[CANCER RESEARCH54, 2895-2899, June 1, 1994@
Enhancement by Organosulfur Compounds from Garlic and Onions of
Diethylnitrosamine-induced Glutathione S@TransferasePositive
Foci in the Rat Liver'
NobuyasuTakada,2TsutomuMatsuda,TatsuyukiOtoshi,YoshihisaYano, ShuzoOtani,Tohru Hasegawa,Dai Nakae,
Yoichi
Konishi,
and Shoji
Fukushinia
First Department of Pathology (N. T., T. M., 7'. 0., S. F.] and Second Department of Biochemistry [V. Y, S. 0.], Osaka City University Medical Schoo4 Asahi-machi
Abeno-kis, Osaka 545, Japan; Department of Community Health Science, Saga Medical Schoo4 Saga 849, Japan [T. H.]; and Department of Oncological Pathology, Cancer
Center, Nara Medical University, Nara 634, Japan ID. N., Y. K]
ABSTRACT
cancer, esophageal
Ten organosulfur compounds from garlic and onions were studied for
their modifying effects on diethylnitrosamine-induced neoplasia ofliver in
male F344 rats using the medium-term bioassay system oflto based on the
two-step model of hepatocarcinogenesis. Carcinogenic potential was
scored by comparing the number and area per cm2 ofinduced glutathione
S-transferase placental form-positive foci in the liver with those of the
corresponding control group given diethylaitrosamine alone. In experi
meats 1 and 2, high doses of diallyl sulfide, diallyl trisulfide, allyl methyl
sulfide, allyl methyl trisulfide, and dipropyl sulfide had enhancing effects
on focus formation. In contrast, high doses ofmethyl propyl disulfide and
propylene
sulfide
significantly
decreased
the
number
of glutathione
S
transferase placental form-positive foci. In the third experiment, corn
biased treatment with the five chemicals that had enhancing activity were
fed at low doses and increased the induction of glutathione S-transferase
placental form-positive foci. To investigate the mechanism of the modify
big effect on hepatocarcinogenesis, ornithine decarboxylase activity was
measured
In diallyl sulfide-,
allyl methyl sulfide-, and dipropyl
sulfide
treated liver tissue without prior initiation with diethylnitrosarnine, and
its activity
was increased
N'-acetyltransferase
compared
to controk
Spermidine/spermine
activity was not significantly changed. Formation of
cancer, pulmonary
adenoma,
and forestomach
tumors in rodents when administered prior to administration of several
carcinogens (13—16).Moreover, DAS inhibited hepatocarcinogenesis
when administered after an initiating procedure, but this has not been
consistently observed (17, 18).
DAS, diallyl disulfide, diallyl trisulfide, allyl methyl sulfide, allyl
methyl trisulfide, and propylene sulfide are richly contained in garlic;
and dipropyl sulfide, dipropyl disulfide, methyl propyl disulfide, and
dimethyl disuffide are contained abundantly in onion (7, 11, 19, 20).
In the present study, the modifying potential in the promoting stage
of ten OSCS contained in garlic and onion was examined in a rat liver
medium-term bioassay system as defined by Ito et aL (5). Moreover,
the enhancement by combined treatment with OSCS was evaluated in
the same system. To begin investigating possible mechanisms that
might be involved, formation of 8-OHdGuo, a DNA adduct generated
by activated oxygen species, and ThA-reacting substances produced
in rat liver were measured (21, 22). In addition, ODC and SAT, which
are rate-limiting enzymes of polyamine biosynthesis and were re
ported to be increased in the promoting stage of skin and bladder
carcinogenesis (23, 24), were also measured.
8-hydroxydeoxyguanosine,a DNAadduct generated by activated oxygen
species, and lipid peroxidation
(2-thiobarbituric
acid-reacting
substance
production) were also not changed. These results suggest that the promot
lag effect could be caused by increased cell proliferation with increased
polyamine biosynthesis. In evaluating relationships between diet and can
car, it is appropriate to consider not only the possible protective role of
garlic and onions but also their enhancing effects.
MATERIALS
Chemicals. Organosulfur compounds used are DAS, DDS, DAT, AMS,
AMT, DPS, DPD, MPD, PS; and DMD (19, 20). DAT and AMT were obtained
from Oxford Chemicals Co., Northhamptonshire, United Kingdom; MPD was
from Aldrich Chemical Co., Milwaukee, WI; and the other OSCs and DEN
from Tokyo Chemical industry Co., Tokyo, Japan.
Animals
INTRODUCTION
Environmental compounds are known to be involved in the gener
ation of many human cancers. Elimination of carcinogenic corn
pounds from our environment would be expected to help prevent
human cancer, but this is not a practical proposition. Therefore, it is
important to discover naturally occurring or synthetic compounds
which can suppress or prevent the process of carcinogenesis (1—12).
We have focused on some OSCs3 which are contained in garlic and
onion. Some OSCS have been proved to be chemopreventive in animal
models. For example, diallyl sulfide inhibits development of colon
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement
in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
I This study was supported by Grant-in-Aid of Cancer Research from the Ministry of
Health and Welfare.
2 To
3 The
whom
requests
abbreviations
for
reprints
used
are:
should
OSCs,
be
addressed.
organosulfur
compounds;
DAS,
diallyl
sulfide;
DDS, diallyl disulfide; DAT, diallyl trisulfide; AMS, allyl methyl sulfide; AMD, allyl
methyl disulfide; AMT, allyl methyl trisulfide; PS, propylene sulfide; DPS, dipropyl
sulfide; DPD, dipropyl disulflde; MPD, methyl propyl disulfide; DMD, dimethyl disul
fide; DEN, N-diethylnitrosamine; 8-OHdGuo, 8-hydroxydeoxyguanosine; ODC, ornithine
and
Treatments.
Male
F344
rats obtained
at 5 weeks
of age
(Charles River Japan, Inc., Hino, Japan) were housed in an air-conditioned
room at 23 ±1°C
(SD), a relative humidity of 36 ±6%, with a 12-h light/12-h
dark cycle, and were given diet (Oriental MF; Oriental Yeast Co., Tokyo,
Japan) and tap water ad libitum. The diet is a commercial diet, which is a
modification of the NIH open formula diet (NIH-07). Rats were acclimatized
for 1 week before use.
Experiments 1 and 2 were performed to investigate modifying effects of
each OSC on hepatocarcinogenesis.
In experiment
1, a total of 150 rats were
divided into 12 groups. The rats in groups 1 to 5 were given a single i.p.
injection of DEN (200 mg/kg body weight) dissolved
in saline to initiate
hepatocarcinogenesis. After 2 weeks on basal diet, they received DAS (100
mg/kg body weight; group 1), DDS (25 mg/kg body weight; group 2), AMS
(150 mg/kg
Received8/18/93;accepted4/5/94.
AND METhODS
body weight;
group
3), DPS (150 mg/kg
body
weight;
group 4),
and DPD (150 mg/kg body weight; group 5) dissolved in corn oil (1 mI/kg) by
i.g. gavage
5 times/week
for 6 weeks.
Animals
were subjected
to PH at week
3 to maximize any interaction between proliferation and the effects of the
compounds
tested. Group 6 was given DEN and PH without administration
of
any test compounds. Animals in groups 7 to 11 received saline instead of DEN
solution but were subjected to administration of test compounds and PH.
Group 12 animals were given saline injections and then subjected to admin
istration of corn oil instead of test compounds; they were also subjected to PH.
Rats in each group were killed for examination
at week 8 (Fig. 1). The livers
acid; i.g., intragastrically; PH, two-thirds partial hepatectomy; GST-P, glutathione 5-
were examined immunohistochemically for GST-P expression.
In experiment 2, a total of 150 rats were divided into 12 groups. Rats in
transferase placental form.
each group were treated by the same medium-term
decarboxylase;
SAT, spermidine/spermine
N1-acetyltransferase;
TBA, 2-thiobarbituric
bioassay
system
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as
ORGANOSULFIDE ENHANCEMENT OF HEPATOCARCINOGENESIS
@3
Groups
@
1—5
Fig. 1. Liver medium-term bioassay protocol. V,
DEN, 200 mg/kg body weight (i.p.); V, saline, 5
6
mI/kg body weight (i.p.), •, partial hepatectomy; 5,
sacrifice;
I
I
8weeks
@iiII—@
—I
@,
test chemicals (i.g.) 5 times/week; 0,
corn oil, 1 mI/kg body weight (i.g.).
7
@.11 [
:_______
I
12
experiment 1. They received DAT (150 mg/kg body weight; group 1), AMT
(100 mg/kg body weight; group 2), MPD (100 mg/kg body weight; group 3),
Measurement of SAT and ODC Activity and Polyamine Biosynthesis.
We measured SAT and ODC activity by the method of Matsui et aL (36) and
PS (50 mg/kg body weight; group 4), and DMD (50 mg/kg body weight; group
5) dissolved in corn oil by i.g. gavage 5 times/week. All rats were killed at
week 8, and livers were examined immunohistochemically for GST-P expres
0.5 ml of 50 mM Tris (pH 7.5) containing
sion, formation
at 100,000
of 8-OHdGuo,
and production
of ThA-reacting
substances.
The dose of DAS and DDS were one-half of the doses (200 and 50 mg/kg
body weight) used in a previous experiment by us (18). The doses of the other
chemicals tested were the same or nearly equimolar concentration
as DAS or
DDS. Each dose is less than the maximum tolerated dose.
In experiment 3, the modifying effects with combined treatment of OSCs on
rat hepatocarcinogenesis modification were examined. A total of 105 rats were
divided into 7 groups. Rats in each group were given DEN and PH with or
without combined treatment of 5 test compounds. The test compounds which
increased
the areas and numbers
and 2 were DAS,
DAT, AMS,
of GST-P-positive
AMT,
foci from experiments
1
and DPS. The doses of the combined
treatment of five OSCs are shown in Table 1. The determination of doses were
based on the ratio of their content in garlic (groups 1—3)
or using equimolar
concentrations
(groups
4—6). For these two combinations,
three dose levels
Otani et a!. (37), respectively. The frozen piece of rat liver was suspended in
0.25 M sucrose
homogenizer for 30 seconds. The homogenized
and disrupted
by a
suspensions were centrifuged
x g for 30 mm, and the supernatant was assayed for ODC and SAT
activity by measurement of the amount of radioactive putrescine produced
from [5-'4C]ornithine and the amount of acetyl moiety transferred from
[1.'4C]acetyl-CoA to spermidine, respectively.
Statistical Analysis. Statistical analysis of the observed values was per
formed using Student's t test or Dunnett's t test.
RESULTS
In DEN-initiated groups of experiments 1 and 2, final body weights
were significantly decreased in rats treated with DPD, DAT, and PS
compared to rats treated without test chemicals. Relative liver weights
were significantly increased in DEN-initiated rats treated with DDS,
were used.
In experiment 4, ODC and SAT activity were measured in the liver follow
ing simple administration
groups.
From
of the OSCs. A total of 20 rats were divided
the beginning
of the experiment,
the rats received
into 4
Table 1 Treatment of 0SCs with two
differentsynergisticcombinationsDoseGroup(mg/kg)DATAMTDASAMSDPS112032122221/2ofgroupl601661131
DAS (100
mg/kg body weight; group 1), AMS (150 mg/kg body weight; group 2), or DPS
(150 mg/kg body weight; group 3) in corn oil (1 mI/kg) by i.g. gavage 5
times/week. Group 4 animals received corn oil alone. All rats were killed at
week 3.
Tissue Processing. At autopsy, livers were excised and sections 2—3
mm
thick
were
cut with
a razor
blade.
Three
slices,
one
each
from
I30830.50.54302020303051/2ofgroup4151010151561/4
of group
the right
posterior, anterior, and caudate lobes, were fixed in ice cold acetone for
47.5557.57.5700000
of group
immunohistochemical
examination of GST-P in experiments 1—3.To measure
the formation of 8-OHdGuo and lipid peroxidation of rat liver in experiment 2
and ODC and SAT activity of rat liver in experiment 4, rat livers were frozen
by liquid nitrogen.
GST-P Immunohistochernistry. The avidin-biotin-peroxidase complex
method was used to demonstrate
GST-P-positive
Table 2Final body and liver weights of rats initiated with DEN in experiment 1
OSCsTest
followed by treatment with various
liver foci, a putative preneo
plastic lesion (5, 25, 26). After deparaffinization, liver sections were treated
sequentially with normal goat serum, rabbit anti-GST-P (1:8000) (provided by
EffectiveRelative
no. of rats
Final wi (g)(g/100
wtGroupchemical
wt)1
Dr. K. Sato, Hirosaki University) biotin-labeled goat anti-rabbit IgG (1:400)
and avidin-biotin-peroxidase
complex. The sites of peroxidase binding were
demonstrated by the diaminobenzidine method. Sections were then counter
stained with hematoxylin
@
for microscopic
examination.
As a negative
control
for the specificity of anti-GST-P antibody binding, preimmune rabbit serum
was used instead of antiserum. The numbers and the areas of GST-P-positive
foci >0.2 mm in diameter and the total areas of the liver sections examined
14
273 ±13―
2DAS
014b3
DDS
14
263±14333
14
268±19
4
5AMS
018b614
DPS
14
268±13
DPD
14
liver
g body
±0.20
3.65±
3.92±O.2l@'
243 ±19―3.32±0.16
3.04 ±
274 ±143.25
±0.17
a Mean ± SD.
b
< 0.01
(versus
group
6, Student's
t test).
were measured using a color video image processor (VIP-21C).
Determination
of 8-OHdGuo
Formation
in DNA.
Liver sample
portions
Table 3Final body and liver weights of
initiated with DEN in experiment 2
followed by treatmentrats
OSCsTest
with various
—2g, wet weight, were used. Liver DNA was isolated and digested as
described
by Takagi et aL (27). The level of 8-OHdGuo
formation
in resulting
samples was determined by high performance liquid chromatography by an
adaptation of the methods of Floyd et a!. (28, 29) and Kasai et a!. (30—32)as
residues
formed/10@ of total deoxyguanosine.
Determination of Lipid Peroxidation (TBA-reacting Substance Produc
tion). Peroxidationof liver lipids was quantitatedby measuringaccumulation
of TBA (Sigma)-reacting
@
substances and expressed as nmol value equivalent
(eq) to a standard amount of malondialdehyde (dimethyl acetal; Aldrich) by an
adaptation of the method of Yagi (34) as described previously (35).
no. of ratsFinal
016b2AMT
±3MPD
0.18c4PS
0.I4'@5DMD
described previously (33). The level of 8-OHdGuo formation was expressed as
the number of 8-OHdGuo
EffectiveRelative
wtGroupchemical
wt)1DAT
0.23c615261
aMean
b
< o.oi
wt (g)(g/100
liver
g body
15232
±14a.b3.36
14256
15261
±103.04
±122.69
14245
±12b2.81
±
±
14262
±102.74
±142.56
±
±0.17
±
± SD.
(versus
group
6, Student's
t test).
Cp < 0.05 (versus group 6, Student's t test).
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ORGANOSULFIDE
@
@
@
ENHANCEMENT
DPS, DAT, AMT, MPD, PS, and DMD and decreased in DPD-treated
group (Tables 2 and 3).
Tables 4 and 5 show the numbers and areas of GST-P-positive foci
per unit area of liver sections after DEN initiation. In experiment 1,
values for both parameters in groups given DAS, AMS, and DPS were
significantly increased over control levels. In particular, DPS was
most effective. In experiment 2, in groups given DAT or AMT, the
number and area of GST-P-positive foci were significantly increased.
In contrast, in groups given MPD or PS, values for the number of foci
were significantly decreased. In experiments 1 and 2, GST-P-positive
foci were not seen, and the histological findings of livers were normal
in groups without DEN.
For experiment 3, final body and relative liver weights are sum
marized in Table 6. Final body weights were significantly decreased
in rats treated concomitantly with high doses oftest chemicals (groups
1 and 4). Relative liver weights were significantly increased in all of
the treated groups, and a dose dependency could be observed. The
proportion of numbers and areas of GST-P-positive foci in the differ
ent groups compared to group 7 (control) are summarized in Fig. 2.
They were significantly increased by the combined treatments corn
pared to the control group. In each group treated with test chemicals,
no significant differences were observed, but there was a tendency
toward a dose-dependent response.
Results for determinations of 8-OHdGuo formation in DNA and
ThA-reacting substances produced in the livers of rats initiated by
DEN in experiment 2 are shown in Table 7. Formation of 8-OHdGuo
tended to decrease in groups treated with MPD or PS. Results for
ThA-reacting substances displayed significant decreases in rat livers
with MPD and PS treatment. In groups treated with DAT or AMT,
formation of 8-OHdGuo and ThA-reacting substances hardly in
creased compared to control group.
For experiment 4, the results of ODC and SAT activity in the liver
are shown in Table 8. ODC activity increased in DAS (42.9 ± 12.5
pmol/mg)-, AMS (137.5 ± 157.6 pmol/mg)-, and DPS (38.7 ±
34.5 pmol/mg)-treated rats over the control level (13.5 ± 6.6 pmol/
mg) and was especially elevated significantly in the group treated with
DAS. SAT activity was not increased in any of the groups.
Table 4 N
and areas of GST-P positive foci in the liver of rats initiated with
EN inOSCsTest
experiment 1 followed by treatment with various
Dumbers
No.Groupchemical
(mm2/cm2)IDAS
of rats
No/cm2
0.17'2DDS
0.173AMS
14
14
6.58 ±2.12'@b
4.39 ±1.81
Area
0.52 ±
0.33 ±
018b4DPS
14
6.14 ±1.71c
0.56 ±
0.29―5DPD
14
8.45 ±2.52―
0.88 ±
0.20614
14
5.53±1.39
0.43±
4.69 ±1.61
0.38 ±0.16
aMean ±SD.
b Significantly
different
from
group
6 at P < 0.05
(Student's
t test).
C Significantly
different
from
group
6
(Student's
t test).
d Significantly
different
from
group
6 at P < 0.001
at
P
<
0.01
(Student's
t test).
and areas of GST-P positive foci in the liver of rats initiated with
Table 5 Nit
OSCsGroupTest
EN in experiment 2 followed by treatment with various
Dmbers
No.
(mmicm2)1DAT
chemical
±2
MPD
3
0.175DMD PS
4AMT
0.16615
aMean
of rats
No./cm2
15
6.62 ±1.89―
Area
0.58
14
8.24 ±2.71c
0.59 ±0.15'
15
14
14
3.28
3.17
4.09
4.71
0.26
0.26
0.35
0.35
±155b
±172b
±1.81
±1.38
± SD.
b Significantly
different
from
group
6 at P < 0.05
(Student's
t test).
C Significantly
different
from
group
6
(Student's
t test).
at
P
<
0.01
±0.17
±
±
±0.14
OF HEPATOCARCINOGENESIS
Table 6 Fina1 body and liver weights of rats initiated with DEN in experiment 3
OSCsEffectiveRelative
followed by treatment with various
WIGroupno.
WI)1
of rats
liver
g body
Final WI (g)(g/l00
zs6±lla.@@
2
14
268±9
3.77±0.10―'
3
14
266± 11
4
14
14
263±11―
269±11
3.60±0.l6@''
3.69±0.12―
5
613
3.47±0.17――715
14
3.59±0.21―
278± 103.94±0.17―
±0.16
287 ± 132.91
a Mean
b
± SD.
< o.oi
(versus
group
7, Student's
t test).
Cp < o.os (versus group 1, Student's t test).
d
< 0.01
(versus
group
4, Student's
t test).
Number (treated/control ratIo)
Area (treated/control ratIo)
2
3
4
5
6
7
3
2
1
u
0
:::j
1
,
2
I
3
I
Fig. 2. Numbers and areas ofGST-P-positive foci in the liver ofrats initiated with DEN
followed by treatment with various OSCs. Significantly different from group 7 at
*P < 0.05, **@ <
Dunnett's t test.
DISCUSSION
Although most OSCs have been shown to be chemopreventive in
previous studies, high doses of DAS, DAT, AMS, AMT, and DPS
were shown to promote preneoplastic lesion development in the rat
liver in the present study. Their combined treatment enhanced GST
P-positive focus development. Their effects indicated a remarkable
dose dependency in the two different combined groups. Therefore,
their combined treatment may exert promoting activities on hepato
carcinogenesis of rats.
In previous studies, the inhibiting effects of OSCS were mainly
examined in the initiation stage (13, 38—42).AMT, AMD, DAT, and
DAS administered 96 and 48 h prior to the carcinogen inhibited
benzopyrene-induced tumors of the forestomach in female A/i mice.
DAS and AMD inhibited pulmonary adenoma formation in female
A/i mice, but neither DAT nor AMT did (41). In those experiments,
the administration of OSCs during the initiation stage inhibited the
metabolic activation of the carcinogens (43, 44); but in our experi
ment, since we administered OSCs during the promotion stage, our
results imply the involvement of a different mechanism.
The selection of the bioassay system in this study was based on
examining the modifying potential of OSCs on the promoting stage of
hepatocarcinogenesis. The data obtained from the present investiga
tions
unexpectedly
showed
enhancing
effects
of five OSCs,
DAS,
DAT, AMS, AMT, and DPS, several of which inhibited carcinogen
esis by administration during the initiation stage. This bioassay system
has been utilized to detect modifying effects of many chemicals on
liver carcinogenesis (5). Moreover Ogiso et a!. (45, 46) indicated that
the degree of induction of GST-P-positive foci and nodules in this
bioassay system for liver carcinogens corresponds with the incidence
of hepatocellular carcinomas revealed in a long-term in vivo assay.
Consequently the present study strongly suggests that these OSCs
promote hepatocarcinogenesis.
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ORGANOSULFIDEENHANCEMENTOF HEPATOCARCINOGENESIS
Table 7 Induction of oxidative DNA damage and ThA-reacting substance production
in rat livers initiated with DEN followed by treatment with various OSCs
substance
production (@mol
mg)1DAT4.5±0.9―8.7±
malondialdehyde/l00
deoxyguanosineThA-reacting
chemical8-OHdGuo/10@
GroupTest
1.52
±0.8
8.0±3.0―
3.1 ±1.4
MPD
3
6.9±l.l@'5DMD4.7±2.08.2±
3.6±1.59.4±0.5
4AMT
PS4.1
1.164.7
±2.211.2
±1.8
aMean ±SD.
b Significantly
different
from
group
6 at P < 0.05
(Student's
t test).
Table 8 ODC and SAT activity in liver of rats treated with OSCs
Test
protein)DAS42.9
chemicalODC
activity
(pmol/hlmg protein)SAT
1.20AMS137.5
0.90DPS38.7
±12.5a0.24
±1576b0.82
±
±
±0.72
0.77 ±1.42
±34.5
13.5 ±6.60.45
aM@
activity
(pmol/10 min/mg
± SD.
b Significantly
different
from
the nontreated
group
at P < 0.05
(Student's
t test).
8-OHdGuo correlates with development of putative preneoplastic
‘y-glutamyltransferase-positive focal lesions using a rat hepatocarci
nogenesis model by a choline-deficient L-amlno acid-defined diet.
Takahashi et aL (55) reported that lipid peroxidation was increased in
gastric mucosa by NaCl, a stomach cancer promoter. That study
clearly demonstrated that the gastric tumor promoter NaCl dose
dependently increased ThA-reacting substances production levels in
both gastric mucosa and urine, supporting a conclusion that there is
enhanced lipid peroxidation in the target tissue. Although increased
formation of 8-OHdGuo in DNA and lipid peroxidation were cx
pected to participate in the promoting effects of OSCs, they were not
significantly involved as determined in the present study.
In this study, the number but not the area of GST-P-positive foci
was significantly decreased in rats given PS or MPD. This suggests
that these compounds might have inhibitory effects on hepatocarci
nogenesis. In addition, the reason why induction of 8-OHdGuo and
lipid peroxidation were decreased in the liver should be clarified with
further experiments.
The test chemicals are sulfur-containing, volatile compounds, and
especially the monosulfides, except PS, and the trisulfides have a
tendency to enhance GST-P-positive focus formation. The number of
sulfur atoms may relate to the mechanism of promotion of hepatocar
cinogenesis by OSCS (56).
Humans are exposed to numerous environmental chemicals during
their life span and the chemicals may act in combination, positively or
antagonistically, to affect cancer production. Moreover synergistic
Most of OSCs used may tend to decrease body weights and increase
relative liver weights. In further studies, we must examine the dose
response for each OSC which enhanced GST-P positive focus forma
tion in order to evaluate the possibility of a practical threshold. In
addition, we must dissociate the effects of enhancing focus formation
effects
of chemicals
on development
of tumors
have been repeatedly
from the toxicity of OSCS.
A modifying response of DAS in the promoting stage was exam
observed in many organs (57, 58). We observed a dose-dependent
tendency in the promoting effects by combined treatment with five
med in a rat multiorgan carcinogenesis model by Jang et a!. (17) and
Takahashi et a!. (18), with different results. Jang et al. reported that
OSCs (DAS, DAT, AMS, AMT, and DPS) in two different combi
DAS administered in the diet significantly decreased the incidence of
nation groups. Since we also found two inhibitors, we need additional
hepatic hyperplastic nodules in the liver. However, Takahashi et a!.
studies to examine the effects of combined treatment with enhancers
reported that DAS demonstrated clear enhancing effects on hepato
and inhibitors. It is possible that low doses of carcinogenic substances
carcinogenesis. In the experiments of Takahashi et aL and in our
in combination are of critical importance in determining effects. This
study, rats received DAS by i.g. intubation, which was quite different
may contribute to the correlation between hepatocarcinogenesis and
from the method of Jang et a!. Moreover, Takahashi et a!. reconfirmed
intake of garlic and onion.
their results in the DEN-initiated liver medium-term bioassay con
In conclusion, many OSCs which exerted promoting effects in
cerning promoting effects of DAS. Our present findings are in agree
hepatocarcinogenesis exist in garlic and onion. ODC activity in
ment with the latter. The reason for this discrepancy with Jang et a!.
creased in the treated groups. Moreover, these data suggest that
is unclear.
cell proliferation in liver tissue is important for the promoting
Cell proliferation has long been suspected of enhancing the fre
effects of OSCs.
quency of tumor initiation in chemical carcinogenesis. In addition,
cell proliferation appears to play an important role in the effects of
ACKNOWLEDGMENTS
tumor promoters or nongenotoxic carcinogens (47, 48). Polyamines
are involved in epithelial cell proliferation (49). ODC, which is a
We extend our gratitude to the late Professor Kiyomi Sato of the Second
biomarker of cell proliferation, increased with epithelial cell prolifer
Department
of Biochemistry,
Hirosaki University,
for the generous provision
ation of skin or bladder when a promoting agent was administered (23,
of purified antibody against GST-P.
24). ODC activity is high in carcinomas induced experimentally by
chemical carcinogens (50, 51) and in carcinomas obtained from pa
tients (52, 53). SAT is a newly established rate-limiting enzyme of
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Enhancement by Organosulfur Compounds from Garlic and
Onions of Diethylnitrosamine-induced Glutathione S
-Transferase Positive Foci in the Rat Liver
Nobuyasu Takada, Tsutomu Matsuda, Tatsuyuki Otoshi, et al.
Cancer Res 1994;54:2895-2899.
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