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Department of Medical Sciences, University of East Piedmont "Amedeo Avogadro," Novara, Italy
2To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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0.05). Putative-initiated
P-GST-positive hepatocytes appeared after administration of 20 mg/kg in
refed rats, and they showed a higher LI (6%) than the surrounding
P-GST-negative cells 3 d after DENA (LI = 2%; P
0.01), while very few P-GST-positive cells were found in
fully-fed rats. These data indicate that starvation-induced
cell loss and the subsequent refeeding trigger cell proliferation that
gives a selective advantage to the cells initiated by 20 mg/kg of DENA
to grow in the livers of refed rats.
KEY WORDS: • diethylnitrosamine • rat liver • apoptosis • cell proliferation • cancer
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
A single dose of either methylnitrosurea
(MNU)3
or dimethylhydrazine (DMH) becomes carcinogenic for the liver when
administered after partial hepatectomy (PH) (Cayama et al. 1978
, Craddock 1973
). Cell replication seems to
be necessary for chemical induction of initiated hepatocytes for at
least two events: interaction of the carcinogen with the main target
macromolecule, DNA, and fixation of the lesions by a round of cell
division, in order to increase the chances of altered nucleotide
sequences in the newly-made DNA (Farber and Sarma 1987
). Nevertheless, initiated hepatocytes also occur when PH
follows the administration of MNU, suggesting that the replication of
carcinogen-damaged DNA before repair is the critical mechanism by
which cell proliferation stimulates the induction of chemical
initiation (Cayama et al. 1978
, Columbano et al. 1981
). Interestingly, several liver carcinogens are necrogenic
and induce compensatory liver cell proliferation without the need for
an exogenous mitogenic stimulus such as PH (Farber and Sarma 1987
). A single 200 mg/kg dose of diethylnitrosamine (DENA)
induces both extensive ethylation of liver DNA and cell necrosis,
resulting in the generation of initiated hepatocytes selectable by a
promoting regimen, eventually leading to liver cancer (Solt and Farber 1976
). A single 10–50 mg/kg dose of DENA is not
necrogenic and does not induce hepatocellular carcinoma
(Columbano et al. 1981
, Ying et al. 1981
). However, the subnecrogenic (20 mg/kg) dose of DENA
becomes an initiating agent following PH, indicating that cell loss can
be induced by either surgery or necrosis, to trigger a compensatory
proliferative response (Ying et al. 1980
).
We recently reported that administration of 20 mg/kg of DENA to
previously starved and refed rats led to hepatocyte foci, nodules and
liver cancer without inducing cell necrosis, while if the rats received
the full 200 mg/kg dose, refeeding had no effect (Tessitore et al. 1996
). This implies that refeeding can substitute for PH or
liver necrosis in inducing initiated hepatocytes by a subnecrogenic
(noninitiating) dose of DENA. Consistently, Grassl-Kraupp et al. (1994)
found that complete starvation of female rats for 8 d eliminated 20–30% of liver cells. Modulations of cell proliferation
and cell death contribute to the growth of liver tumors (Cayama et al. 1978
, Farber and Sarma 1987
,
Farber and Rubin 1991
, Grassl-Kraupp et al. 1994
, James and Muskhelishvili 1994
), and this
might also be true for tumor genesis.
Based on these observations, in the present study we tried to gain further insight into the mechanisms by which starvation leads to the induction of liver cancer by administering a subnecrogenic dose of DENA to refed rats. For this purpose we investigated whether 4 d of starvation followed by 1 d of refeeding induced changes in the rates of active cell death and cell proliferation of rat liver which may be essential for the genesis of initiated hepatocytes following a subnecrogenic dose of DENA. We found that starvation and the carcinogen induced cell death and, as a consequence, the subsequent feeding a basal diet produced a burst of DNA synthesis of the hepatocytes initiated by 20 mg/kg of DENA.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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3H-thymidine (0.74 TBq/mmol) was from New England Nuclear (Boston, MA), Picofluor 40 was from Packard Instruments International (Zurich, Switzerland); calf thymus DNA, diethylnitrosamine and bromodeoxyuridine (BrdU) were from Sigma Chemical Co. (St. Louis, MO); NTB-2 Kodak emulsion was from Kodak (Rochester, NY); the "In situ cell death detection kit, fluorescein" was from Boehringer, Mannheim (Germany); the monoclonal mouse antibody to BrdU was from Amersham (Milan, Italy); the avidin-biotinylated peroxidase-complex and anti-mouse IgG antibody was from Vector (Burlingame, CA) and alkaline phosphatase-conjugated streptavidin and anti-rabbit IgG were from Dako (Carpinteria, CA). The rabbit anti-rat liver 7-7, glutathione S-transferase (placentar isoform of P-GST) was a gift from Dr. Paolo Pani. All other reagents were from Merck (Darmstadt, Germany).
Animals and treatment
Six-wk-old male Fischer-344 rats were obtained from Charles
River (Como, Italy). They were maintained under a 12-h light/12-h dark
cycle, controlled temperature (21 ± 1°C) and humidity
(70–80%) and divided into three main groups. One group of rats was
maintained for 4 d under conditions of total deprivation of food
and then refed with the standard basal diet (AIN-76; Piccioni, Brescia,
Italy; American Institute of Nutrition 1977
). After
1 d of refeeding, 20 of these rats were given a single 20 mg/kg
body wt dose i.p. of DENA. Groups 2 and 3 consisted of fully-fed
control rats that were given either 20 (n = 20;
negative control) or 200 (n = 20; positive control)
mg/kg of DENA. The rats were killed by carbon dioxide asphyxia 1, 3, 5
or 7 d after DENA administration to evaluate the appearance of
P-GST positive-initiated cells and the rates of cell death and
proliferation. For this purpose, the rats were given a single 50 mg/kg
dose of BrdU i.p. 1 h before being asphyxiated. Some experiments
were performed with non-DENA treated rats to analyze cell turnover
exclusively during 4 d starvation and the following refeeding. To
study cell loss, we determined the decay of DNA total radioactivity by
prolonged labeling of DNA in vivo. Osmotic minipumps were implanted
i.p. into 40 rats under light ether anesthesia for administering
3H-thymidine (7.4 MBq/rat) for 1 wk. Rats were then divided
into two groups: one group was fed, and the other group was starved and
both groups of rats were asphyxiated at the time of removing the
minipumps (time 0) or 1, 2, 3 or 4 d after. Because anesthesia
affects hepatocytes, both fed and starved rats were treated in the same
manner. To evaluate whether cell loss produced by starvation elicited a
suitable mitogenic stimulus for liver cell proliferation, DNA synthesis
was first studied by administering the label 1 h before death;
then to confirm this result, DNA synthesis was also measured after
multiple doses of label. For this reason, groups of rats were exposed
to 4 d of starvation and 1 or 2 d of refeeding; they received
i.p. either a single dose of 18.5 MBq/kg body wt.
3H-thymidine 1 h before being asphyxiated or four 5.55
MBq/kg body wt doses of 3H-thymidine, at 6 h intervals
during the 24 h before being asphyxiated. All rats were weighed
each day and food intake was measured.
Histological analyses
For histological/histochemical examination, slices taken from different liver lobes were immediately fixed in 10% phosphate-buffered formalin solution (pH 7) for routine embedding in paraffin and staining with hematoxylin and eosin. The mitotic figures were counted, and the mitotic index was calculated scoring not less than 2,000 cells.
Cell proliferation and initiation
Autoradiography. To calculate the percentage of 3H-thymidine positive cells [3H-thymidine labeling index (LI)] some liver samples were processed for autoradiography: sections were coated with NTB-2 Kodak emulsion, dried and sealed in a dark box at -80°C for 2 wk. Slides were then developed and counterstained with hematoxylin and eosin.
Immunohistochemistry.
Other slices were fixed in ice-cold acetone for double
immunohistochemical staining for BrdU and the placental form of
glutathione S-transferase (P-GST) as described by Li et al. (1993)
.
To calculate the percentage of BrdU positive cells, after deparaffinizing and rehydrating, sections were immersed in 0.88 mol/L of hydrogen peroxide for 10 min to block endogenous peroxidase; they were then immersed in 4 mol/L of HCl for 10 min to partially denature the DNA. The first antibody, anti-BrdU (mouse-indiluted antibody), was applied for 1 h at room temperature, washed in phosphate buffer saline (PBS) and reacted with goat anti-mouse IgG diluted 1:200. After washing, sections were incubated with biotine-avidin-peroxidase complex. Visualization was by reaction with 3-amino-9-ethylcarbazole (AEC) in 2.5% N,N-dimethylformamide and 50 mmol/L of acetate buffer, pH 5.0, containing hydrogen peroxide. The counterstain was hematoxylin. Sections were then washed, dehydrated and mounted. After counterstaining with hematoxylin, nuclei containing BrdU were red while other nuclei were blue. The numbers of BrdU-positive cells were determined by examining not less than 2,000 hepatocyte nuclei and expressed per 100 hepatocyte nuclei (LI).
To evaluate the appearance of P-GST positive cells, some sections were also exposed to P-GST antibody rabbit anti-rat diluted 1:500 for 1 h at room temperature, washed in PBS, then reacted with goat anti-rabbit IgG diluted 1:200. After washing, sections were incubated with alkaline phosphatase-conjugated streptavidin. Visualization was achieved by reaction with 5-bromo-4-chloro-3-indoxyl phosphate and nitro blue tetrazolium chloride (BCIP/NBT). The sites with P-GST antigen were blue. A minimum liver area of 2 cm2 was screened for each sample, using a Universal II microscope with5x objective lens. The number of P-GST positive cells/cm2 and the percentage of BrdU-positive cells were determined by means of an interactive image analysis system (Videoplane Zeiss; Kotron, Munich, Germany).
Cell death: end labeling
Sections 5 µm thick were stained with an enzymatic in situ label of apoptosis-induced DNA strand breaks, known also as the TUNEL (TdT-mediated dUTP nick end labeling) technique. The terminal deoxynucleotidyl transferase enzyme labels free 3'-OH DNA termini with fluorescein-labeled nucleotides. Percentages of apoptosing cells were determined by quantitating the percentage of labeled nuclei by fluorescence microscope, scoring not less than 5,000 adjacent cells (apoptotic index, AI).
Biochemical analyses
The liver was removed and weighed immediately after death. Liver
samples were homogenized (100 g/L in distilled water) with a Polytron
10-ST apparatus (Kinematika, Luzern, Switzerland). Liver DNA content
was determined by the method of Burton (1956)
with calf
thymus DNA as the standard. For radioactivity containing 9.6 µmol/L
sodium-deoxycholate for 3 h. The final hydrolysate was
adjusted to pH 5 with 1 mol/L of HCl and radioactivity counted in a
Beckman LS 1801 spectrometer (Milan, Italy), using Picofluor 40
(Packard, Warrenville RD) as scintillation mixture. The mean cell loss
rates were calculated as fractional rates of DNA degradation
(Kd) by the following equation:
Kd = ln (total DNA radioactivity)/t
as previous reported (Tessitore et al. 1987
).
Statistical analysis
Data are expressed as means ± SD and the data were analyzed by two-way ANOVA, followed by the Bonferroni-corrected t-tests. Differences were considered significant if P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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upper panel). Fully-fed rats receiving 20 mg/kg of DENA increased
body wt normally, while fed rats injected with 200 mg/kg of DENA
stopped growing. The food intake of previously starved rats on the
first day of refeeding did not differ from that before starvation and
to that of the fully-fed rats at the same time (Fig. 1
, middle
panel). While fully-fed rats treated with 20 mg/kg of DENA did not
significantly change food intake (P < 0.001 vs.
controls -5 d), those receiving 200 mg/kg of DENA immediately reduced
food intake dramatically for 2 d (P < 0.001),
then gradually returned to normal intake within 3–4 d. On the
contrary, the previously refed rats progressively increased food intake
after administration of DENA, thereafter remaining steadily at a
significantly higher intake than controls (-5 d) for at least 4 d
(Fig. 1
, middle panel). Treatment with 200 mg/kg of DENA caused about
25% decrease (P < 0.0001 vs. -5 d) in liver weight
while 20 mg/kg induced no significant change (P < 0.001 vs. -5 d) (Fig. 1
, lower panel). Starved rats lost liver weight
then quickly recovered on the first day of refeeding, the subsequent
treatment with 20 mg/kg, producing a significant loss of liver weight
(P < 0.001 vs. 0 d) (Fig. 1
, lower panel).
|
, upper panel). Liver DNA total radioactivity
decreased over 4-d starvation, indicating a mean cell loss of about
18%/d (Fig. 2,
lower panel).
|
). After a multiple dose of thymidine label, both LI and the biochemical
analysis confirmed that, during the first 2 d of refeeding, the
mean levels of DNA synthesis were below the values in fully-fed
rats (Table 1)
.
|
shows the time-course of the growth kinetic parameters during the
initiation phase of liver carcinogenesis. Both 20 and 200 mg/kg of DENA
induced liver apoptosis, with the AI being positively related to the
dose (Fig. 3
, upper panel). Starvation also resulted in liver cell
death by apoptosis, and return to ad libitum consumption of food
reversed the AI to the starvation level. Treatment of refed rats with
20 mg/kg DENA induced the reappearance of apoptosis to a greater extent
than fully-fed rats receiving the same dose (P < 0.05). The LI of hepatocytes after 200 mg/kg of DENA rose to about
eight times the normal levels on d 3 and remained higher until d 5,
while no significant change was observed in the liver of fully-fed
rats after 20 mg/kg of DENA vs. -5 d (Fig. 3
, middle panel).
Interestingly, the rats previously starved and refed also had
significantly greater LI than control levels (-5 d) from d 1 to d 7
after 20 mg/kg of DENA, the difference being even more pronounced vs.
the levels of refed rats (0 d). Consistently, the liver mitotic index
increased 5 and 7 d after administration of 200 mg/kg of DENA vs.
0 d, and also after 20 mg/kg of DENA in refed but not in
fully-fed rats (Fig. 3
, lower panel).
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(upper panel). Single P-GST-positive cells were already evident 1 d after injection of 200 mg/kg of DENA, and their number increased
progressively until d 7, while very few P-GST-positive hepatocytes were
found in the liver of rats treated with 20 mg/kg of DENA. The refed
rats showed a pattern of P-GST-positive cells similar to that of rats
treated with 200 mg/kg of DENA, though to a significantly lesser extent
(P < 0.005). Cell division, as P-GST-negative
(Fig. 4
, lower panel) hepatocytes. Following DENA injection, the BrdU
LI of P-GST-positive hepatocytes in the positive control (rats treated
with 200 mg/kg of DENA) was higher than that of P-GST-negative
hepatocytes on d 3, and remaining significantly higher until d 7, while
in the negative controls (rats treated with 20 mg/kg of DENA) the BrdU
LI of P-GST-negative cells, and that of P-GST-positive cells was always
below 2%. Interestingly, administration of 20 mg/kg of DENA to refed
rats also induced a significant increase of P-GST-positive hepatocytes
in the S phase vs. 0 d (P < 0.01)
although the LI did not reach the high peak observed in the positive
controls on d 3, whereas the LI of the P-GST-negative cells was not
different from that of the negative controls.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The depression of cell division in the liver of rats exposed to
prolonged starvation that we observed is consistent with previous
reports in normal tissue (Goodgame et al. 1979
) as well
as in hepatic foci and their surroundings (Hikita et al. 1997
). The liver of starved rats also underwent apoptosis,
probably as a consequence of the withdrawal of growth factors
(Batistatou and Greene 1991
, Chiao et al. 1995
, Lindenboim et al. 1995
, Straus 1994
, Wang et al. 1995
) or the induction of
growth inhibitory factors, such as transforming growth factor beta
(TGFß) (Oberhammer et al. 1992
). Induction of
apoptosis by starvation was also reported in the liver of female rats
(Grassl-Kraupp et al. 1994
) and in both focal lesions
induced by DENA and phenobarbital and the surrounding liver, again in
female rats, resulting in the loss of detectable foci (Bursh et al. 1990
, Hikita et al. 1997
). Refeeding
following prolonged food deprivation is known to be associated with
enhanced liver cell proliferation (Goodgame et al. 1979
,
Grube et al. 1985
, Hikita et al. 1997
).
Refeeding is the proliferative stimulus which could make the rats
susceptible to liver cancer induced by 20 mg/kg of DENA.
The importance of cell proliferation in the induction of carcinogenesis
was extensively characterized in rat liver (Cayama et al. 1978
, Columbano et al. 1981
, Craddock 1971
and Craddock 1973
,
Kaufmann et al. 1991
, Pitot et al. 1978
).
Adult rat liver is resistant to carcinogenesis; however, it becomes
highly susceptible when hepatocyte proliferation is induced by the
carcinogen itself or by PH. Kaufmann et al. (1991)
studied the susceptibility of proliferating hepatocytes to complete
initiation by an alchylating agent administrated at different times
after partial surgical hepatectomy; they reported that S
phase liver cells appeared to be the most-sensitive targets for the
DNA damage induced by the carcinogen. However, our finding that the
percentage of S cells in refed rats at the time of DENA
administration was lower than that in fully-fed rats rules out the
possibility that a higher number of S cells contributed to
the induction of DNA damaged cells by a subnecrogenic dose of DENA
under our experimental conditions. On the other hand, our data suggest
that rare cycling hepatocytes are sufficient to develop DNA damage,
eventually leading to liver cancer.
The placental isozyme of GST is a marker of liver cancer development,
as is the single hepatocyte that expresses P-GST an initiated cell
(Cameron 1989
, Dragan et al. 1993
,
Moore et al. 1987
).
As previously reported (Dragan et al. 1993
), the small
number of P-GST positive hepatocytes induced by 20 mg/kg of DENA
did not develop into foci, nodules or, overtime, cancer. In contrast, a
relevant number of P-GST positive cells appeared in the liver of
refed rats following injection of the same dose of DENA, consistently
with the development of cancer.
At the same time, a large number of hepatocytes underwent apoptosis
after administration of the carcinogen (Daoust and Morais 1986
). Active cell death can result either in the elimination
of cells with pro-mutational lesions, which may be at risk for
initiation, and of about 80% of the initiated cells, or in the
reduction of preneoplastic and neoplastic lesions
(Schulte-Hermann et al. 1995
). Thus, induction of
apoptosis was proposed as a novel preventive and therapeutic strategy
against cancer. On the other hand, in rat liver, the apoptotic and
proliferative rates increased from foci to adenoma to carcinoma,
providing a selective advantage for growth to cell subpopulations with
high levels of malignancy (Schulte-Hermann et al. 1995
).
Consistently, Grasl-Kraupp et al. (1994)
showed that
food restriction triggered apoptosis, and subsequent refeeding induced
cell proliferation in liver focal proliferative lesions.
Our findings that starvation induced cell loss without triggering liver cell necrosis until 7 d after DENA administration indicates that the apoptosis induced by starvation and then by 20 mg/kg of DENA in refed rats may be responsible for the higher cell division rates during the subsequent refeeding after carcinogen treatment. Thus it appears that the toxicity of 20 mg/kg of DENA is enhanced in refed rats, leading to higher apoptotic rates, probably because the wave of cells triggered by refeeding at the transition GI/S is more susceptible to cell death than the quiescent cells. This suggests that high rates of cell death may mean rates of cell loss sufficient to be the mitogenic stimulus for cell proliferation, favoring genesis and growth of initiated hepatocytes. Finally, our data on the different rates of cell division in initiated hepatocytes and noninitiated liver cell population in refed rats indicate that a low differential is sufficient to activate the selective proliferative advantage necessary for initiation.
The enhanced rates of cell proliferation of initiated hepatocytes
induced by DENA in refed rats further support the concept that
replication of carcinogen-damaged DNA is the critical limiting step
in the initiation of carcinogenesis. The susceptibility to initiation
depends on the replication of damaged DNA in critical cellular target
genes, regulating the cell cycle before the genes’s repair, leading to
activation of the protooncogene and inactivation of the oncosuppressor
genes. Gene alterations presumably occur with high frequency in
S phase cells, replicating the ethylated DNA (Farber and Sarma 1987
, Kaufmann et al. 1991
,
Singer 1986
), probably because protooncogenes and
oncosuppressors are more prone to mutagenesis during their replication
(Li et al. 1993
, Ozturk 1991
). Increased
formation of adducts to DNA may be expected in rats previously exposed
to refeeding; cell division must also occur before the
carcinogen-induced critical lesion(s) in DNA have been repaired
(Kaufmann et al. 1991
). Interestingly, DNA damage
produced in rat liver by DENA appears to be rapidly repaired in
noncycling hepatocytes (Tates and Den Engelse 1989
). The
enhanced cell proliferation of initiated hepatocytes in refed rats did
not rule out a direct effect of food deprivation/refeeding on
metabolism of DENA, which will be the subject of further studies.
These results appear of particular interest for the pathogenesis of the hepatocellular carcinoma in underdeveloped countries, where the tumor is endemic and where aflatoxin B1 and hepatitis B virus are the main factors involved, and a condition of food-deprivation/refeeding may be present. This may also be true in developed countries, for people with unbalanced dietary habits.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: AEC,
3-ammino-9-ethylcarbazole; AI, apoptotic index; BCIP,
5-bromo-4-chloro-3-indoxyl phosphate; BrdU, bromodeoxyuridine; DENA,
diethylnitrosamine; DMN, dimethylhydrazine; LI, labeling index; MNU,
methylnitrosurea; NBT, nitro blu tetrazolium chloride; PBS, phosphate
buffer saline; P-GST, placental form of glutathione
S-transferase; PH, partial hepatectomy; TGFß, transforming
growth factor ß.
Manuscript received April 6, 1999. Initial review completed May 27, 1999. Revision accepted September 28, 1999.
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