ones.
This
last
remark
holds
also
for
the
hepatocytes
which
were
only
hit
by
the
CCl4.
The
fraction
of
2C
cells
which
are
proliferating
is
as
great
as
the
4C
fraction.
This
confirms
the
hypothesis
which
states
that
the
outgrowth
of
2C
cells
(in
the
IS-protocol)
is
only
due
to
the
general
observation
that
the
mitosis
of
diploid
cells
occurs
more
easily
than
the
mitosis
of
tetraploid
cells.
There
must
be
another
factor,
linked
to
the
effects
of
initiation
and
selection,
which
makes
diploid
cells
more
likely
to
evolve
to
precancerous
states
than
tetraploid
ones.
Whether
the
process
of
diploidisation
is
a
general
process,
or
only
confined
to
certain
protocols,
is
an
unsolved
prob-
lem.
It
cannot
be
excluded
that,
in
spite
of
the
fact
that
ploidy
reduction
has
been
observed
in
many
carcinogenic
conditions,
this
phenomenon
is
the
result
of
a
co-selection,
only
linked
to
experimental
set-up.
In
order
to
ascertain
whether
the
polyploidisation-block
described
above
is
a
fun-
damental
process,
or
a
necessary
condition
for
cancer
development,
more
hepatocarcinogenic
protocols
should
be
analysed
for
this
parameter.
Nevertheless,
it
is
possible
that
mainly
diploid
foci
remain
at
later
stages
of
the
hepatocarcinogenic
process,
because
of
a
physiological
advantage
in
their
favour.
Previous
work
(Deleener
et
al.,
1987)
reported
diploidisation
of
early
preneoplastic
nodules
in
the
I-S-P
protocol
(Lans
et
al.,
1983).
It
was
shown
that
this
phenomenon
was
not
transient,
but
lasted
for
months
after
the
onet
of
promotion.
However,
it
must
be
stressed
that
if
foci
and
nodules
are
preferentially
diploid,
this
diploidisation
is
not
necessarily
a
characteristic
of
hepatic
carcinoma. This
means
that
the
process
of
dip-
loidisation
in
the
liver
is
linked
to
the
genetic
instability
of
preneoplastic
lesions,
rather
than
to
the
cancer
phenotype.
One
of
the
possibilities
is
that
the
diploid
cell
population
may
be
at
higher
risk
for
further
carcinogenic
alterations,
because
some
cancer
phenotypes
might
be
expressed
more
easy
in
a
diploid
than
in
a
tetraploid
genome
(on
the
assump-
tion
that
these
cancer
phenotypes
are
the
result
of
recessive
traits).
The
authors
wish
to
thank
Dr
H.
Taper
(UCL)
for
help
in
evaluating
the
histological
alterations,
Dr
J.
de
Gerlache
(UCL)
for
supplying
the
carcinogenic
diets,
and
M.
Vanmechelen
and
Fr.
Raymaekers
for
their
technical
assistance.
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