[CANCER RESEARCH 41, 2096-2102, June 1981]
0008-5472/81 /0041-0000$02.00
Role of Acute Hepatic Necrosis in the Induction of Early Steps in
Liver Carcinogenesis by Diethylnitrosamine1
Thomas S. Ying,2 D. S. R. Sarma, and Emmanuel Farber
Department of Pathology, Faculty of Medicine, University of Toronto, Toronto, Ontario M5S 1A8, Canada
ABSTRACT
Experiments were performed to assess the role of liver cell
necrosis in the induction of early steps in liver carcinogenesis
with diethylnitrosamine, as measured by the appearance of foci
of resistant hepatocytes that stain for y-glutamyl transpeptidase
and that are presumptive preneoplastic lesions in the rat. With
the use of a necrogenic dose of diethylnitrosamine
and an
assay for the carcinogen-induced
early stages or resistant
hepatocytes, the number of enzyme-altered foci was decreased
to a major extent (up to 62%) by posttreatment with diethyldithiocarbamate, a derivative of disulfiram. Such posttreatment
decreased to a large degree (78%) the cumulative labeling
index of hepatocytes following an initial exposure to diethylni
trosamine. The performance of partial hepatectomy up to 68
hr after such posttreatment restored the level of the induction
of the resistant hepatocytes. Nonnecrogenic doses of diethyl
nitrosamine or dimethylnitrosamine induced virtually no foci of
resistant hepatocytes but did so when coupled with cell prolif
eration. These results establish clearly an important role for
liver cell necrosis in the production of early steps in liver
carcinogenesis in one model. The mechanism for this effect
appears to be by the induction of compensatory liver cell
proliferation.
INTRODUCTION
The possible relationship of acute and chronic liver injury to
the pathogenesis of liver cancer in humans and in experimental
animals has been discussed for many years (1, 7-9, 11, 12,
20, 22, 26, 42, 44, 47). An association between cirrhosis of
the liver and liver cancer has been known for a long time. More
recently, hepatitis and chronic liver injury related to hepatitis B
virus have been receiving increasing attention as factors that
are probably important in the genesis of human liver cancer (1,
2, 24, 33, 48). Also, many of the chemical carcinogens that
induce liver cancer in experimental animals are acutely hepatotoxic.
Despite the general interest in this area, little of a definitive
nature concerning the possible role of liver cell injury in one or
more steps in the carcinogenic process in the liver has been
documented. A commonly associated cell response, cell prolif
eration, has been implicated in the overall process of liver
cancer development with several chemicals, especially ones
that are not ordinarily considered to be hepatocarcinogens (6,
7, 25, 42). Also, cell proliferation has been shown to be
' This research was supported by research grants from the Medical Research
Council of Canada (MT-5994), the National Cancer Institute of Canada, and the
National Cancer Institute, USPHS United States Department of Health, Education,
and Welfare (CA-21157 and CA-23958).
2 Recipient of a studentship from the Medical Research Council of Canada. To
whom requests for reprints should be addressed.
Received December 12. 1979; accepted February 13, 1981.
2096
important or essential in the genesis of 2 types of very early
lesions in the liver: (a) enzyme-deficient islands (28-30) and
(b) carcinogen-resistant
hepatocytes (3, 37-39, 45), each
induced by many carcinogens. However, what role, if any, is
played by acute liver cell injury in the early steps or in the
subsequent development of liver cancer has not been clarified.
Recently, 2 developments in our laboratory have permitted
us to formulate an approach to the question, does liver cell
death play any role in the initial steps of liver carcinogenesis,
and if so, what role?
One development is a new model for liver carcinogenesis
that enables an analysis of the first few steps in the process,
including an assay for one type of carcinogen-altered
cell, a
resistant hepatocyte (34, 35). This is based on the hypothesis,
for which there is increasing evidence (3, 12, 23, 34, 35, 3739, 45), that one form of early change may consist of the
induction by an initiating dose of a carcinogen of altered
hepatocytes resistant to the inhibitory effects of 2-AAF3 on cell
proliferation. Such resistant hepatocytes can be selectively
stimulated to proliferate rapidly to produce nodules by the use
of an appropriate selection pressure, such as a short exposure
to 2-AAF in combination with a stimulus for hepatocyte prolif
eration such as PH or CCU administration (3, 23, 35, 37-39,
45). In this model, with DENA as the initiating carcinogen, a
linear continuity (12, 13) between early resistant hepatocytes,
nodules, and metastasizing hepatocellular carcinoma has been
established (35), thus suggesting that the foci of resistant
hepatocytes, as a group, are early precursor lesions in liver
carcinogenesis. Since histochemical y-GT activity appears in
90 to 95% of the early hyperplastic foci and nodules under
these circumstance (23), this is a convenient marker for their
quantitation.
The second development is the dissociation of some bio
chemical and metabolic effects of DENA and DMN, including
alkylation of DNA and RNA, from their cytocidal effects by the
use of posttreatment with DEDTC (46). By administering
DEDTC at 4 or 8 hr after the administration of a usually
necrogenic dose of DENA (or of DMN), liver cell necrosis can
be prevented almost completely without inhibiting at least some
of the known interactions of these nitrosamines with cellular
nucleic acids. Thus, if such interactions with DNA or other
cellular components are important in the initiation of carcino
genesis with chemicals, it might be possible to study the role
of liver cell necrosis in the beginning of liver cancer develop
ment.
Using these combined approaches, it has been shown that
liver cell necrosis can play an important role in the induction of
early changes in liver carcinogenesis with DENA and DMN.
3 The abbreviations used are: 2-AAF, 2-acetylaminofluorene;
PH. partial hep
atectomy; DENA, diethylnitrosamine;
y-CT, y-glutamyl transpeptidase;
DMN,
dimethylnitrosamine;
DEDTC. diethyldithiocarbamate;
i.g., intragastric.
CANCER
RESEARCH
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VOL.
41
Liver Necrosis and Early Steps in Liver Carcinogenesis
The experimental evidence in support of this conclusion and
its possible mechanism are the subjects of this paper.
*
4hr- KM *
ExperimentalDControl
"jH**D'DTC
MATERIALS
AND METHODS
C10.9«'.:Î&NâCI
PH
Chemicals. DENA was obtained from Eastman Kodak
Co., Rochester, N. Y., and DMN was obtained from MerckSchuchardt, Munich, West Germany. Their chemical purities
were routinely checked by UV spectrophotometry and gas
chromatography prior to use. Sodium salt of DEDTC and all
other biochemicals were obtained from Sigma Chemical Co.,
St. Louis, Mo. [/T7efhy/-3H]Thymidine(specific activity, 40 Ci/
mmol) was purchased from New England Nuclear, Montreal,
Quebec, Canada.
Animals. Male Fischer344 rats (MicrobiologicalAssociates,
Walkersville, Md.), weighing 175 to 200 g, were maintained on
a semisynthetic diet containing 24% protein (Bio Serv, Frenchtown, N. J) and a daily cycle of alternating 12-hr periods of
light and darkness. The animals were given food and water ad
libitum and were acclimatized to their environment for 1 week
before the start of the experiment.
Experimental Regimen. In Regimen 1 (Chart 1), freshly
dissolved DENA in 0.9% NaCI solution was injected i.p. at a
dose of 100 mg/kg body weight. DEDTC was dissolved in
0.9% aqueous NaCI solution and was administered i.p. at a
dose of 50 mg/kg 3 times at 4-hr intervals, beginning at various
time periods from 4 hr to 10 days after DENA administration.
In control groups, rats received equivalent volumes of 0.9%
NaCI solution instead of DENA (Group C2) or DEDTC (Group
C1). After a 2-week recovery period, the animals were assayed
for resistant cells in the liver (34, 35, 39) by being placed on
a basal diet containing 0.02% 2-AAF in order to create an
inhibitory effect on cell proliferation (34) and 1 week later being
subjected to a standard two-third PH procedure (16). After a
total period of 14 days on the 2-AAF diet, the animals were
sacrificed, and liver samples were processed for histological
examination and for quantitative histochemical analysis.
In Regimen 2 (Chart 2), PH was performed 3 days following
the administration of DENA and DEDTC. Because the dose of
DENA might inhibit the normal regenerating potential after PH
(34, 35), the animals were given no further treatment for 4
weeks. At that time, the animals were assayed for resistant
hepatocytes by placing them on a diet containing 0.02% 2AAF for 7 days and then subjecting them to a liver cell prolif
eration stimulus generated by a single dose of carbon tetrachloride (2 ml/kg body weight in corn oil) i.g. (3, 38, 45). After
a total period of 2 weeks on the 2-AAF diet, the rats were kept
on the basal diet devoid of 2-AAF for 7 days and were then
sacrificed. The 3 control regimens, as depicted in the charts,
lacked PH, DEDTC, and DENA.
In Regimen 3 (Chart 3), a single nonnecrogenic dose of
either DMN (10 mg/kg body weight) or DENA (15 mg/kg body
weight) was administered ¡.p.,and at 4 or 24 hr later, respec
tively, the rats were subjected to PH or sham hepatectomy
(Group C1). Resistant hepatocytes were stimulated selectively
to grow according to the procedure described previously in
Regimen 2. Control Group C2 received vehicle plus PH.
All animals were fasted overnight before sacrifice.
Incorporation of [3H]Thymidine into DNA. Groupsof 4 rats
were given DENA (100 mg/kg ¡.p.),and 4 hr later they were
given injections of either DEDTC (50 mg/kg) 3 times at 4-hr
Control C2
3
Weeks
0 4
Hours
Chart 1. Schematic representation of experimental and control protocols for
the determination of the effect of posttreatment with DEDTC on the induction of
foci of resistant hepatocytes by a necrogenic dose of DENA. Experimental group
had 4 subgroups (E1, E2, E3, and E4) which received 3 doses of DEDTC (50
mg/kg ¡.p.at 4 hourly intervals) beginning at 4, 8, or 24 hr or 10 days (d)
respectively, following administration of DENA (100 mg/kg i.p.). Open bars,
basal diet; hatched bars, diet containing 0.02% 2-AAF.
PH
DOExperimentalDEIControl
1"l,«DEDTC
III
<JA 111
SH
1
C9''
|
C1onControl
09S'*
IUCI
»j»»»*PH
C2
DBControl
09***j!1|C(TINaCI
C30.9".Control
CDEDTC
PH
t)
It
»
C4I
4*irsu172
He*DEDTC
1
4
5
Week
6
7
Chart 2. Experimental design to illustrate the influence of PH in reversing the
inhibitory effect of DEDTC on the development of DENA-induced y-GT-positive
foci of resistant hepatocytes. Control regimens consisted of C1 lacking PH. C3
lacking DEDTC and PH, and C4 lacking DENA. SH, sham hepatectomy.
intervals or 0.9% NaCI solutions. From 1 to 14 days, [methyl3H]thymidine was given i.p. at 8 a.m., 12 noon, 4 p.m., 8 p.m.,
and 12 midnight at a dose of 0.2 /iCi/g body weight to label
proliferating hepatocytes. At the time of sacrifice, part of the
liver was processed for the determination of the specific activity
of DNA. DNA was isolated and purified by the procedure of
Kirby and Cook (18). Appropriate aliquots from the remainder
of the liver were processed for autoradiographic analysis of
labeled cells.
For autoradiographic analysis, multiple sections of the liver
approximately 1 mm thick were obtained and placed in 4%
formalin fixative for 24 hr. They were then processed and
stained with hematoxylin and eosin. Additional contiguous par
affin sections were cut for autoradiographic analysis. These
were deparaffinized, stained with eosin or periodic acid-Schiff,
air dried, coated with Kodak NTB3 emulsion, and stored des
iccated at 4°for at least 2 weeks. They were then developed
and counterstained with hematoxylin for microscopic exami
nation.
Histochemical Localization of y-GT. Liversharvestedon the
final day of each regimen were studied histochemically for the
localization of y-GT using the method described by Rutenberg
ef al. (27) as modified by Ogawa ef al. (23). The number of yGT-positive foci was counted by low-power microscopy and
expressed as foci per sq cm of section area. The long and
short axes of the foci were measured with the use of a microm-
JUNE 1981
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2097
T. S. Ying et al.
DMN or DENÀ
T
I 7
Experimental
DMN 01 DENA
T
Control
itrol C11
C1 f
0.9SNÃŽCT
t
Control C2
0 4 or 24 1
Hours
3
Weeks
Chart 3. Experimental protocols to show the effect of PH on the induction of
•^GT-positivefoci by nonnecrogenic doses of DMN or DENA. Control Group C1,
animals which received either DMN or DENA and SH; Control Group C2. animals
which received vehicle plus PH; SH, sham hepatectomy.
éterin an eyepiece, and the average of the 2 values was used
as the focus ' 'diameter.'' The area of the section was measured
with a planometer.
RESULTS
Inhibition of DENA-induced y-GT-positive Foci by DEDTC.
The results presented in Table 1 show that DEDTC, when given
at 4 or 8 hr after DENA, decreased to a considerable degree
(62 and 38%, respectively) the number of y-GT-positive foci
induced by a necrogenic dose (100 mg/kg) of DENA (Fig. 1).
It was shown previously that a 4-hr posttreatment with DEDTC
effectively prevented almost all necrosis and that a 8-hr posttreatment prevented a considerable amount of necrosis with
the same dose of DENA (46). The prevention of necrosis was
not complete but almost so, as indicated by the degrees of
elevation in serum sorbito! dehydrogenase and glutamate-pyruvate transaminase and by histological examination (46). The
inhibitory effect on the genesis of foci of y-GT-positive and
-resistant hepatocytes does not appear to be related to any
effect of DEDTC per se, since this compound had no effect
when given at 24 hr or at 10 days, times well before the
beginning of the assay procedure for resistant foci. DEDTC
had no effect on liver cell necrosis when given at 24 hr (46).
The range in the diameters of all the y-GT-positive foci in
representative sections (110 to 1380 (im) and the mean diam
eters under each condition were essentially the same (Table
2). The findings validate the use of the number of foci per area
as a quantitative measure.
Since cell proliferation is required for the induction of resist
ant hepatocytes during initiation by many carcinogens (3, 3739, 45), it is possible that the role of liver cell necrosis in
initiation is in the stimulation of cell proliferation as a regener
ative response. This was tested by determining whether PH
performed after the administration of DEDTC would reverse the
inhibitory effect on the development of DENA-induced y-GTpositive foci of resistant hepatocytes. The experimental design
is illustrated in Chart 2.
As is evident from the results in Table 2, PH reversed
completely the decrease in the number of foci seen with posttreatment with DEDTC. The 67% decrease seen in the group
with DENA plus DEDTC became a 41% increase when PH
followed the DEDTC. This "overshooting"
with PH was also
seen in the group treated with DENA only, which showed a
74% increase in the level of induction of foci of resistant
hepatocytes.
If the role of necrosis in the induction of resistant hepatocytes
2098
with DENA is mainly to stimulate cell proliferation, posttreat
ment with DEDTC should decrease, if it does not prevent
completely, the postnecrotic proliferation of hepatocytes. In
order to test for this possibility, the cumulative labeling index
of hepatocytes was measured over a 2-week period after the
administration of DENA with and without posttreatment with
DEDTC. As may be seen in Table 3, the cumulative labeling
index with DENA alone was 13%, and this was decreased to a
large degree (78%) by the 4-hr posttreatment with DEDTC.
These observations are consistent with the suggestion that the
DEDTC might be inhibiting the induction of early changes in
liver carcinogenesis by decreasing secondarily the regenera
tive hepatocyte proliferation as a response to its effect in
decreasing the degree of cell necrosis. It should be pointed
out that theoretically some of the decrease in labeling index
seen in the DENA-DEDTC-treated animals could be due to an
inhibition of thymidine incorporation by the DEDTC. This pos
sibility is very unlikely, since DEDTC had no effect on liver
regeneration following DENA-induced necrosis (46) when given
24 hr after the DENA. Also, the 14-day period of accumulation
of labeled hepatocytes makes any such inhibitory effect of
DEDTC very improbable, since DEDTC must be given repeat
edly every few hr if measurable effects studied are to be
sustained even for a few hr (46).
Effect of PH on the Induction of Foci of Resistant HepaTable 1
Inhibition of DENA-induced putative preneoplastic
posttreatment with DEDTC
of
or0.9%
DEDTC
NaCIsolution
ad
Group0C1E1E2E3E4C2Treatment6DENA
ministration4
NaCIsolutionDENA
+ 0.9%
hr4
DEDTCDENA
+
DEDTCDENA
+
DEDTCDENA
+
DEDTC0.9%
+
solution+
NaCI
DEDTCTime
hr8
hr24
hr10
days4
hrNo.
lesions in rat liver by
T^GT-positivefoci/sq
of
cm29.1
(8)d1 ±1.9°
(9)18.1
1.0 ±1.2
(9)27.1±2.1
(7)24.7±2.6
(6)0.5±2.0
±0.2
(8)%ofdecrease62387IS
E1 and E2 are significantly different from C1 and C2 (p < 0.001). E3 and
E4 are not significantly different from C1 (p = 0.39 and p = 0.1, respectively)
(Student's ( test).
" Experimental details are given in Chart 1.
c Mean ±S.E.
d Numbers in parentheses, number of animals used.
Table 2
Reversal by PH of the inhibitory effect of DEDTC on the appearance of DENAinduced y-GT-positive focal lesions in rat liver
GroupC301ElC2C4Treatment3DENA
+ 0.9% NaCI
SH0DENA
solution +
SHDENA
+ DEDTC +
PHDENA
+ DEDTC +
NaCIsolution
+ 0.9%
PH0.9%
+
+DEDTC
NaCI solution
+ PHNo.
of f-GT-poçitive
cm26.7
foci/sq
(9)'9.3
±1.8e
(12)37.8
±0.7
(13)47.4
±6.0
(8)0.6±6.4
±0.3
of foci
danf434
22439±
28498±
47576±
76428±
(8)Diameter
Experimental details are given in Chart 2.
6 E1 is significantly different from C1 (p < 0.001), C1 is significantly
±21
different
from C3 (p < 0.01), and C2 is significantly different from C3 (p < 0.005).
c The average size of the foci is not significantly different between groups (p
d SH, sham hepatectomy.
9 Mean ±S.E.
' Numbers in parentheses,
effective number of rats.
CANCER
RESEARCH
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VOL. 41
Liver Necrosis and Early Steps in Liver Carcinogenesis
Table 3
Effect of DEDTC on the hepatic hyperplasia induced by DENA
Groups81
+ 0.9% NaCI solution
23Treatment"DENA
DENA + DEDTC
0.9% NaCI solution + DEDTCIndex
of labeled
(%)13.0
nuclei
±1.8C (4)"
2.9 ±1.4 (4)
<0.1 (4)
Group 1 is significantly different from Group 2 (p < 0.01). Groups 1 and 2
are significantly different from Group 3(p < 0.001 ) (Student's i test).
After the appropriate treatment, animals were given [3H]thymidine injections
(0.2 iiC\/g bodyweight) i.p. beginning at Day 1 and continuing to Day 14. Each
animal received 5 doses daily (8 a.m., 12 noon, 4 and 8 p.m., and midnight) for
13 days and was killed on the 14th day. The livers were processed for autoradiography. Only the hepatocytes with heavily labeled nuclei were counted using
a magnification of 400-fold. The heavily labeled cells represent proliferating cells.
In contrast, cells with sparsely labeled nuclei (<30 grains), probably representing
repair replication, were not counted. This type of cutoff may eliminate some bona
fide hepatocytes in S phase, but this was deliberate. Labeling indices for
individual livers were taken as the average value determined from counts of 10
random high-power fields, each consisting of 150 to 200 cells.
c Mean ±S.E.
Numbers in parentheses, effective number of rats.
tocytes by Nonnecrogenic Doses of DENA or DMN. If necrosis
is playing a role in the initiation with DENA or with DMN by
associated
regenerative
cell proliferation,
nonnecrogenic
doses of each nitrosamine should induce few if any foci unless
coupled with a stimulus for cell proliferation. As seen in Table
4, PH performed at 4 hr after DMN or at 24 hr after DENA
increased considerably the number of foci induced with the
compounds. It should be pointed out that the number of foci
induced by carcinogens coupled with PH varies considerably
with the timing of each procedure (3, 39, 45) and that the time
sequence as used here does not lead to a maximum induction
of foci. This is why the number of foci in these experiments is
considerably less than in other types of experiments, such as
in those included in Tables 1 and 2. In these experiments,
times for PH were selected at which no detectable levels (less
than 5 to 10 ng/ml) of DMN (4 hr) or of DENA (24 hr) were
found in the blood and liver (46).
It should be noted that in Experimental Regimens 2 and 3,
CCI4 was used instead of PH as a liver cell proliferative stimulus
in the assay for resistant hepatocytes (3, 38, 39, 45). The foci
obtained under these conditions were indistinguishable from
those seen when 2-AAF plus PH is used as the selecting
environment (Fig. 2). This similarity was evident with respect to
all parameters including vesicular nuclei with prominent nu
cleoli, pseudoacinar arrangement of the hepatocytes, and tend
ency to accumulate glycogen (Fig. 3). However, with the 2AAF plus CCLt, an extra week on stock diet after the 2-week
exposure to dietary 2-AAF was allowed in order to ensure the
production of grossly visible grayish-white elevated nodules
(38, 39). It should be emphasized that the control rats exposed
to 2-AAF plus PH or 2-AAF plus CCU without prior treatment
with DENA developed very few if any resistant foci (Tables 1,
2, and 4).
DEDTC suggests that those interactions of DENA with the
target molecule(s) essential for the development of preneoplas
tic foci were not affected by such treatment. This supposition
is further strengthened by the results of a previous study in
which it was shown that DEDTC posttreatment did not affect
the total extent of ethylation of liver nucleic acids although the
rates of alkylation were different (46). The results presented
also suggest that the key role played by necrosis in the induc
tion of early events in liver carcinogenesis may be by stimulat
ing compensatory cell proliferation. However, it is not yet
unequivocally established whether compensatory cell replica
tion is the only mechanism through which necrosis may exert
its effect on the induction of preneoplastic lesions. Suffice it to
say that DENA-induced liver cell necrosis is important but not
optimum for the induction of preneoplastic lesions, since sup
plementation with PH resulted in a further increased appear
ance of y-GT-positive foci (Table 2, compare Group C2 with
C3). Even though the mechanism responsible for stimulation of
cell proliferation by the toxic effects of the carcinogen may or
may not be identical to that operating after PH, it has been
shown that the proliferative response resulting from acute
exposure to DENA is less effective in the induction of putative
preneoplastic foci than is two-thirds surgical removal of liver.
The importance of cell proliferation in the induction of pre
neoplasia is further substantiated by the observation that a
nonnecrogenic dose of DMN or DENA induced preneoplastic
lesions only in rats that were subjected to PH but not in those
that received SH following the administration of the carcinogen.
The mechanism whereby cell proliferation participates in the
early events in the carcinogenic process is not known. How
ever, an attractive possibility is the replication of carcinogendamaged DNA, prior to its repair, with "fixation" of the damage
in the daughter strands (7, 25, 42).
Several studies have shown that DEDTC or disulfiram, its
parent compound, inhibited the development of tumors in var
ious organs induced by a variety of carcinogens (31, 32, 43).
However, in most of these instances, the protective agent was
given either along with or prior to the administration of the
carcinogen. Under these experimental conditions, it is possible
that DEDTC or disulfiram may inhibit the activation of the
carcinogen and thus may reduce the tumor incidence by de
creasing the generation of the ultimate form(s) of the carcino
gen. The mechanism whereby posttreatment with DEDTC at 4
Table 4
Influence of PH on the induction of y-GT-positive foci of resistant hepatocytes
by nonnecrogenic dose of DMN and DENA
of -y-GT-positive
Group"EC1C2Treatment"DMN
cm12.4
foci/sq
±1.8°(15)d
+ PH
DENA +
PHDMN
+ SHe
DISCUSSION
The results of this study established for the first time an
essential role of liver cell necrosis in the development of early
putative preneoplastic liver lesions induced by a necrogenic
dose of DENA.
The observation that cell proliferation induced by an exoge
nous stimulus such as PH could reverse the inhibitory effect of
JUNE
SH0.9%
DENA +
NaCI solution + PHNo.
8.0
(18)1.8
±1.0
±0.5
(19)0.8
1.1 ±0.3
(8)
±0.2 (12)
Experimental Groups E are significantly different from Control Groups C1
and C2 (p < 0.001). Groups C1 are not significantly different from C2. (p = 0.48
for DMN and p = 0.68 for DENA) (Student's ( test).
6 Experimental details are given in Chart 3.
c Mean ±S.E. for 2 separate experiments.
d Numbers in parentheses, effective number of rats.
8 SH, sham hepatectomy.
1981
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2099
r. S. Ying et al.
or 8 hr largely prevents hepatocyte necrosis is not known.
Since later treatment with DEDTC does not appear to inhibit
necrosis, it is unlikely that the DEDTC is merely interfering in
some way with the process of cell autolysis (necrosis) that
characterizes cells following their death. Rather, it would ap
pear that DEDTC somehow reverses some reversible step in
the multistep sequence that seems to be characteristic of cell
death in many cell types including the liver (46). Conceivably,
its role as a free radical "scavenger" or as a chelating agent
for some ions may be important in this context.
The results of this study offer a possible explanation for the
effects of liver injury with cell death in various infections in the
genesis of liver cancer. For example, hycanthone, an antischistosomal drug and a nonliver carcinogen, becomes carci
nogenic in mice infected with schistosomes (14, 15) or in
noninfected mice if coupled with PH (40). Infection with schis
tosomes or other trematodes is associated with liver cell death
and liver cell proliferation (4, 10,14,15,
19, 36, 40). Thamavit
et al. (36) reported that an otherwise noncarcinogenic dose of
DMN induced cholangiocarcinoma
in Syrian golden hamsters
when they were infected with the parasite Opisthorchis viverrini. Similarly, Domingo et al. (10) observed that low doses of
2-amino-5-azotoluene
produced a much higher incidence of
hepatomas in mice infected with Schistosoma mansoni than in
uninfected mice. Equally convincing is the fact that several
carcinogens that normally do not induce liver cancer in adult
animals, especially with a single dose, induce preneoplastic
and neoplastic liver lesions when given after PH (5-7, 17, 21,
38, 41).
Finally, and most importantly, the results of the present study
implicate liver cell injury as a possible factor in the genesis of
human liver cancer. The increasing suspicion of a relationship
between hepatitis B-virus and liver cancer (1, 2, 24, 33, 48)
and the long-known association between nonneoplastic liver
disease, including cirrhosis, and liver cancer could be in part
explained by a role for liver cell death in the induction of very
early changes in liver carcinogenesis by chemicals.
ACKNOWLEDGMENTS
The authors wish to thank Esther Roberts for her excellent technical assist
ance, Esther Deak and Patrick Home for the preparation of histological and
histochemical specimens, and Hélène
Robitaille for the careful preparation of the
manuscript.
REFERENCES
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2. Blumberg, B. S. Australia antigen and the biology of hepatitis B. Science
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3. Cayama, E. Tsuda. H., Sarma. D. S. R., and Farber, E. Initiation of chemical
carcinogenesis
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Liver Necrosis and Early Steps in Liver Carcinogenesis
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Fig. 1. Histochemical staining for y-GT of a liver section from a rat showing y-GT-positive foci of resistant proliferating hepatocytes distributed randomly throughout
the lobe. The animal received DENA (100 mg/kg), followed by a selection procedure as shown in Chart 1. x 10.
Fig. 2. Photomicrograph of a liver section showing a y-GT-positive focus of proliferating hepatocytes. The rat was treated with DENA (100 mg/kg) and DEDTC 4
hr later with PH 72 hr later, followed by a selection procedure as shown in Chart 2. H & E, X 40.
Fig. 3. Higher magnification of Fig. 2 showing portions of the hyperplastic focus with one arrow of at least 3 mitotic figures seen in the whole focus. The
architecture of the hepatocytes is not evident in this particular focus since the sinusoids are collapsed. The liver cells show vacuolization largely because of periodic
acid-Schiff-positive
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JUNE
1981
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VOL. 41
Role of Acute Hepatic Necrosis in the Induction of Early Steps
in Liver Carcinogenesis by Diethylnitrosamine
Thomas S. Ying, D. S. R. Sarma and Emmanuel Farber
Cancer Res 1981;41:2096-2102.
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