Carcinogenesis vol.20 no.7 pp.1169–1175, 1999
Resistance to the promotion of glutathione S-transferase 7-7positive liver lesions in Copenhagen rats
Geoffrey A.Wood1, Dittakavi S.R.Sarma2 and
Michael C.Archer1,3,4
1Department
of Medical Biophysics, 2Department of Laboratory Medicine
and Pathobiology and 3Department of Nutritional Sciences, University of
Toronto, Toronto M5S 3E2, Canada
4To whom correspondence should be addressed at: Department of
Nutritional Sciences, Faculty of Medicine, FitzGerald Building, 150 College
Street, Toronto M5S 3E2, Canada
Email: [email protected]
Previously, we have shown that Copenhagen (Cop) rats are
highly resistant to the induction of putative preneoplastic,
glutathione S-transferase 7-7 (GST 7-7)-positive liver
lesions following treatment with a modified resistant hepatocyte protocol. The objective of the current study was to
establish the time course for the development of resistance
and examine potential resistance mechanisms in Cop rats
using F344 rats as susceptible controls. Male Cop and F344
rats (n J 25), 7–8 weeks of age, were initiated with
diethylnitrosamine (200 mg/kg) and promoted 3 weeks later
with four doses of 2-acetylaminofluorene (20 mg/kg) and a
2/3 partial hepatectomy (PH). Groups of rats from each
strain were killed on days 2, 4, 7, 14 and 21 post-PH, 2 h
after receiving bromodeoxyuridine. Cop livers contained
similar numbers of GST 7-7-positive lesions to F344 livers
on days 2 and 4 post-PH. The percent volume of liver
occupied by these lesions did not differ between the strains
on days 2, 4 and 7 post-PH. On day 14, however, ~29% of
the liver volume in F344 rats was occupied by lesions,
whereas in Cop rats this was significantly less (~9%, P <
0.001). On day 21, lesions occupied ~58% of F344 rat livers
and only ~6% of Cop livers. Despite these differences, the
labeling index of hepatocytes was not significantly different
between the strains at any time point, either within lesions
or within surrounding normal liver. Furthermore, the
apoptotic indices were not different between the strains at
any time. However, differences were found in the extent of
lesion remodeling (redifferentiation) and in the pattern of
oval cell response following PH in Cop livers. By day 14
post-PH, ~76% of Cop liver lesions showed evidence of
remodeling, compared with only ~14% of F344 lesions.
The oval cell response to PH was equivalent in the two
strains up to day 4 post-PH but by day 7, in F344 livers
there was extensive migration of these cells into the liver
parenchyma, whereas in Cop livers, the response remained
localized to the portal regions. These results suggest that
Cop resistance occurs at the promotion stage and not the
initiation stage of carcinogenesis. Resistance appears not
to be due to a lower proliferation rate nor to a higher
apoptotic rate within Cop lesions. Precocious remodeling
Abbreviations: 2-AAF, 2-acetylaminofluorene; AI, apoptotic index; BrdU,
59-bromodeoxyuridine; Cop, Copenhagen; DEN, diethylnitrosamine; GST 7-7,
glutathione S-transferase 7-7; HGF, hepatocyte growth factor; LI, labeling
index; PBS, phosphate-buffered saline; PH, 2/3 partial hepatectomy; RH,
resistant hepatocyte.
© Oxford University Press
and/or a diminished oval cell response, however, may
contribute to the resistance of Cop rats to the growth of
GST 7-7-positive hepatic lesions.
Introduction
Previously, we have shown that Copenhagen (Cop) rats are
highly resistant to the formation of preneoplastic liver lesions
induced by diethylnitrosamine (DEN) and promoted using a
modified resistant hepatocyte (RH) protocol (1). However,
since these lesions were measured 3 weeks after the start of
promotion, we do not know when during this process resistance
occurs or what mechanism(s) confers resistance. In the present
study, we established the time course for the development of
resistance and examined potential resistance mechanisms in
Cop rats using F344 rats as susceptible controls.
In the RH model of hepatocarcinogenesis, the initiating
agent results in the formation of single cells and small foci of
putative initiated hepatocytes (2,3). Phenotypic markers for
these lesions include altered expression of a number of enzymes
that can be detected histochemically, such as the placental
form of glutathione S-transferase (GST 7-7) (4,5). The lesions
are then promoted with three doses of 2-acetylaminofluorene
(2-AAF) followed by a partial hepatectomy (PH) and then a
fourth dose of 2-AAF (6). 2-AAF is mito-inhibitory to normal
hepatocytes but does not inhibit a subpopulation of the GST
7-7-positive hepatocytes. The PH acts as a mitotic stimulus
that results in the rapid growth of this subpopulation into
larger GST 7-7-positive lesions, while the surrounding liver
remains quiescent. Some lesions eventually develop into
grossly visible nodules and, over months, tumors arise (6).
Two other phenomena that occur following the promotional
stimulus of the RH protocol are remodeling (or redifferentiation) of lesions and the proliferation and migration of oval
cells. Hepatocytes within remodeling lesions lose expression
of GST 7-7 and other enzymes common to preneoplastic liver
lesions and also revert to a more normal liver architecture
(7,8). Remodeling occurs in most lesions over the months
following promotion. Lesions that remain are one site within
which hepatocellular carcinoma can develop. Proliferation and
migration of oval cells occurs in rat liver following treatment
with a variety of chemical compounds (9). Oval cells are
thought by some to be bipotential cells that can differentiate
into either bile duct cells or hepatocytes (10–12). Oval cells
have been proposed to play a role in this model of hepatocarcinogenesis since their proliferation and migration may be an
attempt by the liver to regenerate under conditions where
hepatocytes cannot divide, such as occurs during the RH
protocol (13,14).
In addition to establishing the time course of resistance in
Cop rats, in the present study we have also explored potential
mechanisms of resistance. The proliferation of hepatocytes
within lesions and in surrounding normal liver as well as
apoptosis in lesions of both strains were examined. A lower
1169
G.A.Wood, D.S.R.Sarma and M.C.Archer
proliferation rate and/or higher apoptotic rate in lesions of
Cop rats could reduce their overall growth rate. We also
determined whether Cop rats have a greater proportion of
lesions undergoing remodeling and/or a diminished oval cell
response compared with susceptible F344 rats.
Materials and methods
Chemicals
DEN (Eastman Kodak, Rochester, NY) was .98% pure by gas chromatography. Normal swine serum and swine anti-rabbit biotinylated antibody were
from DAKO (Mississauga, Ontario, Canada). 2-AAF, 3,39-diaminobenzidine
tetrahydrochloride (DAB) and X-Gal were from Sigma (St Louis, MO). The
rabbit anti-rat GST 7-7 antibody was a gift from Dr Tom Rushmore
(4). 59-Bromodeoxyuridine (BrdU), the monoclonal antibody to BrdU and
streptavidin-β-galactosidase conjugate were purchased from Boehringer
Mannheim (Dorval, Quebec, Canada). The peroxidase-conjugated sheep antimouse antibody was from Santa Cruz Biotechnology (Santa Cruz, CA).
Animals
Cop and F344 rats, purchased from Harlan Sprague–Dawley (Indianapolis,
IN), were allowed to acclimatize for 1 week prior to the start of experiments
at which time they were 7–8 weeks old. Food (Harlan Teklad, 6% fat,
Madison, WI) and acidified water (pH 2.8) were provided ad libitum and a
12 h light–dark cycle was maintained automatically.
Animal treatments
Twenty-five Cop and 25 F344 rats were administered a single i.p. dose of
200 mg/kg DEN dissolved in 0.9% NaCl solution. All rats were then treated
using a modified RH protocol (6). Briefly, 18 days after DEN, three daily
gavages of 20 mg/kg 2-AAF in DMSO and corn oil (1:29, vol:vol) were
given followed by a PH. A fourth 20 mg/kg dose of 2-AAF was given the
day after PH. Groups of five rats from each strain were killed on days 2 and
4 post-PH, six rats from each strain on day 7, and four Cop and three F344
rats on day 14 post PH. Representative liver samples were fixed in 5% acetic
acid in methanol. Using this regime we found that a number of rats of both
strains became moribund around day 11 post-PH such that there were too few
rats to make comparisons at our intended 21 day time point. Therefore, to
obtain data at 21 days, a group of five Cop and eight F344 rats were treated
as above except the fourth dose of 2-AAF was given 4 days after PH and
was lowered to 5 mg/kg. This is a commonly used regime that has a
promoting effect equivalent to the protocol that we used for the rats killed on
days 2–14 (6). The broken line connecting points in Figures 2, 3, 4 and 7
indicates this change in protocol.
To label newly synthesized DNA, 100 mg/kg BrdU dissolved in phosphatebuffered saline (PBS) was injected i.p. 2 h before killing, and a sample of
duodenum that normally has a high rate of cell division, was taken as positive
control for BrdU incorporation.
Immunohistochemical staining
After fixing overnight, tissues were embedded in paraffin wax and 2 µm
sections were prepared on microscope slides. Double immunohistochemical
staining was carried out for GST 7-7 using X-Gal as a chromogen, and for
BrdU using DAB as a chromogen, as described by Stinchcombe et al. (15).
Briefly, following deparaffinization and rehydration, sections were blocked
with 10% normal swine serum for 10 min, then rabbit anti-GST 7-7 antibody
was applied (1:2000) overnight at 4°C. The following were then applied to
the sections at room temperature, washing with PBS between steps: biotinylated
swine anti-rabbit antibody at 1:500 for 2 h, streptavidin-β-galactosidase
complex at 1:200 for 2 h and X-Gal substrate for 1 h. All dilutions were
made in 1% normal swine serum in PBS.
Sections were then dehydrated and placed in 1% hydrogen peroxide in
methanol for 15 min to block endogenous peroxidase activity, rehydrated and
incubated in 4 N HCl for 15 min followed by rinsing in water and then PBS.
BrdU staining was carried out by blocking with 1% bovine serum albumin in
PBS, applying primary antibody (1:200) at 4°C overnight, rinsing in PBS,
and then applying secondary antibody conjugated to peroxidase (1:200) for
2 h at room temperature. Sections were washed again and DAB was used as
a chromogen. Dilutions for BrdU staining were made in 0.1% bovine serum
albumin in PBS. Sections were finally counterstained with hematoxylin and
eosin following the method described by Stinchcombe et al. (15).
Analysis of stained sections
The areas occupied by GST 7-7-positive lesions in liver sections were
measured using a morphometric analysis machine (Bioquant IV; Zeiss, Wetzler,
Germany) and converted to the percent of liver volume occupied by lesions
and the number of lesions per liver using the method of Enzmann et al. (16).
1170
Only lesions with radii .35 µm in diameter were included in the analysis
(17,18). Lesions undergoing remodeling often had indistinct borders but their
areas were measured in the same manner as non-remodeling lesions, tracing
the outermost edge of the lesion.
Random microscope fields from all lobes were scored for BrdU-positive
hepatocytes within GST 7-7-positive lesions. The labeling index (LI) in each
rat was calculated as the percent of hepatocytes within lesions that were
BrdU-positive after at least 1000 hepatocytes were counted per rat. The same
procedure was used to obtain an LI for surrounding GST 7-7-negative
hepatocytes.
The mean apoptotic indices (AI) within lesions of each group of rats were
expressed as the percent of apoptotic cells after at least 2000 hepatocytes
within lesions had been counted per rat. Lesions in F344 rats on days 14 and
21 post-PH were very large, so at least 5000 hepatocytes within lesions were
examined in these livers. Apoptotic cells within lesions were identified using
the property of eosin fluorescence of apoptotic bodies (15) in random
microscope fields, and their identity was confirmed using normal transmitted
light.
Lesions undergoing remodeling or redifferentiation were identified by their
patchy GST 7-7 staining and indistinct borders (7). The extent of remodeling
in each rat was expressed as the percent of all lesions in a section undergoing
remodeling. Oval cells were identified by their characteristic ovoid nucleus
and scant cytoplasm (9).
Since individual lesions may have differences in LI or AI that could
influence their growth rate, we measured these parameters as well as the
extent of remodeling in 20 randomly selected individual lesions in each rat.
Statistical tests
Results were analyzed using a 2-way ANOVA, and a Tukey post-test was
used to compare one group of animals to another. P-values ,0.05 were
considered significant.
Results
Gross appearance
Cop and F344 rats were initiated with DEN and promoted
using a modified RH protocol (6). Groups of rats of each
strain were killed on days 2, 4, 7, 14 and 21 post-PH. No
lesions were grossly visible in any of the livers until day 14
when all F344 livers had many pale yellowish-white nodules
clearly visible on the surface and in sections. By day 21 these
lesions occupied much of the liver (Figure 1A). In contrast,
Cop livers were essentially free of gross lesions at every time
point, with only two livers having two small pale spots each
on day 14. There were no liver lesions in any of the Cop rats
killed on day 21 (Figure 1B).
Number of GST 7-7-positive lesions per liver
We quantified the number of lesions per liver by multiplying
the estimated number of GST 7-7-positive lesions/cm3 by the
weight of the liver (in g). Calculated in this manner, Cop and
F344 rats had the same number of lesions per liver on days 2
and 4 post-PH, whereas from day 7 onwards, Cop rats had
significantly more lesions per liver than F344 rats (Figure 2).
It should be noted, however, that on days 14 and 21 F344
livers contained large lesions that were coalescing.
Percent liver volume occupied by GST 7-7-positive lesions
Following immunohistochemical analysis for GST 7-7-positive
lesions, no difference in the percent of liver volume occupied
by lesions was seen between strains until day 14, when a clear
difference between the Cop and F344 rats became evident
(Figure 3). This difference was even greater on day 21.
BrdU labeling index
To estimate cell proliferation rates, BrdU was injected into
rats 2 h before killing. The labeling indices of hepatocytes
within GST 7-7-positive lesions and in the surrounding normal
parenchyma are shown in Figure 4. There were no differences
in hepatocyte labeling indices between rat strains, either within
lesions or surrounding normal liver, at any time point during
Resistance to promotion of liver preneoplasia
Fig. 3. Percent of liver volume occupied by GST 7-7-positive lesions in
Cop and F344 rats treated as in Figure 1. There were no differences
between strains from the time of PH up to day 7 post-PH. On days 14 and
21 Cop rats had a significantly smaller percent of liver volume occupied by
GST 7-7-positive lesions than F344 rats (*P , 0.001).
Fig. 1. Gross appearance of (A) F344 and (B) Cop rat livers following
initiation with DEN and promotion using a modified RH protocol. Rats
were killed 21 days following PH. All F344 rat livers had numerous pale
yellowish-white nodules on their surface (arrows) whereas all Cop rat livers
appeared grossly normal.
Fig. 4. LI of hepatocytes within GST 7-7-positive lesions and in the
surrounding parenchyma in Cop and F344 rats as measured by BrdU
incorporation. There were no significant differences in LI between the
strains at any time point, either within lesions or surrounding parenchyma.
using transmitted light. The apoptotic indices were not significantly different between the strains at any time point
(Figure 5).
Fig. 2. Estimated number of GST 7-7-positive lesions per liver in Cop and
F344 rats treated as in Figure 1. As in all figures, each value is the mean 6
SEM of five F344 or five Cop rats, except for day 14 (three F344 and four
Cop rats) and day 21 (eight F344 rats). Cop rats had significantly more
lesions than F344 rats on days 7, 14 and 21 post-PH (*P , 0.05).
the experiment. The intestinal epithelial cells of the duodenum
used as a positive control had a high LI in all of the rats (data
not shown).
Apoptotic index
Apoptotic cells within lesions were identified using eosin
fluorescence of apoptotic bodies followed by confirmation
Remodeling
The percent of lesions undergoing remodeling was not significantly different between the strains on days 4 and 7 postPH. On days 14 and 21, however, liver sections from Cop rats
appeared to contain many more remodeling lesions than F344
rats (Figure 6F and H versus E and G). When this was
quantified it became apparent that remodeling in Cop rats
was significantly more extensive than in F344s (Figure 7).
Individual lesion measurements
BrdU labeling and apoptotic indices determined in individual
lesions showed no differences between strains. Lesions undergoing remodeling did not have a lower mean LI or a higher
mean AI than non-remodeling lesions.
1171
G.A.Wood, D.S.R.Sarma and M.C.Archer
Fig. 5. Apoptotic index of hepatocytes within GST 7-7-positive lesions in
Cop and F344 rats. There were no differences in the apoptotic index
between strains at any time point.
Oval cell response
Figure 6 shows the pattern of oval cell response from day 4
to day 21 post-PH. On day 4 post-PH, in both strains, there
is a small amount of oval cell proliferation with little or no
migration of these cells (Figure 6A and B). On days 7 and 14,
however, F344 livers had extensive infiltration of oval cells
from the portal areas into the liver parenchyma (Figure 6C
and E), whereas in Cop livers this response remained localized
to the portal areas (Figure 6D and F). On day 21, much of
the liver volume was occupied by nodules in F344 livers, and
the surrounding parenchyma had fewer oval and duct-like cells
than on day 14 (Figure 6G). In Cop livers the oval cell
response appeared to reach a maximum around day 7 and
declined thereafter to levels similar to day 4.
Discussion
Initiation followed by promotion using the RH protocol is a
well established model of liver carcinogenesis in rats that has
consistently been shown to produce many large GST 7-7positive lesions in susceptible rats (4,5). Previously, we have
shown that Cop rats are resistant to the formation of GST 77-positive liver lesions at 21 days following PH using this
treatment, having 9- to 27-fold less of the area of liver sections
occupied by lesions than F344 rats (1). In the present study,
resistant Cop rats and susceptible F344 rats were treated using
a similar protocol but were killed at earlier times post-PH to
establish the time course of the development of resistance in
the Cop rat.
The number of lesions per liver was the same in Cop and
F344 rats on days 2 and 4 post-PH, but on days 7, 14 and 21,
Cop rats had more lesions per liver than F344 rats (Figure 2).
Cop rats may indeed have more lesions per liver at these time
points, but it is possible that the stereological method we used
may have produced incorrect estimates of this parameter. In
particular, on days 14 and 21 post-PH, F344 livers contained
large lesions that were coalescing, such that several lesions
may have been counted as one. This would lead to an
underestimation of the number of individual lesions present in
F344 rats. Regardless of the possible errors in the estimates
of lesions per liver, Cop rats are clearly not resistant to the
initiation of lesions and may actually be more susceptible to
initiation than F344 rats.
The percent of the liver volume occupied by GST 7-7positive lesions was the same in both strains up to day 7 post1172
PH (Figure 3). On day 14, however, the two strains diverged
such that Cop rats showed no significant increase in percent
volume occupied by lesions compared with day 7, while F344
rats had ~8-fold more liver occupied by lesions on day 14
than day 7. By day 21 post-PH, the percent of liver volume
occupied by lesions in Cop rats had not changed compared
with day 14, whereas F344 liver lesions continued to grow,
approximately doubling the percentage of liver volume the
lesions occupied on day 14.
The overall rate of lesion growth during promotion is
influenced by the rate of cell division and the rate of cell loss
(19,20). During the RH protocol, GST 7-7-positive hepatocytes
can be lost either through cell death or through loss of GST
7-7 expression (remodeling) (21). We investigated these factors
in both strains by measuring the BrdU LI of lesions and
surrounding liver, the AI in lesions and the extent of lesion
remodeling.
In order for liver lesions to grow rapidly during the RH
protocol, 2-AAF must have a mito-inhibitory effect on normal
hepatocytes. The PH then provides a potent proliferative
stimulus to which only the ‘resistant’ hepatocytes that make
up the lesions respond (22,23). Previously, we have shown
that the degree of 2-AAF-induced mito-inhibition of normal
hepatocytes following a PH is the same in Cop and F344 rats
(1). The current study corroborates this finding by showing
that surrounding normal hepatocytes have the same LI in both
strains at all time points examined (Figure 4). It is also evident
from Figure 4 that GST 7-7-positive hepatocytes are resistant
to 2-AAF mito-inhibition to the same extent in both strains.
These ‘resistant’ hepatocytes had the same high LI in both
strains even on day 2 post-PH and maintained this growth
advantage over surrounding hepatocytes to the same extent in
both strains at least until day 7. By day 14 post-PH, there was
no growth advantage for GST 7-7-positive hepatocytes in
either strain.
Another factor influencing lesion growth is the rate of cell
death. Some promoting agents, such as phenobarbital (24,25)
and 2,3,7,8-tetrachlorodibenzo-p-dioxin (15) are thought to
act, at least in part, by inhibiting apoptosis. Dietary restriction
and S-adenosyl-L-methionine are both potent inhibitors of
carcinogenesis and at least some of their anti-carcinogenic
effects appear to be mediated by an increase in apoptosis
within liver lesions (21,26). Although we used the same
methods as the above studies to quantify apoptotic cells, we
were unable to show a higher AI in Cop compared with
F344 lesions.
There are several shortcomings of the methods used to
quantify apoptosis in the liver. The popular TUNEL method
has been shown to label cells undergoing necrosis as well as
apoptosis and also does not detect apoptotic bodies that lack
chromatin (27). The technique we have used that detects
apoptotic bodies by eosin fluorescence, takes advantage of the
eosinophilia of apoptotic bodies in the liver and is thought to
increase the sensitivity and speed of detection (15). It is also
relatively easy to check that a fluorescent structure represents
an apoptotic body by switching to normal transmitted light to
confirm its identity. Detection of apoptotic cells by any method,
however, has a limited window of time in which these cells
are visible. Using the gold standard of morphology, apoptotic
cells are only visible for ~3h in the liver before they are
completely degraded by other cells (25). Given these limitations, it is possible that a small difference in AI between
lesions in Cop and F344 rats may be undetectable. A small
Resistance to promotion of liver preneoplasia
Fig. 6. Micrographs of liver sections from Cop and F344 rats, stained for GST 7-7 and counterstained with hematoxylin and eosin (463 final magnification).
Days post-PH: (A and B) 4; (C and D) 7; (E and F) 14; (G and H) 21. (A, C, E and G) F344 livers; (B, D, F and H) Cop livers. Large arrow, GST 7-7positive focus; medium arrow, proliferating duct-like cells and oval cells; small arrows, migrating duct-like cells and oval cells; N, GST 7-7-positive nodule
with no evidence of remodeling; R, lesions undergoing remodeling.
difference maintained over a long period of time, however,
could potentially result in a significant difference in the growth
rate of lesions in the two strains.
Another factor that might explain the difference in lesion
growth between the strains is the proportion of lesions that
are resistant to 2-AAF mito-inhibition. Such resistant lesions
have previously been defined as having a higher LI than the
highest LI in the surrounding liver (28). Using this criterion,
1173
G.A.Wood, D.S.R.Sarma and M.C.Archer
Fig. 7. The percent of GST 7-7-positive lesions undergoing remodeling in
Cop and F344 rats. Cop rats had a significantly higher percent of lesions
undergoing remodeling than F344 rats on days 14 and 21 post-PH
(*P , 0.001).
we found no differences between the strains in the percent of
lesions that were resistant on days 4, 7 or 14 post-PH (data
not shown).
Remodeling or redifferentiation has been characterized in
the RH model of hepatocarcinogenesis (7). Typically, ù95%
of hepatic lesions induced by the RH protocol eventually
undergo remodeling (8). Our results show that a significantly
higher percent of lesions in Cop rats undergo remodeling early
after PH compared with F344s, the difference becoming
significant between 7 and 14 days post-PH (Figure 7). This
precocious remodeling could account for the small volume of
liver occupied by GST 7-7-positive hepatocytes in the Cop
rats following the RH protocol, even though the proliferative
and apoptotic indices in the lesions of the two strains were
indistinguishable. Brown Norway rats that are also known to
be resistant to hepatocarcinogenesis, likewise also have a
greater proportion of their liver lesions remodeling compared
with F344 rats following an RH protocol (18). Cop and Brown
Norway rats may, therefore, share a common mechanism of
resistance.
Other groups have found a lower LI and a higher AI in
remodeling compared with non-remodeling lesions (19,21).
There were, however, no such differences in our animals. A
possible explanation is that we examined lesions very early
after the PH in the RH protocol when the promotional stimulus
was still present, while the other studies examined lesions
many weeks after PH. Furthermore, in our study, almost all
of the remodeling lesions were in Cop rats and the process by
which these lesions remodel may be very different from that
in F344 rats.
2-AAF administration followed by PH in rats is known to
stimulate proliferation and migration of small duct-like epithelial cells, called oval cells (9), that originate in portal areas
and express GST 7-7 (29). On day 7 post-PH in F344 rats,
oval cells can be found invading far into the parenchyma and
on day 14 surround GST 7-7-positive lesions (Figure 6C and
E). In contrast, although we showed the presence of oval cells
in the portal regions of Cop livers, these cells failed to migrate
and remained localized to the periportal areas (Figure 6D and
F). The process of oval cell proliferation and migration is
associated with the production of several growth factors
1174
including hepatocyte growth factor (HGF), transforming
growth factor α, acidic fibroblast growth factor and stem
cell factor (30,31), and the production of urokinase-type
plasminogen activator (32,33) which can cleave pro-HGF to
produce active HGF (34,35). Although the role of oval cells in
hepatocarcinogenesis is currently being debated (13,14,36,37),
local production of growth factors associated with the oval
cell response could contribute to the growth of preneoplastic
lesions. Thus, the weak oval cell response that we observed
in Cop rats may play a role in their resistance. This is the first
report, to our knowledge, where 2-AAF/PH treatment resulted
in mito-inhibition without an extensive oval cell response. Cop
rats appear to be unique in this respect and thus provide a
model to study the potential independence of these events in
the RH protocol.
In summary, we have shown that Cop rats form similar
numbers of GST 7-7-positive lesions as F344 rats following
DEN initiation. However, Cop rats are resistant to the promotion of growth of these lesions using a modified RH protocol.
The proliferative rates within lesions were indistinguishable
in Cop and F344 rats, as were the proliferative rates in the
surrounding normal hepatocytes. The apoptotic indices within
lesions were also not different between strains. Two characteristics of Cop rats that were different from F344s were
precocious remodeling of liver lesions and a diminished oval
cell response. These differences can potentially account for
the resistance of Cop rats to the growth of GST 7-7-positive
hepatic lesions.
Acknowledgements
The authors thank Dr Ezio Laconi for valuable discussions. This investigation
was supported by a grant from the Canadian Breast Cancer Research Initiative.
M.C.A. is the recipient of a National Sciences and Engineering Research
Council of Canada Industrial Research Chair, and acknowledges support from
the member companies of the Program in Food Safety (University of Toronto).
References
1. Wood,G.A., Korkola,J.E., Lee,V.M., Sarma,D.S. and Archer,M.C. (1997)
Resistance of Copenhagen rats to chemical induction of glutathione Stransferase 7-7-positive liver foci. Carcinogenesis, 18, 1745–1750.
2. Moore,M.A., Nakagawa,K., Satoh,K., Ishikawa,T. and Sato,K. (1987)
Single GST-P positive liver cells—putative initiated hepatocytes.
Carcinogenesis, 8, 483–486.
3. Cameron,R.G. (1989) Identification of the putative first cellular step of
chemical hepatocarcinogenesis. Cancer Lett., 47, 163–167.
4. Rushmore,T.H.,
Harris,L.,
Nagai,M.,
Sharma,N.,
Hayes,M.A.,
Cameron,R.G., Murray,R.K. and Farber,E. (1988) Purification and
characterization of P-52 (glutathione S-transferase-P or 7-7) from normal
liver and putative preneoplastic liver nodules. Cancer Res., 48, 2805–2812.
5. Satoh,K., Kitahara,A., Soma,Y., Inaba,Y., Hatayama,I. and Sato,K. (1985)
Purification, induction, and distribution of placental glutathione transferase:
a new marker enzyme for preneoplastic cells in the rat chemical
hepatocarcinogenesis. Proc. Natl Acad. Sci. USA, 82, 3964–3968.
6. Semple-Roberts,E., Hayes,M.A., Armstrong,D., Becker,R.A., Racz,W.J.
and Farber,E. (1987) Alternative methods of selecting rat hepatocellular
nodules resistant to 2-acetylaminofluorene. Int. J. Cancer, 40, 643–645.
7. Tatematsu,M., Nagamine,Y. and Farber,E. (1983) Redifferentiation as a
basis for remodeling of carcinogen-induced hepatocyte nodules to normal
appearing liver. Cancer Res., 43, 5049–5058.
8. Enomoto,K. and Farber,E. (1982) Kinetics of phenotypic maturation of
remodeling of hyperplastic nodules during liver carcinogenesis. Cancer
Res., 42, 2330–2335.
9. Farber,E. (1956) Similarities in the sequence of early histological changes
induced in the liver of the rat by ethionine, 2-acetylaminofluorene, and
39methyl-4-dimethylamino-azobenzene. Cancer Res., 16, 142–148.
10. Evarts,R.P., Nagy,P., Marsden,E. and Thorgeirsson,S.S. (1987) A precursorproduct relationship exists between oval cells and hepatocytes in rat liver.
Carcinogenesis, 8, 1737–1740.
Resistance to promotion of liver preneoplasia
11. Fausto,N. (1990) Hepatocyte differentiation and liver progenitor cells.
Curr. Opin. Cell. Biol., 2, 1036–1042.
12. Lemire,J.M., Shiojiri,N. and Fausto,N. (1991) Oval cell proliferation and
the origin of small hepatocytes in liver injury induced by D-galactosamine.
Am. J. Pathol., 139, 535–552.
13. Thorgeirsson,S.S. (1996) Hepatic stem cells in liver regeneration. Faseb.
J., 10, 1249–1256.
14. Alison,M.R., Golding,M. and Sarraf,C.E. (1997) Liver stem cells: when
the going gets tough they get going. Int. J. Exp. Pathol., 78, 365–381.
15. Stinchcombe,S., Buchmann,A., Bock,K.W. and Schwarz,M. (1995)
Inhibition of apoptosis during 2,3,7,8-tetrachlorodibenzo-p-dioxinmediated tumour promotion in rat liver. Carcinogenesis, 16, 1271–1275.
16. Enzmann,H., Edler,L. and Bannasch,P. (1987) Simple elementary method
for the quantification of focal liver lesions induced by carcinogens.
Carcinogenesis, 8, 231–235.
17. Buchmann,A., Stinchcombe,S., Korner,W., Hagenmaier,H. and Bock,K.W.
(1994) Effects of 2,3,7,8-tetrachloro- and 1,2,3,4,6,7,8-heptachlorodibenzop-dioxin on the proliferation of preneoplastic liver cells in the rat.
Carcinogenesis, 15, 1143–1150.
18. Pascale,R.M., Simile,M.M., DeMiglio,M.R., Muroni,M.R., Gaspa,L.,
Dragani,T.A. and Feo,F. (1996) The BN rat strain carries dominant
hepatocarcinogen resistance loci. Carcinogenesis, 17, 1765–1768.
19. Schulte-Hermann,R., Timmermann-Trosiener,I., Barthel,G. and Bursch,W.
(1990) DNA synthesis, apoptosis, and phenotypic expression as
determinants of growth of altered foci in rat liver during phenobarbital
promotion. Cancer Res., 50, 5127–5135.
20. Schulte-Hermann,R., Bursch,W., Kraupp-Grasl,B., Oberhammer,F.,
Wagner,A. and Jirtle,R. (1993) Cell proliferation and apoptosis in normal
liver and preneoplastic foci. Environ. Health Perspect., 101, 87–90.
21. Garcea,R., Daino,L., Pascale,R., Simile,M.M., Puddu,M., Frassetto,S.,
Cozzolino,P., Seddaiu,M.A., Gaspa,L. and Feo,F. (1989) Inhibition of
promotion and persistent nodule growth by S-adenosyl-L-methionine in
rat liver carcinogenesis: role of remodeling and apoptosis. Cancer Res.,
49, 1850–1856.
22. Farber,E., Parker,S. and Gruenstein,M. (1976) The resistance of putative
premalignant liver cell populations, hyperplastic nodules, to the acute
cytotoxic effects of some hepatocarcinogens. Cancer Res., 36, 3879–3887.
23. Solt,D.B., Medline,A. and Farber,E. (1977) Rapid emergence of carcinogeninduced hyperplastic lesions in a new model for the sequential analysis of
liver carcinogenesis. Am. J. Pathol., 88, 595–618.
24. Bursch,W., Lauer,B., Timmermann-Trosiener,I., Barthel,G., Schuppler,J.
and Schulte-Hermann,R. (1984) Controlled death (apoptosis) of normal
and putative preneoplastic cells in rat liver following withdrawal of tumor
promoters. Carcinogenesis, 5, 453–458.
25. Bursch,W., Paffe,S., Putz,B., Barthel,G. and Schulte-Hermann,R. (1990)
Determination of the length of the histological stages of apoptosis in
normal liver and in altered hepatic foci of rats. Carcinogenesis, 11, 847–853.
26. Grasl-Kraupp,B., Bursch,W., Ruttkay-Nedecky,B., Wagner,A., Lauer,B.
and Schulte-Hermann,R. (1994) Food restriction eliminates preneoplastic
cells through apoptosis and antagonizes carcinogenesis in rat liver. Proc.
Natl Acad. Sci. USA, 91, 9995–9999.
27. Grasl-Kraupp,B., Ruttkay-Nedecky,B., Koudelka,H., Bukowska,K.,
Bursch,W. and Schulte-Hermann,R. (1995) In situ detection of fragmented
DNA (TUNEL assay) fails to discriminate among apoptosis, necrosis, and
autolytic cell death: a cautionary note. Hepatology, 21, 1465–1468.
28. Yusuf,A., Backway,K.L., Laconi,E., Rao,P.M., Rajalakshmi,S. and
Sarma,D.S.R. (1998) Development of resistance to the mitoinhibitory
effects of orotic acid during experimental liver carcinogenesis. In
Bannasch,P., Kanduc,D., Papa,S. and Tager,J.M. (eds) Cell Growth and
Oncogenesis. Birkhauser Verlag, Basel, Switzerland, pp. 177–188.
29. Nakatsukasa,H., Evarts,R.P., Burt,R.K., Nagy,P. and Thorgeirsson,S.S.
(1992) Cellular pattern of multidrug-resistance gene expression during
chemical hepatocarcinogenesis in the rat. Mol. Carcinog., 6, 190–198.
30. Evarts,R.P., Hu,Z., Fujio,K., Marsden,E.R. and Thorgeirsson,S.S. (1993)
Activation of hepatic stem cell compartment in the rat: role of transforming
growth factor alpha, hepatocyte growth factor, and acidic fibroblast growth
factor in early proliferation. Cell Growth Differ., 4, 555–561.
31. Fujio,K., Evarts,R.P., Hu,Z., Marsden,E.R. and Thorgeirsson,S.S. (1994)
Expression of stem cell factor and its receptor, c-kit, during liver
regeneration from putative stem cells in adult rat. Lab. Invest., 70, 511–516.
32. Nagy,P., Bisgaard,H.C., Santoni-Rugiu,E. and Thorgeirsson,S.S. (1996) In
vivo infusion of growth factors enhances the mitogenic response of rat
hepatic ductal (oval) cells after administration of 2-acetylaminofluorene.
Hepatology, 23, 71–79.
33. Bisgaard,H.C., Santoni-Rugiu,E., Nagy,P. and Thorgeirsson,S.S. (1998)
Modulation of the plasminogen activator/plasmin system in rat liver
regenerating by recruitment of oval cells. Lab. Invest., 78, 237–246.
34. Naldini,L.,
Tamagnone,L.,
Vigna,E.,
Sachs,M.,
Hartmann,G.,
Birchmeier,W., Daikuhara,Y., Tsubouchi,H., Blasi,F. and Comoglio,P.M.
(1992) Extracellular proteolytic cleavage by urokinase is required for
activation of hepatocyte growth factor/scatter factor. Embo. J., 11, 4825–
4833.
35. Mars,W.M., Zarnegar,R. and Michalopoulos,G.K. (1993) Activation of
hepatocyte growth factor by the plasminogen activators uPA and tPA. Am.
J. Pathol., 143, 949–958.
36. Aterman,K. (1992) The stem cells of the liver—a selective review. J.
Cancer Res. Clin. Oncol., 118, 87–115.
37. Alison,M.R., Golding,M.H. and Sarraf,C.E. (1996) Pluripotential liver
stem cells: facultative stem cells located in the biliary tree. Cell. Prolif.,
29, 373–402.
Received August 5, 1998; revised January 27, 1999; accepted March 17, 1999
1175