Persistent chemopreventive effect of 5-adenosyl-L

Carclnogenesls vol.17 no.7 pp. 1533-1537, 1996
SHORT COMMUNICATION
Persistent chemopreventive effect of 5-adenosyl-L-methionine on
the development of liver putative preneoplastic lesions induced by
thiobenzamide in diethylnitrosamine-initiated rats
Maria M.Simile, Michela Saviozzi1, Maria R.De Miglio,
Maria R.Muroni, Alessandra Nufris, Rosa M.Pascale,
Gino Malvaldi1 and Francesco Feo2
Istituto di Patologia Generale e Centro di Ricerche Oncologiche, Universita
di Sassari, Via P.Manzella 4, 07100, Sassari and 'Dipartimento di
Biomedicina, Sezione di Patologia Generale, Universita di Pisa, Italy
2
To whom correspondence should be addressed
5-Adenosyl-L-methionine (SAM) is a strong chemopreventive agent of rat liver carcinogenesis. Examination
was made to determine whether inhibition by SAM of the
development of preneoplastic liver lesions persists to SAM
withdrawal in diethylnitrosamine-initiated F344 rats promoted with thiobenzamide (TB). The rats were subjected,
2 weeks after initiation, to 5 weeks feeding with a 0.1%
TB diet followed by a TB-free diet for 6 weeks and then
by a second TB treatment for 3 weeks. SAM (384 ujnol/
kg/day) was injected i.m. during the first TB cycle (treatment A) or for 6 weeks after the first TB cycle (treatment
B). Many yglutamyltranspeptidase (GGT)-positive lesions
developed in initiated rats after the first TB cycle. They
decreased in number after TB withdrawal, while partial
recovery of lesion number and a great increase in volume
occurred after the second TB cycle. Liver ornithine
decarboxylase (ODC) activity and c-myc and c-Ha-ras
mRNAs increased during the TB cycles and returned to
normal liver values after TB withdrawal. Number and size
of GGT-positive lesions, DNA synthesis of GGT-positive
cells, liver ODC activity and c-myc and c-Ha-ras mRNA
levels decreased as a consequence of SAM treatment A.
The recovery of these parameters, induced by a second TB
cycle in rats not treated with SAM, was prevented by SAM
treatment B. These results suggest that SAM causes a
persistent decrease in growth capacity of preneoplastic
liver lesions in rats subjected to a diethylnitrosamine/TB
protocol.
5-Adenosyl-L-methionine (SAM*) and its precursors Lmethionine, choline and betaine strongly prevent experimental
liver carcinogenesis (1). Chronic treatment of rats with SAM
during the development of preneoplastic lesions inhibits growth
and stimulates apoptosis and remodeling (2) of preneoplastic
cells (3,4). This results in long-term tumor chemoprevention
(5). SAM is a non-toxic compound which seems to enter
liver cells (1). It reconstitutes the endogenous SAM pool
in preneoplastic hepatocytes, which exhibit decreased SAM
content (3,6). Its chemopreventive effect correlates with inhibition of polyamine synthesis, overall DNA methylation and
methylation and decrease in expression of c-myc, c-Ha-ras
and c-Ki-ras genes in preneoplastic liver lesions (6—8). It is
not yet clear, however, if SAM induces a transient decrease
•Abbreviations: SAM, s-adenosyl-L-methionine; TB, thiobenzamide; ODC,
ornithine decarboxylase; LI, labeling index; GTT, y-glutamyltranspeptidase,
BrdU, 2-bromo-3'-deoxyuridine.
© Oxford University Press
in the growth rate of preneoplastic cells resulting in a decreased
probability of further evolution to malignancy. This effect
could be reversed by additional promoting treatments after
SAM withdrawal, while a persistent decrease in growth capacity of preneoplastic cells should result in a chemoprevention
resistant to further mitogenic stimuli. A suitable experimental
model to test these possibilities is represented by promotion
of hepatocarcinogenesis with thiobenzamide (TB) in diethylnitrosamine-initiated rats (9,10). In this model preneoplastic
lesions grow asynchronously and tend to disappear after TB
cessation, but they recover after a second treatment (11). In
this paper diethylnitrosamine-initiated rats were subjected to
two TB cycles and SAM was given between these cycles
to investigate the persistence of its effect at the cellular,
biochemical and molecular levels.
Male Fisher 344 rats (140-160 g) were housed three
per suspended wire-bottomed cage in a room with constant
temperature (22°C) and humidity (55%) and with a 12 h light
(6 a.m.-6 p.m.)/dark (6 p.m-6 a.m.) cycle. The rats were
randomly divided into nine groups (Figure 1). Groups C, Cl
and C2 (10 rats each) were normal rats, initiated rats without
TB and TB-treated uninitiated rats respectively. TB treatment
consisted of 5 weeks feeding with a standard diet containing
0.1% TB (11), followed by 6 weeks feeding with a standard
diet without TB and then by a second TB cycle for 3 weeks.
All other groups (10-15 rats) were initiated by a single i.p.
dose of diethylnitrosamine (150 mg/kg). Two weeks later the
animals were fed the TB diet for 5 weeks, followed in groups
3-6 by a 6 week period of TB-free diet and in groups 5 and
6 by a second 3 week TB cycle after a 6 week period of TBfree diet. The rats received four daily i.m. injections (96 mmol/
kg each) of SAM bisulfate p-toluene sulfonate (98.3% pure;
BioResearch SpA, Liscate, Milano, Italy; 5) in 0.05 rrd/100 g
body wt lysine buffer containing 25 (ig lidocaine-HCl (final
pH 6.9) into the hind legs (Figure 1). Groups 1 and 3 received
solvent alone. Surviving rats were killed by bleeding through
the abdominal aorta under ether anesthesia and the livers were
resected and used for histology/histochemistry and ornithine
decarboxylase (ODC) activity determination, or were frozen
in liquid nitrogen and stored at -170°C prior to use for
molecular biological determinations. Five liver sections from
each of five or six rats were used for stereological and labeling
index (LJ) determinations. ODC activity was evaluated in
one of these rats. Determination of ODC activity and RNA
extraction were performed in another set of four or five rats.
Acetone-fixed and paraffin-embedded 5 (im serial sections
were processed for hematoxylin/eosin staining and y-glutamyltranspeptidase (GGT) histochemistry and subjected to morphometric analysis as published (8). Number of lesions per liver
(lesions/cm3 X liver weight, since the density of liver is
~1 g/cm3), mean volume of lesions and volume fraction
(percentage of liver volume occupied by glutathione S-transferase, placental form-positive lesions, calculated by the
Delesse method) were determined according to Pugh et al. (19).
1533
M.M.Simile <•/ al
Table I. Quanlilkation ol GGT-posiine lesions
Rat group
Treatment1"
No/Lner
[)hNA/TB
[)ENA/TB(SAMl
DENA/TB/BD
l)ENA/TB/BD(SAMl
DENA/TB/BD/TB
l)ENA/TB/BD/TB(SAMi
II SI2
4678
4982
4675
±
±
±
±
1468
802"
1870"1022
11 202 1 28(K)d
6KX) ± 94(f'
Volume (cnv'x KV4)
Volume fraction ('/! )
0416
0301
0 370
0 240
0 750
0 359
464 ± 7 0
±
i
±
±
±
±
0 12
0 02"
0 04
0 06d
0 I6d
0(W '
11 3
14 0
12 2
79 0
213
± I 21
+ 2 6'
1 0 9'1
± 18 8'1
+
2 ft1'1
J
Thc r.ils were subletted lo the lollowing treatment gioups I anil 2 TB for 5 weeks, groups 3 and 4 TB + basal diet (BD) lor 6 weeks, groups 5 and 6
TB ' BD i TB lor ' weeks The rals were killed al the end ol treatments Group 2 was treated with SAM during the tirst TB cycle, and groups 4 and 6
received SAM during HD leeding. then group 6 was led TB up to killing Groups I. 3 and 5 received solvent alone during the same periods of time Data are
means ' SI) ol live experiments
Percentage ot luer volume occupied hs GGT-positixe lesions
'Different from group I al least P • 0 02
''Diflerenl Irom group < al leasl P • 0(M)l
"Different Irom group 4 at leasl P
0 01
'Different Irom group 5 at least P • 0 02
Group
C Normal rats
Standard dm
• 0 IX JB
Table 11. Labeling index ol preneoplastic hepatocytes in rals treated with
TB and SAM
DENA
I
I
I
I
I
I
I
I I
I
I
I
I
I I
Rat group
C2|
DENA
1
1
I
2
1 1 1 1
3
4
1 [
1 I
I
5
]
1 I
1
6
"j 1
I" I
1
Week
i
8
9
SAM
10
11 12
i
r "]" " T i
r •••j-" T
13 14 15 16
Fig. I. Schematic representation ot the.' experimenta
cxpe rimen tal mod el The arrows
indicate the killing tunes DENA. diethylnilrosamme
Transections with a radius > 3 5 (im were reliably identified and
included in the analysis. To determine the LI rats of groups
I, 2, 5 and 6 received, 2 h before killing, an i.p. injection
of 5 mg/100 g body wt 2-bromo-3'-deoxyundine (BrdU).
lmmunohistochemical staining of BrdU incorporated into
nuclei was performed using a Cell Proliferation Kit (Amersham
International). ODC activity was determined using 30 000 g
supernatants of liver homogenates as described (12). Proteins
were determined as in Lowry et al. (13). Total RNA was
extracted by a guanidium thiocyanate/lithium chloride procedure (14). Poly(A) + RNAs were purified on an oligo(dT)—
cellulose column (15). RNA was electrophoresed under denaturating conditions, transferred onto Highbond nylon membranes
and fixed with a UV Stratalinker (12). Pre-hybridization,
hybridization and autoradiography were performed according
to Garcea et al. (6). Probes were 1.2, 0.46 and 1.15 kb
fragments respectively of the following clones: pRYC 7.4 for
c-myc (16); pBs9 for c-Ha-ra.v (17); 91 for a-actin (18). The
probes were radiolabeled with 1 : P to a specific activity of 1.22X10'' d p.m./(ig DNA using a random primer DNA kit
(Boehnnger Mannheim). Data are expressed as means ± SD.
Differences between the means were evaluated by Student's
Mest.
Food intake and body and liver weights were not influenced
by TB and SAM treatments throughout the experiment (data
not presented). Table I shows the presence of many GGT-
1534
Labeling index.h
DBNA/TB
DBNA/TBlSAM)
DBNA/TB/BDATB
DENA/TB/BD/TB(SAM)
5 20 -1 I (•/•
14 3 1 J 3.V
7 62 -> I 3 d
8 12 - 22
J
SAM
I
Treatment'1
The same treatments as in Table I BD. basal diel without TB SAM
(384 (jmol/kg/day) was injected i m during TB feeding in rals ot group 2.
or during BD leeding in rats ol group 6. The nits ol groups I and 5
received solvent alone LI of normal liver. 0 37 - 0 08
b
2(KX)-4()fK) preneoplastic hepatocytes per liver were counted
"Different from group I for at least P < 0 05
d
Difterent trom group 5 lor P < (MX) I
Table HI. Etlect ot SAM on ODC activity of rat liver during
hepatocarcinogenesis promotion by TB
Rat group
Treatment'1
ODC activity*
Normal liver
DENA/TB
DENA/TB(SAMl
DENAATB/BD
DENAATB/BDtSAMl
DENAyTB/BDn~B
DENA/TB/BD/TB(SAM)
31
43
35
32
29
57
37
9
3
3
3
4
5
4
± 10
± 5 T
± 1 6'1
: 2 I'1
i 0 4l
-n 3 Sl
± I ()'
J
The same treatments as in Table I BD. basal diet without TB SAM
(384 nmol/kg/day) was ui|eeted i m dunng TB feeding in rats ot group 2.
or dunng BD feeding in rats of groups 4 and 6 The rats of groups I 3 and
5 received solvent alone
b
Data are means ± SD ol 6 rats ODC activity is expressed as pmol ol
14
COi released trom | l- l4 C]ornithine per h. mg protein Normal rats were
killed 7 weeks after initiation No significant variations in ODC activity
occurred in these rats 13 and 16 weeks after initiation (data not included in
the Table)
^Different from C for P < 0 001
d
Different from group I lor P < 0 01
'Different from group 3 for P < (MX) I
'Different from group 4 for P < 0(X)l
^Different from group 5 for P < (MK)I
positive lesions, occupying 46.4% of liver, in initiated rats,
subjected to one TB cycle (group 1). A decrease in number/
liver of these lesions, leading apparently to a 59% decrease in
volume fraction, occurred 6 weeks after arresting TB treatment
S-Adenosyl-L-methionine chemoprcvcntlve effect
kb
5
6
2.5
-c-myc
c-Ha-ras
co
1.4
*» V
•
-c-Ha-rat
3 4
CD
OC
19
-»
•
• . « • « . _
-a-actin
1
1JJJ
-jj
Fig. 2. Autoradiograms from representative Northern blot of 2 u.g poly(A)+ RNA and densitometric analysis of four different experiments (mean ± SD) with:
liver of normal rats (lane I); uninitiated rats subjected to two TB cycles (group C2, lane 2); initiated rats subjected to one TB cycle without (lane 3) or with
(lane 4) SAM; initiated rats subjected to two TB cycles without (lane 5) or with SAM (lane 6). Densities of c-myc and c-Ha-ras mRNAs were normalized to
the density of a-actin mRNA. c-myc, one TB cycle and two TB cycles (lanes 3 and 5) versus normal liver (lane \), P < 0.001; versus uninitiated rats (lane
2), at least P < 0.01 (f-test). SAM-treated, lane 4 versus lane 3 and lane 6 versus lane 5, P < 0.001. c-Ha-ras, uninitiated (lane 2) versus normal liver (lane
I). P < 0.01; one TB cycle and two TB cycles (lanes 3 and 5) versus normal liver (lane 1), P < 0.001, versus uninitiated rats (lane 2), P < 0.01.
SAM-treated, lane 4 versus lane 3 and lane 6 versus lane 5, at least P < 0.001.
(group 3). Partial recovery of lesion number and increases in
volume and volume fraction apparently occurred after a second
TB cycle (compare group 5 with group 3). However, since the
rats of various groups were killed at different times after
initiation, the decrease in lesion number (group 3 versus group
4) and the recovery after the second TB cycle could be underor overestimated. GGT-positive lesions were absent in normal
rats (group C), whereas a few GGT-positive minifoci (6-10
cells each) developed in initiated rats without TB (group Cl)
and in TB-treated uninitiated rats (group C2; not shown). SAM
treatment during the first TB cycle (group 2, treatment A)
caused 60, 27.6 and 75.6% decreases in number, volume and
volume fraction of GGT-positive lesions respectively. SAM
treatment after the first TB cycle (group 4, treatment B) caused
a decrease in size of these lesions and prevented increases in
number and size in rats again fed the TB diet either for 3
(compare group 6 with group 5) or 5 weeks (data not presented).
To exclude the possibility that SAM merely affected GGT
expression in preneoplastic cells, cytologically recognizable
lesions were counted in serial sections stained with hematoxylin
and eosin. Clear/eosinophilic, mixed and basophilic cell lesions
represented 86.5, 11.7 and 0.8% of all lesions respectively in
initiated rats subjected to one TB cycle. These percentages
were not significantly affected by SAM treatments A and B,
which, however, induced a decrease in lesion number/liver
from 14 780 ± 2864 and 12 680 ± 1992 in groups 1 and 5
(untreated) to 3970 ± 724 and 5400 ± 862 in groups 2 and
6 (SAM-treated; mean ± SD, n = 5, P < 0.001).
LI of preneoplastic hepatocytes (Table IT) was 76% higher
in the rats subjected to two TB cycles than in those subjected
to one cycle. SAM treatments A and B caused 36 and 48%
inhibition respectively.
As shown in Table UJ, ODC activity was 36 and 80% higher
in the liver of initiated rats subjected to one and two TB cycles
than in normal liver. No variations in ODC activity occurred
in initiated controls without TB 7-16 weeks after initiation
(not shown), while an 8-10% increase was found in uninitiated
controls subjected to one or two TB cycles (ODC activity
34.5 ± 1.3 at the end of the first cycle and 35.1 ± 1.5 at the
end of the second cycle; mean ± SD, n = 4, P < 0.01 versus
normal liver). ODC activity returned to normal values after
TB withdrawal. SAM treatment A caused an 18% decrease in
ODC activity, while treatment B induced a small (9%) but
significant inhibition of enzymatic activity (compare group 4
with group 3) and largely prevented the rise in ODC activity
induced by the second TB cycle.
Figure 2 shows a representative Northern blot hybridizing
with c-myc, c-Ha-ras and a-actin probes, with the mean
densitometric values of four experiments. It appears that c-myc
and c-Ha-ras mRNAs increased in the liver of initiated rats at
the end of the first TB cycle (lane 3) with respect to normal
liver (lane 1) and uninitiated controls treated with two TB
cycles (lane 2). A second TB cycle further increased c-myc
and c-Ha-ras mRNA levels in initiated rats (lane 5). SAM
treatment A (lane 4) strongly decreased c-myc and c-Ha-ras
mRNA levels in these rats and treatment B (lane 6) prevented
the rise in mRNA levels induced by the second TB cycle.
TB, a non-necrogenic xenobiotic at the doses used in the
present paper, induced the development of many GGT-positive
lesions in initiated rats. Previous observations (11) and the
data in the present paper indicate that TB withdrawal is
followed by the disappearance of many preneoplastic lesions,
probably dependent on phenotypic reversion (see 11), while
most initiated cells acquire a preneoplastic phenotype and are
stimulated to grow by a second TB cycle. In agreement with
previous results (3,4), SAM treatment A (during the first TB
cycle) induced a decrease in number and size Of preneoplastic
lesions. However, our results clearly show that although SAM
treatment B only slightly enhanced spontaneous disappearance
of preneoplastic lesions after cessation of the first TB cycle,
1535
M.M.Simile ft al
it largely prevented recovery of number and volume induced
by the second TB cycle, after SAM withdrawal. This excludes
a marked induction of cell death by SAM under the present
experimental conditions. A persistent decrease in DNA synthesis after SAM withdrawal, probably associated with an
inability of phenotypically reversed cells to re-express the
preneoplastic phenotype. occurs in SAM-treated rats.
One of the main features of fast growing cells is a high
activity of ODC. a key enzyme of polyamine synthesis (20).
This activity and polyamine synthesis are high in the liver
during the development of preneoplastic lesions and correlate
with growth rate of these lesions (21.22). TB induced a slight
increase in ODC activity in the liver of uninitiated rats, whereas
large increases occurred in initiated rats subjected to TB. This
indicates a possible contribution of preneoplastic cells to the
ODC increase, as is also suggested by the coincidence of the
highest increase in ODC activity with maximum development
of GGT-positive lesions SAM treatments A and B caused a
decrease in ODC activity to near to normal liver values and
SAM treatment B prevented the enhancement of this activity
by a second TB cycle. Although ODC activity was determined
in whole liver, the rough correspondence between this activity
and the relative percentage of preneoplastic lesions suggests
that the effect of SAM, at least in part, depends on a fall in
the volume fraction of GGT-positive lesions and on a lowered
ODC activity in the remaining lesions. Indeed, these lesions
had a poor growth capacity, as indicated by a relatively low
level of DNA synthesis and a slight size increase at the end
of the second TB treatment.
Analogous behavior was found on evaluating c-wyc and cHa-ra.v mRNA levels. These oncogenes are overexpressed in
the liver during the development of preneoplastic lesions and
immunohistochemically recognizable gene products have been
found almost exclusively in these lesions (6.8.23,24). TBinduced overexpression of these oncogenes occurred only in
initiated rats, suggesting that this effect was linked to the
presence of growing preneoplastic lesions, while no evident
increase in gene expression occurred in initiated rats receiving
two TB cycles plus SAM Polyamines and c-myc and c-Ha-ra.v
gene products play a role in the control of cell proliferation
(20,25). Previous work showed inhibition by SAM of c-mvc
and c-Ha-ra.v expression and ODC activity in preneoplastic
liver lesions (6.8,22). The present results indicate that this
inhibition persists on stimulation by a promoter This should
lead to a decrease in growth signal transduction and cell cycle
progression and could explain the persistently scarce tendency
of initiated cells to respond to TB by enhancing DNA synthesis
in SAM-treated rats. Since active growth is necessary for the
evolution of initiated cells to malignancy, these cells should
not be propelled by mitogenic stimuli to more advanced stages
of the tumorigenesis process in SAM-treated rats, even after
the cessation of SAM administration.
Acknowledgement
This work was supported by tunds Irom (he CNR (prog Mil ACRO)
As.socia/ione hah.ma Riccrca sul Cancro. M t ' R S T (progr 60'^ and 40'^f )
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Received on December 12, 1995; revised on March 18, 1996: accepted on
March 26, 1996
1537

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