Full text of DOI:10.1207/S15327914nc391_13

Nutrition and Cancer
ISSN: 0163-5581 (Print) 1532-7914 (Online) Journal homepage: http://www.tandfonline.com/loi/hnuc20
Effects of Lycopene and Sho-saiko-to on
Hepatocarcinogenesis in a Rat Model of
Spontaneous Liver Cancer
Seishiro Watanabe , Yukihiro Kitade , Tsutomu Masaki , Mikio Nishioka ,
Kimihiko Satoh & Hoyoku Nishino
To cite this article: Seishiro Watanabe , Yukihiro Kitade , Tsutomu Masaki , Mikio Nishioka ,
Kimihiko Satoh & Hoyoku Nishino (2001) Effects of Lycopene and Sho-saiko-to on
Hepatocarcinogenesis in a Rat Model of Spontaneous Liver Cancer, Nutrition and Cancer, 39:1,
96-101, DOI: 10.1207/S15327914nc391_13
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NUTRITION AND CANCER, 39(1), 96–101
Copyright © 2001, Lawrence Erlbaum Associates, Inc.
Effects of Lycopene and Sho-saiko-to on Hepatocarcinogenesis in a
Rat Model of Spontaneous Liver Cancer
Seishiro Watanabe, Yukihiro Kitade, Tsutomu Masaki,
Mikio Nishioka, Kimihiko Satoh, and Hoyoku Nishino
Abstract: The Long-Evans Cinnamon (LEC) rat is a well-
characterized model of spontaneous hepatocarcinogenesis.
It has been shown that dietary administration of lycopene or
the herbal medicine Sho-saiko-to (TJ-9) has anticarcino-
genic activity, although the mechanism by which these prod-
ucts protect against carcinogenesis is not well known. We
investigated the outcome of administration of lycopene and
TJ-9 on the occurrence of hepatic neoplasia in LEC rats. A
diet containing 0.005% lycopene (originally the product of
tomato oleoresin containing 13% lycopene) and 1% TJ-9
Introduction
Epidemiological studies have suggested that increased
vegetable intake lowers the incidence of cancer deaths in the
elderly (1,2). Lycopene is one of the major carotenoids in to-
matoes and is known to have a protective effect against can-
cer with its high single oxygen-quenching capability (3).
Low plasma lycopene levels have been demonstrated in pa-
tients with hepatitis and cirrhosis (4). Sho-saiko-to (TJ-9) is
a Chinese herbal medicine widely used in Japan. The active
components of TJ-9, baicalin and baicalein, were reported to
have an antioxidative activity (5). TJ-9 has been shown to
prevent hepatic fibrosis (5) and growth of hepatocellular car-
cinoma (HCC) experimentally (6) and clinically (7). A high
incidence of HCC in aging Long-Evans Cinnamon (LEC)
rats has been reported (8). Approximately 30–40% of LEC
rats die at around four months of age from acute liver failure;
the remaining rats survive with chronic hepatitis and de-
velop preneoplastic and neoplastic liver lesions (9). Almost
100% of these rats older than 1.5 years have liver cancer (8).
LEC rats show a pattern of hepatocarcinogenesis similar to
the Solt-Farber model (10), and glutathione S-transferase
placental form (GST-P)-positive staining has been detected
in LEC rats without administration of exogenous carcino-
gens (11). GST-P is a preneoplastic and neoplastic marker
enzyme of rat hepatocarcinogenesis (12).
Inhibition of the development of HCC and cholangio-
fibrosis has been demonstrated in LEC rats with the use of
the copper-chelating agent trientine dihydrochloride (13).
Feeding the LEC rats a copper-deficient diet completely
suppressed the manifestation of the chronic liver cell injury
(14). Accumulation of copper may act as a promoting factor
for the development of liver cancer in LEC rats (9). Iron
deprivation also has been reported to inhibit liver tumor de-
velopment, but not to inhibit the development of cholangio-
fibrosis (15). Accumulation of copper and iron, therefore,
appears to play a role in hepatocarcinogenesis in LEC rats.
(
crude extracts of 7 herbs: bupleurum root, pinellia tuber,
scutellaria root, jujube fruit, ginseng root, glycyrrhiza root,
and ginger rhizome) was administered from 6 weeks of age
until the rats were sacrificed at 76 weeks of age, at which
time most of the nontreated animals were known to have
hepatocellular carcinoma (HCC). Development of HCC in
treated groups was analyzed histologically by comparison
with untreated controls. Glutathione S-transferase placental
form (GST-P) was analyzed by an immunohistochemical
method. Concentration of copper, iron, and zinc, which ap-
pear to play a role in hepatocarcinogenesis in LEC rats, was
analyzed. The percent areas of HCC in the liver specimens
of control, lycopene, and TJ-9 groups were 17.9 ± 17.1%,
2
7.2 ± 20.8%, and 27.6 ± 18.4%, respectively. These inter-
group differences were not significant. The percent area,
number of areas, and mean size of area staining posi-
tively for GST-P revealed no significant differences between
the groups. The number of GST-P-positive areas within the
HCC lesions was greater in the TJ-9 group than in the con-
trol or lycopene group (p = 0.024 and p = 0.012, respec-
tively). The study also demonstrated a lower concentration
of iron in livers of the lycopene group than the control group
(
p = 0.019). There were no differences in serum a-feto-
protein levels or the cumulative survival rates between the
groups. In conclusion, long-term administration of lycopene
or TJ-9 did not reduce the risk of hepatocarcinogenesis in
LEC rats.
S. Watanabe, Y. Kitade, T. Masaki, and M. Nishioka are affiliated with the Third Department of Internal Medicine, Kagawa Medical University, Kagawa
61-0701, Japan. K. Satoh is affiliated with the Second Department of Biochemistry, Hirosaki University, School of Medicine, Hirosaki, Japan. H. Nishino is
7
affiliated with the Department of Biochemistry, Kyoto Prefectural University of Medicine, Kyoto, Japan.

To elucidate the effect of the heavy metals on hepatocar-
cinogenesis, copper, iron, and zinc in the liver, serum, and
diet were analyzed. The present study aimed to clarify the
effects of lycopene and TJ-9 on the spontaneous develop-
ment of HCC in LEC rats.
chen, Germany) without knowledge of the group of origin.
Areas of cholangiofibrosis in each liver specimen were ex-
cluded from the analysis of the number and the area of GST-
P-positive lesions.
Serum a-fetoprotein level at autopsy was measured by a
sensitive sandwich enzyme immunoassay method using an
affinity-purified antibody to rat a-fetoprotein (SRL, Tokyo,
Japan) in 31 rats (17).
Materials and Methods
About 0.5 g of liver was digested with a mixture of
perchloric acid and nitric acid (1:5), and the digest was di-
luted with deionized water. The copper, iron, and zinc con-
tents were measured with an atomic absorption/flame
emission spectrophotometer (type AA-630-01, Shimadzu,
Kyoto, Japan). Serum and diet were assayed directly in the
equipment after dilution with deionized water (18).
Values are means ± SD. Differences from the control
group were assessed using one-factor analysis of variance.
Intergroup differences were assessed with Fisher’s protected
least significant difference test. For comparison of the sur-
vival rate among the three groups, the Kaplan-Meier method
Male LEC rats (n = 105) bred at Charles River Japan
Kanagawa, Atsugi, Japan) were transported to our univer-
(
sity at six weeks of age and housed at 24 ± 1°C and 55 ± 5%
humidity. Rats were separated into three groups. The control
group (n = 40) was fed a conventional basal diet (Charles
River Formula-1, Charles River Japan, and Oriental Yeast,
Tokyo, Japan). Composition of the diet per 100 g was as fol-
lows: 22.6 g protein, 5.6 g fat, 3.3 g fiber, 1.27 g calcium,
.32 g sodium, 0.85 g potassium, 4.10 g other minerals, 53.8
g soluble nonnitrogen component, 8.1 g water, 16.6 mg iron,
.92 mg copper, 6.9 mg zinc, 4.2 mg aluminum, 0.28 mg co-
0
0
balt, 7.78 mg manganese, 3,783 IU vitamin A, 503 IU vita-
(log rank Mantel-Cox test) was used; p < 0.05 was consid-
min D , 21.2 mg vitamin E, 4.44 mg vitamin B-1, 3.06 mg
3
ered statistically significant.
vitamin B-2, 1.26 mg vitamin B-6, 12.2 mg vitamin B-12, 14
mg vitamin C, 0.31 g choline, 0.25 mg folic acid, and 356
kcal. The lycopene group (n = 40) was fed a prepared diet
containing 0.005% (wt/wt) lycopene, which was manufac-
tured as a 0.005% mixture of lycopene-enriched tomato
oleoresin (product of concentrated tomato oleoresin contain-
ing 13% lycopene; Lyc-O-Mato, RicoRed, Natural Products
Industries, Beer-sheva, Israel) with a powdered basal diet at
Results
The 76-week survival rate was 50% (20 of 40) among
control rats, 50% (20 of 40) among lycopene-treated rats,
and 64% (16 of 25) among TJ-9-treated rats. Mean body
weight decreased 16–20 weeks after birth as a result of
fulminant hepatitis. The body weight of most of the rats sur-
viving the fulminant hepatitis continuously increased until
76 weeks of age. The final body weight was not affected by
long-term administration of lycopene or TJ-9. On gross ex-
amination of the liver, lesions of cholangiofibrosis showed
irregularly shaped whitish nodules with firm consistency,
whereas some large HCCs showed reddish tumors with un-
clear margins. Both lesions were scattered on the irregular
surface of the liver.
<
50°C (16). The TJ-9 group (n = 25) was fed a prepared diet
containing 1% TJ-9 (Xiao-chai-hu-tang, Tsumura, Tokyo,
Japan) (7). All rats were sacrificed at 76 weeks of age for
evaluation of the incidence of HCC; pentobarbital sodium
(
0.04 mg/g body wt ip) was used for anesthesia. Histo-
pathological examination of all animals was performed. Sera
and part of each liver were frozen and stored at -80°C for
further examination. Sections of liver were taken from the
left and right lateral lobes. The numbers of liver specimens
from the untreated, lycopene, and TJ-9 groups were 105, 74,
and 85, respectively. Specimens were fixed in 10% forma-
lin, embedded in paraffin, cut into 4-mm-thick sections, and
stained with hematoxylin and eosin, silver stain, Azan stain,
Victoria blue stain for copper-associated protein, or Berlin
blue stain for iron and examined histologically by light mi-
croscopy.
For GST-P immunohistochemical staining, GST-P (12)
was applied to the liver specimen for 30 minutes at room
temperature. After several washes with phosphate-buffered
saline, the sections were incubated with goat-anti-rabbit im-
munoglobulin-conjugated peroxidase-labeled dextran poly-
mer (EnVision, DAKO, Carpinteria, CA) for 30 minutes at
room temperature. After several washes, the sections were
incubated with diaminobenzidine solution (DAB stain kit,
Muto Chemical, Tokyo, Japan) for five to eight minutes.
GST-P-positive lesions were measured using a computer-
assisted morphometric analyzer (EIVASS, Zeiss, Oberko-
Histopathological Observations
Histopathological observation at sacrifice disclosed that
the livers in all rats were occupied by multiple macronodules
of well-differentiated HCCs, hyperplastic nodules, and cho-
langiofibrosis. Some well-differentiated HCCs compressed
surrounding hepatocytes and were demarcated from the
chronic hepatitis lesion, but many of them invaded into the
hepatic parenchyma. Hyperplastic nodules or foci were
composed of compactly arranged small hepatocytes having
clear cytoplasm and round nuclei with one or two nucleoli.
The chronic hepatitis lesion showed fibrosis and many en-
larged hepatocytes having large nuclei with infiltration of a
few inflammatory cells. Iron and copper accumulation was
observed diffusely in nonparenchymal and parenchymal
cells. Areas of cholangiofibrosis, characteristic in aging
LEC rats, were observed. These features were observed in
Vol. 39, No. 1
97

all three groups. The HCC in all three groups was predomi-
nantly of a thin, trabecular pattern with wide blood spaces or
sinusoids. Occasional lesions demonstrated a thick trabecu-
lar pattern, compactly arranged small HCC cells with
steatosis, or large HCC cells. Hyperplastic nodules showed
various degrees of dysplasia in all groups. No effect of
lycopene or TJ-9 administration on the livers was detected
by histological examination.
different among the three groups and was larger in the TJ-9
group than in the control or the lycopene group (p = 0.024, p =
0.012, respectively).
The percent area of cholangiofibrosis in the liver speci-
mens of control, lycopene, and TJ-9 groups was 4.8 ± 4.5%,
12.2 ± 13.2%, and 5.3 ± 6.0%, respectively. The percent area
of cholangiofibrosis in the liver specimens was not signifi-
cantly different among the three groups.
All three groups of rats had HCC. The percent volume
of HCC in the liver specimens of control, lycopene, and TJ-9
groups was 17.9 ± 17.1%, 27.2 ± 20.8%, and 27.6 ± 18.4%,
respectively. These intergroup differences were not signifi-
cant.
Concentrations of Copper, Iron, and Zinc Ions
Concentrations of copper, iron, and zinc ions in the diet
of control rats were 0.23, 2.93, and 4.14 ppm, respectively.
Concentrations of copper, iron, and zinc ions in the diet of
the lycopene group were 0.20, 2.91, and 4.41 ppm, respec-
tively. Concentrations of copper, iron, and zinc ions in the
diet of the TJ-9 group were 0.23, 2.48, and 3.75 ppm, respec-
tively. These concentrations did not differ significantly
among the groups.
Concentrations of copper, iron, and zinc ions in the liver
are shown in Table 2. Concentrations of iron ion in the livers
of animals in the lycopene group were lower than in the con-
trol group (p = 0.019).
Immunohistochemical Staining of GST-P
GST-P-positive lesions in the liver specimen: GST-P-
positive lesions were stained and irregularly scattered in the
liver (Table 1). The percent area of GST-P-positive lesions in
the liver specimens, the mean sizes of the GST-P-positive ar-
eas, and the number of lesions stained for GST-P per square
centimeter of liver specimen were not significantly different
among the three groups.
Concentrations of copper, iron, and zinc ions in the serum
are shown in Table 2. These concentrations did not differ
significantly among the groups.
Serum a-fetoprotein levels of control, lycopene, and TJ-9
groups were 331.0 ± 249.1 (n = 9), 559.4 ± 1,211.4 (n = 11),
and 588.3 ± 653.4 (n = 11) ng/ml, respectively. There were
no significant differences in serum a-fetoprotein levels
among the three groups.
GST-P-positive lesions within HCC lesions: HCC le-
sions were partially GST-P positive in the control, lycopene,
and TJ-9 groups. The nuclei of the HCC cells stained most in-
tensely, although there was some cytoplasmic staining. The
percentage of GST-P-positive area in HCC lesions was not
significantly different among the three groups. The number
of GST-P-positive areas per square millimeter of HCC was
Table 1. Quantitative Value for GST-P-Positive Focal Lesions in the Liver and Hepatocellular Carcinoma Lesions at
a–c
7
6 Weeks of Age
GST-P-Positive Focal Lesions in Liver Specimen
GST-P-Positive Focal Lesions in HCC
Group
n
No./cm2
Mean area, mm2
Area, %
No./mm2
Mean area, mm2
Area, %
Control
Lycopene
TJ-9
20
20
16
16.0 ± 6.5
12.1 ± 7.2
15.1 ± 4.1
1.2 ± 1.3
1.5 ± 2.3
1.3 ± 1.0
8.1 ± 6.6
8.3 ± 7.3
8.9 ± 7.2
0.048 ± 0.066*
0.021 ± 0.038
0.063 ± 0.060*
1.1 ± 1.8
0.7 ± 1.6
1.2 ± 1.1
7.7 ± 15.8
3.1 ± 5.5
10.6 ± 13.1
†
,
†
a: Values are means ± SD; n, number of rats.
b: Intergroup differences are not significant. The glutathione S-transferase placental form (GST-P)-positive area within hepatocellular carcinoma (HCC) is
greater in Sho-saiko-to (TJ-9) group than in control or lycopene group.
c: Statistical significance is as follows: *, p = 0.024; †, p = 0.012.
Table 2. Concentration of Metals in the Liver and Serum at 76 Weeks of Agea–c
Concentration in Liver, ppm
Concentration in Serum, ppm
Group
n
Copper
Iron
Zinc
n
Copper
Iron
Zinc
Control
Lycopene
TJ-9
14
10
8
4.84 ± 1.26
5.08 ± 1.31
5.61 ± 0.90
4.78 ± 1.32*
4.14 ± 0.49*
4.68 ± 0.38
1.60 ± 0.27
1.60 ± 0.33
1.83 ± 0.29
10
8
8
0.28 ± 0.05
0.34 ± 0.32
0.22 ± 0.05
0.92 ± 0.12
0.96 ± 0.23
0.97 ± 0.32
0.83 ± 0.09
0.93 ± 0.27
0.98 ± 0.23
a: Values are means ± SD; n, number of rats.
b: Concentration of iron ion is lower in livers of animals in lycopene group than in control group.
c: Statistical significance is as follows: *, p = 0.019.
9
8
Nutrition and Cancer 2001

butyl)nitrosamine treatment in male Fischer 344 rats (25). A
prospective cohort study suggested an association of dietary
intake of tomato-based foods and the reduction of prostate
cancer risk (26). Tomato juice rich in lycopene, but not puri-
fied lycopene, has a protective effect against colon carcino-
genesis in rats (27). Vainio and co-workers (28) reported
that, pending further research, lycopene should not be rec-
ommended for cancer prevention in the general population.
In experimental hepatocarcinogenesis, it is generally ac-
cepted that the size of GST-P-positive lesions correlates
with the intensity of the promotion effect, whereas the num-
ber of lesions in rat livers correlates with the intensity of the
initiation effect (14). The mean number of GST-P-positive
lesions per square centimeter in male LEC rats continuously
increased until 80 weeks of age (29). Although the method
of counting GST-P-positive lesions is useful for the short-
term study (30), this method is also practical to use for
assessment of initiation of hepatocarcinogenesis in a long-
term follow-up study. We utilized the GST-P assay for eval-
uating initiation and promotion (31) in this study, because
this assay is one of the most widely used methods for the
quantitative study of hepatocarcinogen-associated preneo-
plastic lesions (32). Twelve weeks of administration of
Figure 1. Cumulative survival of control, Sho-saiko-to (TJ-9), and lyco-
pene groups. Intergroup differences are not significant.
0
.0025% lycopene in drinking water did not decrease the in-
The cumulative survival rates were better for the TJ-9 and
lycopene groups than for the control group, but the differ-
ence was not significant (Figure 1).
cidences of bladder carcinomas in male rats after treatment
with N-butyl-N-(4-hydroxybutyl)nitrosamine (33). Dietary
lycopene (0.03%) has been shown to decrease the initiation
of preneoplastic foci in the liver by administration of di-
ethylnitrosamine in rats (34). Forty weeks of administration
of 50 ppm lycopene in drinking water decreased the inci-
dence of liver carcinomas in male C3H/He mice after com-
bined treatment with 4-nitroquinoline-1-oxide and glycerol
Discussion
To analyze the effect of lycopene or TJ-9 on hepato-
carcinogenesis in LEC rats, we sacrificed all rats at 76 weeks
of age, when most of the animals were known to have HCC
(16). Also, administration of 50 ppm lycopene in drinking
water decreased the incidence of lung adenomas plus carci-
nomas in mice (24). We decided to use a diet containing
0
mopreventive efficacy of lycopene. Another report showed
that b-carotene was protective against aflatoxin B -induced
(
19). Hepatocarcinogenesis in the three groups was similar
with respect to the incidence of HCC, serum a-fetoprotein
level, and histological grade of HCC. Oxygen radicals play
an important role in the development of hepatitis and subse-
quent liver cancer in the rat (20). However, neither agent in-
fluenced hepatocarcinogenesis in this study. Antioxidative
activities of lycopene (3) and TJ-9 (5) to prevent HCC may
be insufficient for preventing hepatocarcinogenesis in the
LEC rat. Lycopene, as well as a- and b-carotene, has been
shown to inhibit the production of chemically induced trans-
formed foci of 10T1/2 cells in a dose-dependent manner
.005% lycopene in these earlier rodent studies of the che-
1
initiation of hepatocarcinogenesis, whereas lycopene was
not (35).
In our study, HCC lesions were partially GST-P positive
in all three groups. GST-P is a marker of preneoplastic and
neoplastic cells (36,37), not only at the protein level but also
at the mRNA level (38). GST-P tends to decrease with cellu-
lar dedifferentiation in HCC (36). TJ-9 inhibits proliferation
of cancer cell lines by inducing apoptosis and arrest at the
G /G phase (39), and it also prevents liver fibrosis and de-
velopment of preneoplastic lesions in choline-deficient diet-
induced liver cirrhosis in male Wistar rats (40).
In our study, concentrations of copper in the livers of the
rats in all three groups were similar, and the promoting ef-
fect of copper in the liver may have occurred equally. The
concentration of copper, iron, and zinc ions in the diet did
not differ among the groups. The reason for the low concen-
tration of iron ion in the livers of the animals in the lycopene
(
(
21). Lycopene inhibits endometrial (Ishikawa), mammary
MCF-7), and lung (NCI-H226) cancer cell growth in cul-
ture more effectively than a- or b-carotene but did not in-
hibit the growth of human fibroblasts effectively (22). The
anticarcinogenic activity of intraperitoneal injection of lyco-
pene-enriched tomato oleoresin has been reported in 7,12-
dimethylbenz[a]anthracene-induced rat mammary tumors
0
1
(
23). The anticarcinogenic activity of lycopene toward lung
tumors after combined initiation treatment with diethylnitro-
samine, N-methyl-N-nitrosourea, and 1,2-dimethylhydra-
zine in male B6C3F mouse has been reported (24). Tomato
1
c-src
juice exerts an inhibitory effect on the development of uri-
group is unknown. Kinase activity of pp60 has been re-
nary bladder carcinoma induced by N-butyl-N-(4-hydroxy-
ported to increase proportionally with the development of
Vol. 39, No. 1
99

active site hydrophobicity of isoenzymes. Carcinogenesis 19, 1665–
671, 1998.
HCC in LEC rats (41), but the mechanisms of hepatocar-
cinogenesis in LEC rats have not been well delineated.
In conclusion, no influence on the incidence of HCC or
serum a-fetoprotein levels in LEC rats was noted after the
administration of lycopene or TJ-9. The concentration of
iron ion was lower in the livers of animals in the lycopene
group than in the control group. No statistical difference was
found in the volume, mean size, or number of GST-P-
positive areas in the liver specimens. The number of GST-P-
positive areas in HCC lesions was larger in the TJ-9 group
than in the control and lycopene groups.
1
1
3. Sone, H, Maeda, M, Wakabayashi, K, Takeichi, N, Mori, M, et al.:
Inhibition of hereditary hepatitis and liver tumor development in Long-
Evans Cinnamon rats by the copper chelating agent trientine dihydro-
chloride. Hepatology 23, 764–770, 1996.
14. Sawaki, M, Hattori, A, Tsuzuki, N, Sugawara, N, Enomoto, K, et al.:
Chronic liver injury promotes hepatocarcinogenesis of the LEC rat.
Carcinogenesis 19, 331–335, 1998.
1
5. Kato, J, Kobune, M, Kohgo, Y, Asamoto, M, Iigo, M, et al.: Hepatic
iron deprivation prevents spontaneous development of fulminant hepa-
titis and liver cancer in Long-Evans Cinnamon rats. J Clin Invest 98,
923–929, 1996.
1
6. Nishino, H: Cancer prevention by natural carotenoids. J Cell Biochem
Suppl 27, 86–91, 1997.
Acknowledgments and Notes
17. Nishita, T, and Hibi, N: Enzyme immunoassay of rat a-fetoprotein, a
highly sensitive method by using peroxidase labeled antibody. The
physico-chemical biology. Jpn J Electrophoresis 29, 369–374, 1985.
This work was supported by Grant-in-Aid 10670480 for scientific re-
search from the Ministry of Education, Science, and Culture of Japan. Ad-
dress correspondence to Dr. Mikio Nishioka, Third Dept. of Internal
Medicine, Kagawa Medical University, 1750-0157, Miki-Cho, Kita-Gun,
Kagawa, Japan. Phone: +81-0878-91-2156. FAX: +81-0878-91-2158. E-
mail: seishiro@kms.ac.jp.
1
8. Sugawara, N, Sugawara, C, Katakura, M, Takahashi, H, and Mori, M:
Copper metabolism in the LEC rat: involvement of induction of
metallothionein and disposition of zinc and iron. Experientia 41,
1
060–1063, 1991.
1
2
2
2
2
2
9. Masuda, R, Yoshida, M, Sasaki, M, Dempo, K, and Mori, M: High sus-
ceptibility to hepatocellular carcinoma development in LEC rats with
hereditary hepatitis. Jpn J Cancer Res 79, 828–835, 1988.
Submitted 19 October 2000; accepted in final form 10 January 2001.
0. Yamamoto, Y, Sone, H, Yamashita, S, Nagata, Y, Niikawa, H, et al.:
Oxidative stress in LEC rats evaluated by plasma antioxidants and free
fatty acids. J Trace Elem Exp Med 10, 129–134, 1997.
References
1. Bertram, JS, Pung, A, Churley, M, Kappock, TJ, Wilkins, LR, et al.:
Diverse carotenoids protect against chemically induced neoplastic
transformation. Carcinogenesis 12, 671–678, 1991.
1
2
3
4
. Colditz, GA, Branch, LG, Lipnick, RL, Willett, WC, Rosner, B, et al.:
Increased green and yellow vegetable intake and lowered cancer deaths
in an elderly population. Am J Clin Nutr 41, 32–36, 1985.
. Franceschi, S, Bidoli, E, Vecchia, CL, Talamini, R, D’Avango, B, et
al.: Tomatoes and risk of digestive-tract cancers. Int J Cancer 59,
2. Levy, J, Bosin, E, Feldman, B, Giat, Y, Miinster, A, et al.: Lycopene is
a more potent inhibitor of human cancer cell proliferation than either
a-carotene or b-carotene. Nutr Cancer 24, 257–266, 1995.
3. Scharoni, Y, Giron, E, Rise, M, and Levy, J: Effects of lycopene-
enriched tomato oleoresin on 7,12-dimethyl-benz[a]anthracene-in-
duced rat mammary tumors. Cancer Detect Prev 21, 118–123, 1997.
4. Kim, DJ, Takasuka, N, Kim, JM, Sekine, K, Ota, T, et al.: Chemo-
prevention by lycopene of mouse lung neoplasia after combined initia-
tion treatment with DEN, MNU, and DMH. Cancer Lett 120, 15–22,
1
81–184, 1994.
. Di Mascio, P, Kaiser, S, and Sies, H: Lycopene as the most efficient bi-
ological carotenoid single oxygen quencher. Arch Biochem Biophys
2
74, 532–538, 1989.
. Yamamoto, Y, Yamashita, S, Fujisawa, A, Kokura, S, and Yoshikawa,
T: Oxidative stress in patients with hepatitis, cirrhosis, and hepatoma
evaluated by plasma antioxidants. Biochem Biophys Res Commun 247,
1
997.
2
5. Okajima, E, Tsutsumi, M, Ozono, S, Akai, H, Denda, A, et al.: Inhibi-
tory effect of tomato juice on rat urinary bladder carcinogenesis after
N-butyl-N-(4-hydroxybutyl) nitrosamine initiation. Jpn J Cancer Res
1
66–170, 1998.
5
6
7
8
9
. Shimizu, I, Ma, Y-R, Mizobuchi, Y, Liu, F, Miura, T, et al.: Effects of
Sho-saiko-to, a Japanese herbal medicine, on hepatic fibrosis in rats.
Hepatology 29, 149–160, 1999.
. Okita, K, Li, Q, Murakamio, T, and Takahashi, M: Anti-growth effects
with components of Sho-saiko-to (TJ-9) on cultured human hepatoma
cells. Eur J Cancer Prev 2, 169–176, 1993.
. Oka, H, Yamamoto, S, Kuroki, T, Harihara, S, Marumo, T, et al.: Pro-
spective study of chemoprevention of hepatocellular carcinoma with
Sho-saiko-to (TJ-9). Cancer 76, 743–749, 1995.
. Sasaki, M, Yoshida, MC, Kagami, K, Takeichi, N, Kobayashi, H, et
al.: Spontaneous hepatitis in an inbred strain of Long-Evans rats. Rat
News Lett 14, 4–6, 1985.
8
9, 22–26, 1998.
2
2
6. Giovannucci, E, Ascherio, A, Rimm, EB, Stampfer, MJ, Colditz, GA,
et al.: Intake of carotenoids and retinol in relation to risk of prostate
cancer. JNCI 87, 1767–1776, 1995.
7. Narisawa, T, Fukaura, Y, Hasebe, M, Nomura, S, Oshima, S, et al.:
Prevention of N-methylnitrosourea-induced colon carcinogenesis in
F344 rats by lycopene and tomato juice in lycopene. Jpn J Cancer Res
8
9, 1003–1008, 1998.
2
8. Vainio, H, and Rautalahti, M: An international evaluation of the cancer
preventive potential of carotenoids. Cancer Epidemiol Biomarkers
Prev 7, 725–728, 1998.
. Sawaki, M, Enomoto, K, Hattori, A, Tsuzuki, N, Sugawara, N, et al.:
Role of copper accumulation and metallothionein induction in sponta-
neous liver cancer development in LEC rats. Carcinogenesis 15,
29. Sawaki, M, Enomoto, K, Takahashi, H, Nakajima, Y, and Mori, M:
Phenotype of preneoplastic liver lesions during spontaneous liver car-
cinogenesis of LEC rats. Carcinogenesis 11, 1857–1861, 1990.
30. Tsuda, H, Uehara, N, Iwahori, Y, Asamoto, M, Iigo, M, et al.: Chemo-
preventive effects of b-carotene, a-tocophenol and five naturally oc-
curring antioxidants on initiation of hepatocarcinogenesis by 2-amino-
3-methylimidazo(4,5-f)quinoline in the rat. Jpn J Cancer Res 85,
1214–1219, 1994.
1
833–1837, 1994.
1
1
0. Solt, D, and Farber, E: New principle for the analysis of chemical
carcinogenesis. Nature 263, 701–703, 1976.
1. Oyamada, M, Dempo, K, Fujimoto, Y, Takahashi, H, Satoh, MI, et al.:
Spontaneous occurrence of placental glutathione S-transferase-positive
foci in the livers of LEC rats. Jpn J Cancer Res 79, 5–8, 1988.
2. Satoh, K: Weak electrophile selective characteristics of the rat pre-
neoplastic marker enzyme glutathione S-transferase P-form, GST-P (7-
1
31. Tsuda, H, Matsumoto, K, Ogino, H, Asamoto, M, Iigo, M, et al.: Dem-
onstration of initiation potential of carcinogens by induction of pre-
neoplastic glutathione S-transferase P-form-positive liver cell foci:
7
): a theory of linear free energy relationships for evaluation of the
1
00
Nutrition and Cancer 2001

possible in vivo assay system for environmental carcinogens. Jpn J
Cancer Res 84, 230–234, 1993.
37. Tatsuta, M, Iishi, H, Baba, M, Yano, H, Iseki, K, et al.: Enhancement
by ethyl alcohol of experimental hepatocarcinogenesis induced by N-
nitrosomorpholine. Int J Cancer 71, 1045–1048, 1997.
38. Imai, T, Masui, T, Ichinose, M, Nakanishi, H, Yanai, T, et al.: Reduction
of glutathione S-transferase P-form mRNA expression in remodeling
nodules in rat liver revealed by in situ hybridization. Carcinogenesis
18, 545–551, 1997.
3
3
2. Mutai, M, Tatematsu, M, Wada, S, and Ito, N: Modulatory interaction
between initial clofibrate treatment and subsequent administration of 2-
acetylaminofluorene or sodium phenobarbital on glutathione S-trans-
ferase-positive lesion development. Cancer Lett 49, 127–132, 1990.
3. Okajima, E, Ozono, S, Endo, T, Majima, T, Tsutsumi, M, et al.:
Chemopreventive efficacy of piroxicam administered alone or in com-
bination with lycopene and b-carotene on the development of rat uri-
nary bladder carcinoma after N-butyl-N-(4-hydroxybutyl) nitrosamine
treatment. Jpn J Cancer Res 88, 543–552, 1997.
39. Yano, Y, Mizoguchi, A, Fukuda, K, Haramaki, M, Ogasawara, M, et
al.: The herbal medicine Sho-saiko-to inhibits proliferation of cancer
cell lines by inducing apoptosis and arrest at the G /G phase. Cancer
0
1
Res 54, 448–454, 1994.
3
3
4. Astorg, P, Gradelet, S, Berges, R, and Suschelet, M: Dietary lycopene
decreases the initiation of liver preneoplastic foci by diethylnitro-
samine in the rat. Nutr Cancer 29, 60–68, 1997.
40. Sakaida, I, Matsumura, Y, Akiyama, S, Hayashi, K, Ishige, A, et al.:
Herbal medicine Sho-saiko-to (TJ-9) prevents liver fibrosis and en-
zyme-altered lesions in rat liver cirrhosis induced by a choline-
deficient L-amino acid-defined diet. J Hepatol 28, 298–306, 1998.
5. Gradelet, S, Le Bon, AM, Berges, R, Suschelet, M, and Astorg, P: Di-
etary carotenoids inhibit aflatoxin B -induced liver preneoplastic foci
41. Masaki, T, Okada, M, Shiratori, Y, Rengifo, W, Matsumoto, K, et al.:
1
c-src
and DNA damage in the rat: role of the modulation of aflatoxin B me-
pp60
Activation in hepatocellular carcinoma of humans and LEC
1
tabolism. Carcinogenesis 19, 403–411, 1988.
rats. Hepatology 27, 1257–1264, 1998.
3
6. Sato, K: Glutathione transferases as markers of preneoplasia and neo-
plasia. Adv Cancer Res 52, 205–255, 1989.
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