Toxicologic Pathology

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Modifying Effects of 1'-Acetoxychavicol Acetate (ACA) and the Novel Synthetic Retinoids Re-80, Am-580
and Am-55P in a Two-Stage Carcinogenesis Model in Female Rats
Shinichiro Orita, Masao Hirose, Satoru Takahashi, Katsumi Imaida, Nobuyuki Ito, Koichi Shudo, Hajime Ohigashi, Akira
Murakami and Tomoyuki Shirai
Toxicol Pathol 2004 32: 250
DOI: 10.1080/01926230490274425
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Toxicologic Pathology, 32:250–257, 2004
C by the Society of Toxicologic Pathology
Copyright ISSN: 0192-6233 print / 1533-1601 online
DOI: 10.1080/01926230490274425
Modifying Effects of 1 -Acetoxychavicol Acetate (ACA) and the Novel
Synthetic Retinoids Re-80, Am-580 and Am-55P in a Two-Stage
Carcinogenesis Model in Female Rats
SHINICHIRO ORITA,1 MASAO HIROSE,2 SATORU TAKAHASHI,1 KATSUMI IMAIDA,3 NOBUYUKI ITO,4 KOICHI SHUDO,5
HAJIME OHIGASHI,6 AKIRA MURAKAMI,7 AND TOMOYUKI SHIRAI1
1
Department of Experimental Pathology and Tumor Biology, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan
2
Division of Pathology, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan
3
Onco-Pathology, Department of Pathology and Host-Defense, Kagawa Medical University, 1750-1 Ikenobe,
Miki-cho, Kita-gun, Kagawa 761-0793, Japan
4
Nagoya City University Medical School, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan
5
Research Foundation Itsuu Laboratory, 2-28-10, Tamagawa, Setagaya-Ku, Tokyo 158-0094, Japan
6
Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Oikawa-cho, Kitashirakawa,
Sakyo-ku, Kyoto 606-8502, Japan, and
7
Department of Biotechnological Science, Faculty of Biology-Oriented Science and Technology, Kinki University,
Iwade-Uchita-cho, Naka-gun, Wakayama 649-6493, Japan
ABSTRACT
1 -acetoxychavicol
Effects of dietary administration of
acetate (ACA) and the novel synthetic retinoids 4-[1-hydroxy-3-oxo-3(5,6,7,8-tetrahydro-3-hydroxy-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid (Re-80); 4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2naphthalenyl)carboxamido]benzoic acid (Am-580); and 6-[(3,5-di-tert-butylphenyl) carbamoyl]nicotinic acid (Am-55P) were examined using a
two-stage rat carcinogenesis model. A total of 190 female SD rats was treated sequentially with 1,2-dimethylhydrazine (DMH, s.c.); 7,12dimethylbenz(a)anthracene (DMBA, i.g.); and 2,2 -dihydroxy-di-n-propylnitrosamine (DHPN, in the drinking water) during the first three weeks
(DDD-initiation), and an additional 60 rats received the vehicle alone (non-initiation). One week after the completion of the initiation period, they
were divided into nine groups and administrated Re-80 (at dose levels of 1.0 or 0.4 ppm), Am-580 (20 or 4 ppm), Am-55P (20 ppm), ACA (100
ppm), all-trans-retinoic acid (10 or 2 ppm) or no supplement in the diet for 33 weeks, until survivors were euthanatized at week 37 weeks. After
DDD-initiation, all-trans-retinoic acid at the high dose delayed the development of mammary tumors. The multiplicity of colon tumors in the group
fed Am-55P and the incidences of nephroblastomas with ACA or Am-580 were decreased as compared with the control values, but the other chemicals
had no modifying effects on tumor development in any organs. Thus, among ACA and the novel synthetic retinoids tested, only Am-55P showed a
weak inhibitory effect on a neoplasm of general interest under the present experimental conditions.
Keywords. Synthetic retinoids; 1 -acetoxychavicol acetate; chemoprevention; rat; Re-80; Am-580; Am-55P.
In this study, one natural compound—1 -acetoxychavicol
acetate (ACA)—and 3 synthetic retinoids—Re-80 (4-[1hydroxy-3-oxo-3-(5,6,7,8-tetrahydro-3-hydroxy-5,5,8,8tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid); Am580 (4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carboxamido]benzoic acid); and Am-55P (6-[(3,5-ditert-butylphenyl)carbamoyl]nicotinic acid)—were therefore
evaluated for chemopreventive potential using a 2-stage
carcinogenesis model in female rats undergoing multiple
initiation.
ACA is found in edible plants, some seeds and a rhizome of
Languas galanga (Zingiberaceae) that is used as a ginger substitute and a stomach medicine in Thailand and other countries of Southeast Asia (Murakami et al., 1995). It has been
shown to inhibit the activity of xanthine oxidases, including
a nitric oxide (NO) synthase (Noro et al., 1998; Ohata et al.,
1998). Reactive oxygen species are known to participate in
all stages of carcinogenesis including initiation, promotion,
and progression (Pence and Reiners, 1987; Cerutti, 1994),
and therefore, xanthine oxidase inhibitors are expected to be
chemopreventors.
INTRODUCTION
Cancer prevention is a key strategy in controlling malignant tumor development and maintaining a good quality of
life. Chemoprevention of cancer may be possible employing
certain natural or synthetic chemicals. Numerous compounds
have been evaluated already for their preventive potential
in in vivo and in vitro studies, and some have been recognized as promising chemopreventive agents (Chemoprevention Working Group, 1999). Many of these compounds,
have a significant downside in benefit-versus-risk analysis,
so chemicals that possess chemopreventive potency without
adverse effects are urgently needed. For the identification of
these chemicals, in vivo animal experimentation is essential.
Since it has been clearly shown that chemopreventive effects
of most chemicals are organ-specific, effects on multiple organs should be examined to screen for preventive efficacy.
Address correspondence to: Masao Hirose, Division of Pathology,
Biological Safety Research Center, National Institute of Health Sciences, 1-18-1, Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan; e-mail:
[email protected]
250
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Vol. 32, No. 2, 2004
ACA AND NOVEL SYNTHETIC RETINOIDS
Actually, ACA has been shown to decrease cancer risk in
skin, liver and digestive organs in rodents (Tanaka and Mori,
1995; Murakami et al., 1996; Ohnishi et al., 1996; Tanaka
et al., 1997a, 1997b; Kobayashi et al., 1998; Nakamura et al.,
1998; Miyauchi et al., 2000), but there have been no reports of
chemopreventive effects in other organs, including the mammary gland. ACA was reported to have a cancer chemopreventive effect at 100 ppm or 500 ppm in diet, and the mean
body weights of 500 ppm groups were significantly smaller
than that of each control group (Kagechika et al., 1989b;
Ohnishi et al., 1996; Tanaka et al., 1997a).
Re-80 is a synthetic analog of retinoid, named retinobenzoic acid, and a retinoic acid receptor (RAR)-pan-agonist
that does not bind to the retinoic X receptor (RXR) (Shudo,
unpublished results). It has differentiation-inducing activity in the human promyelocytic leukemia cell line, HL60 (Kagechika, 1994). Am-580, and Am-55P are aromatic amides: Am-580 is a selective agonist of RAR-alpha
(Fukushima et al., 1991), and Am-55P is speculated to be an
RAR-agonist predominantly acting on RAR-alpha because
of the profiles of similar compounds (Shudo, unpublished
results). Vitamin A and its metabolites are known to have
important roles in the growth and the differentiation of many
kinds of cell lines (Kagechika, 1994). They also have been
shown to inhibit cancer development in many organ sites, including the breast (Moon, 1989; Bollag and Holdener, 1992;
Tallman and Wiernik, 1992; Lippman et al., 1995; Laura et al.,
2000).
Nevertheless, retinoids achieved limited therapeutic success of certain cancer in clinically (Bollag and Holdener,
1992). It is partly considered that the selectivity of binding
affinity of retinoids and each retinoic receptor is low, and
the use of high-dose and/or long-term requirements led to
undesirable physiological side effects. The novel synthetic
retinoids used in this study had high affinity to RARs, especially RAR-alpha, and would overcome the problem. In
addition, there have been few reports of effects of the novel
retinoids on tumor development with long-term administration in in vivo models. Re-80, Am-580, and Am-55P were
not reported in the result of the rat carcinogenesis model;
they were set enough doses to get an expected result, based
on the doses of previous date and relative activities of these
compounds (Welsch and DeHoog, 1983; Jetten et al., 1987;
Kagechika et al., 1989a; Oikawa et al., 1993; Kagechika,
1994; Lee et al., 1995).
In the present study, the 4 compounds described above
were examined for inhibitory activity in a 2-stage carcinogenesis model in female rats initiated with 3 different carcinogens. A similar multi-organ carcinogenesis model in
male rats has already been established in our laboratory
and used to detect chemopreventors as well as carcinogens
(Fukushima et al., 1991; Hirose et al., 1991, 1993; Kimura
et al., 1996). This model uses several carcinogens to initiate
carcinogenesis in multiple organs so that inhibitory and/or
enhancing influence of a test compound on many sites can
be detected in a single experiment (Hirose et al., 1993).
We continued to use various approaches to study agents
having promising chemopreventive effects with an ultimate
goal of clinical applications. The multi-organ carcinogenesis model is considered to be a good in vivo model for this
purpose.
251
MATERIALS AND METHODS
Chemicals
1,2-Dimethylhydrazine (DMH) was purchased from
Tokyo Kasei Kogyo Co., Ltd. (Tokyo, Japan), 7,12dimethylbenz-(a)anthracene (DMBA) and 2,2 -dihydroxydi-n-propylnitrosamine (DHPN) were purchased from
Nacalai Tesque Inc. (Kyoto, Japan). All-trans-retinoic acid
was purchased from Sigma Chemical Co. (St. Louis, MO).
ACA was supplied by the Department of Food Science and
Technology, Faculty of Agriculture, Kyoto University, Japan.
The novel synthetic retinoids (Re-80, Am-580, Am-55P)
were synthesized in the Faculty of Pharmaceutical Sciences,
University of Tokyo, Japan. The structures of ACA, Re-80,
Am-580, Am-55P, and all-trans-retinoic acid are shown in
Figure 1.
Animal Treatment
A total of 250 female SD rats, aged 5 weeks, were obtained
from Charles River Japan Inc. (Kanagawa, Japan), and they
were housed five to a plastic cage with hard wood chips for
bedding in an air-conditioned room at 24 ± 2◦ C and 55 ± 5%
humidity with a 12-hour light/dark cycle. They were maintained on Oriental MF basal diet (Oriental Yeast Co., Tokyo,
Japan) and tap water ad libitum. The experimental design is
presented in Figure 2.
One hundred and ninety rats were given DMH (40 mg/kg
body wt., s.c., five times), DMBA (40 mg/kg body wt., i.g.,
single dosage) and DHPN (0.1% in drinking) during the first
3 weeks (DDD-initiation). At week 4, these DDD-initiated
rats were randomly divided into 9 groups of 17 or 18 animals each, and fed 1.0 or 0.4 ppm of Re-80, 20 or 4 ppm of
Am-580, 20 ppm of Am-55P, 100 ppm of ACA, 10 or 2 ppm
of all-trans-retinoic acid or no-chemical compound in powder basal diet, to which 2% corn oil was added, for 33 weeks.
The other 60 rats were not treated with carcinogens (noninitiation). They were divided into 6 groups of 10 animals each
at week 4, fed 1.0 ppm of Re-80, 20 ppm of Am-580, 20 ppm
of Am-55P, 100 ppm of ACA, 10 ppm of all-trans-retinoic
acid or no-chemical compound in a similar manner. Diets including test chemicals were consumed within 2 weeks after
preparation. The doses of ACA and novel synthetic retinoids
were set based on previous data (Welsch and DeHoog, 1983;
Jetten et al., 1987; Kagechika et al., 1988, 1989a, 1989b;
Oikawa et al., 1993; Kagechika et al., 1994; Kulesz-Martin
et al., 1995; Lee et al., 1995; Ohnishi et al., 1996; Tanaka et al.,
1997; Kobayashi et al., 1998). All animals were weighed and
palpated weekly, and dead or moribund animals were autopsied immediately. At week 37, all surviving animals were
sacrificed by exsanguination under ether anesthesia and subjected to complete autopsy (Figure 2).
Histopathological Examination
At autopsy, the liver and kidneys were excised and
weighed, and the relative organ weights were calculated on
the basis of the final body weights. The size of each mammary
tumor was measured, and the volume was given by calculation as oval sphere. Neutral buffered formalin solution was
injected into the lungs, esophagus, stomach, intestines, and
urinary bladder. The major organs, including whole skin with
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252
ORITA ET AL.
TOXICOLOGIC PATHOLOGY
FIGURE 1.—Chemical structures of ACA, Re-80, Am-580, Am-55P and all-trans-retinoic acid.
mammary tumors, were fixed in buffered formalin. Swiss roll
preparations were made from the large and small intestines.
These organs and all tumors were embedded in paraffin and
sectioned. These sections were stained with hematoxylin and
eosin (H&E) for histopathological examination. Proliferative
lesions were distinguished according to the criteria used in
our previous studies (Hirose et al., 1988, 1991, 1993; Kimura
et al., 1996).
Statistical Analysis
Two-way analysis of variance followed by Scheffe’s multiple comparison test was applied to parametric data such as
body weights, organs weights, tumor volume, and multiplicity. The significance of differences in incidences of proliferative lesions was evaluated using the chi-square and cumulative chi-square tests. For statistical analysis of differences
between test chemical treated and basal diet groups, the criterion for significance was set at p < 0.05.
RESULTS
General Signs, Body and Organ Weights, Food and
Chemical Compound Intake
Deformation of the forefoot or foot was observed in 2 out
of 18 animals in the 1 ppm Re-80 group and 4 out of 18 in the
20 ppm Am-580 group given DDD-initiation, as well as 3 out
of 10 in the 1 ppm Re-80 without initiation. The deformation
occurred mainly in the carpal and the tarsal joints, which were
adducted. These animals also showed emaciation and poor
hair quality.
Some rats given DDD-initiation deteriorated and died due
to mammary tumors. The numbers of surviving animals, final
mean body, liver, and kidneys weights of each group, and
intake data are shown in Table 1. In the DDD-initiated groups,
the mean body weights of rats receiving 20 ppm Am-580
group were significantly smaller than the basal diet values.
The 1 ppm Re-80 groups, with or without DDD-initiation,
and the 20 ppm Am-580 non-initiated group demonstrated
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Vol. 32, No. 2, 2004
ACA AND NOVEL SYNTHETIC RETINOIDS
253
FIGURE 2.—Experimental design of the present rat two-stage carcinogenesis model. DDD-initiated groups were given DMH (arrows) and DMBA (diamond) at
weeks 0–1, and DHPN (black portion) during weeks 1–3. They were not treated in the following weeks, then divided into 9 groups at week 4 and fed test chemicals
or basal diet (shaded portion) for 33 weeks ad libitum. Non-initiated groups were not treated with carcinogens. They were divided into 6 groups at week 4 and fed
test chemicals or basal diet in a similar manner.
nonsignificant reduction of mean body weights. The other
groups showed no significant body weight changes during the
experimental period. Relative liver weights in the 0.4 ppm Re80 and 20 ppm Am-580 groups given DDD-initiation were
significantly larger than those in the basal diet group.
Incidences and Multiplicities of Tumors in Different Organs
of DDD-Initiated Rats
Data for palpable mammary tumor development in DDDinitiated groups are shown in Figure 3. The first tumors in
the 10 ppm all-trans-retinoic acid group were observed at
week 20; the first tumors in the other groups were observed
at weeks 9 through 12. The 10 ppm all-trans-retinoic acid
group showed late increase compared with the other groups,
especially from week 31. Am-580 and 1.0 ppm Re-80 groups
generally showed higher incidences than the basal diet group.
Nevertheless, there were no significant differences in final
mammary tumor incidences among the groups.
The results of histopathological examination of mammary
tumors are shown in Table 2. Re-80 increased the volume
of benign mammary tumors at both low and high doses, but
there were no differences in the multiplicities or volumes of
adenocarcinomas and total tumors. The other groups did not
show remarkable differences from the basal diet group.
Histopathological evaluation of colon tumors revealed that
Am-55P reduced the multiplicity of total tumors (benign tumors and adenocarcinomas). On the other hand, high-dose
treatment with all-trans-retinoic acid increased the colonic
tumor incidence. There were no other significant differences
in colonic tumor incidences or multiplicities (Table 3).
In the liver, the small intestine and the Zymbal’s gland,
the tumor incidences were low and no intergroup differences
TABLE 1.—Body and liver weights.
Relative weight (% B.W.)
Treatment
DDD-initiated groups
Re-80
Re-80
Am-580
Am-580
Am-55P
ACA
All-trans-retinoic acid
All-trans-retinoic acid
Basal diet
Non-initiated groups
Re-80
Am-580
Am-55P
ACA
All-trans-retinoic acid
Basal diet
∗
Dose (ppm)
Intake of compounds (µg/rat/day)
Initial number of rats
Survival rats
Body weight (g)
Liver
Kidneys
1
0.4
20
5
20
100
10
2
—
19.2
7.5
340.1
90.0
429.6
1980
230.1
42.8
—
18
18
18
18
18
18
17
18
18
10
12
9
13
14
13
13
12
15
327 ± 43
346 ± 85
300 ± 48∗
369 ± 42
347 ± 46
388 ± 41
359 ± 54
394 ± 48
359 ± 63
4.1 ± 1.2
3.6 ± 0.5∗∗
3.7 ± 0.4∗∗
3.7 ± 0.8
3.5 ± 0.4
3.5 ± 0.5
3.8 ± 0.6
3.6 ± 0.6
3.3 ± 0.3
1.3 ± 1.35
0.7 ± 0.12
0.7 ± 0.12
0.6 ± 0.09
0.9 ± 0.87
0.6 ± 0.05
0.7 ± 0.25
0.6 ± 0.07
1.1 ± 1.69
1
20
20
100
10
—
18.1
442.8
468.0
2210
245.0
—
10
10
10
10
10
10
9
10
10
10
10
10
313 ± 63
327 ± 77
416 ± 60
410 ± 31
411 ± 50
405 ± 59
3.4 ± 0.4
3.5 ± 0.6
3.0 ± 0.2
3.0 ± 0.5
3.0 ± 0.3
3.6 ± 2.8
0.7 ± 0.20
0.7 ± 0.10
0.5 ± 0.14
0.5 ± 0.06
0.6 ± 0.07
0.6 ± 0.26
p < 0.05, ∗∗ p < 0.01 vs basal diet group of DDD-initiated group.
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254
TOXICOLOGIC PATHOLOGY
ORITA ET AL.
FIGURE 3.—Sequential change in incidences of mammary tumors revealed by palpation in DDD-initiated groups.
were noted. In the kidneys, ACA and the high dose of Am-580
significantly decreased the incidences of nephroblastomas,
but the other chemicals had no effects on neoplastic and
preneoplastic lesion development (Table 4). In non-initiation
groups, tumors were not observed in any organ.
DISCUSSION
ACA has been found to be a potential inhibitor of tumorpromotion in an in vitro Epstein-Barr virus activation test
for screening of edible plants from Thailand (Kondo et al.,
1993; Murakami et al., 1995), and ACA showed chemopreventive or anti-tumor promotion activities in vivo studies in
rats or mice treated with chemical carcinogens (Tanaka and
Mori, 1995; Murakami et al., 1996; Ohnishi et al., 1996;
Nakamura et al., 1998; Tanaka et al., 1997a, 1997b). ACA
inhibits xanthine oxidase activity and nitric oxide production
(Noro et al., 1988; Ohata et al., 1998) involved in tumorigenesis (Pence and Reiners, 1987; Cerutti, 1994). Oxygen
radicals, especially nitric oxide, cause p53 gene mutations,
TABLE 2.—Data for incidences, multiplicities and volumes of mammary tumors in DDD-initiated groups.
Treatment
(DDD-initiated groups)
Dose
(ppm)
No. of
rats
Re-80
Re-80
Am-580
Am-580
Am-55P
ACA
All-trans-retinoic acid
All-trans-retinoic acid
Basal diet
1
0.4
20
5
20
100
10
2
—
18
18
18
18
18
18
17
18
18
a
∗
Benign tumorsa
Adenocarcinomas
Incidence (%) Multiplicity
Volume (cm3 )
2.0 ± 3.1
0.9 ± 1.9
0.2 ± 0.4
0.7 ± 1.0
1.2 ± 2.6
1.9 ± 2.6
1.4 ± 2.1
1.7 ± 2.4
1.3 ± 2.3
6.8 ± 18.0∗
2.3 ± 3.4∗
0.2 ± 0.1
0.4 ± 0.4
6.9 ± 17.1
0.7 ± 0.8
0.5 ± 0.9
1.3 ± 3.0
0.3 ± 0.6
10 (55.6)
6 (33.3)
3 (16.7)
8 (44.4)
7 (38.9)
12 (66.7)
10 (58.8)
11 (61.1)
8 (44.4)
Total tumors
Incidence (%) Multiplicity Volume (cm3 ) Incidence (%) Multiplicity Volume (cm3 )
12 (66.7)
9 (50.0)
15 (83.3)
15 (83.3)
8 (44.4)
10 (55.6)
10 (58.8)
14 (77.8)
13 (72.2)
1.7 ± 1.9
1.6 ± 3.3
3.1 ± 2.6
3.3 ± 3.1
2.2 ± 3.6
1.4 ± 1.9
1.1 ± 1.4
2.6 ± 3.2
1.8 ± 1.6
Benign tumors: adenoma, fibroadenoma, or fibroma.
p < 0.05 vs basal diet group.
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4.2 ± 11.7
8.7 ± 18.5
9.9 ± 5.3
5.5 ± 5.3
2.2 ± 7.7
6.1 ± 11.5
3.1 ± 7.5
6.1 ± 13.1
6.6 ± 15.9
15 (83.3)
12 (66.7)
15 (83.3)
16 (88.9)
12 (66.7)
16 (88.9)
14 (82.4)
17 (94.4)
14 (77.8)
3.7 ± 3.9
2.4 ± 4.0
3.3 ± 2.8
4.1 ± 3.8
3.4 ± 4.8
3.3 ± 3.0
2.5 ± 2.6
4.2 ± 3.9
3.1 ± 2.8
5.5 ± 15.4
6.4 ± 15.1
4.4 ± 5.2
4.6 ± 8.5
3.8 ± 10.8
2.9 ± 8.3
1.7 ± 5.2
4.2 ± 10.8
4.0 ± 13.0
Vol. 32, No. 2, 2004
ACA AND NOVEL SYNTHETIC RETINOIDS
255
TABLE 3.—Data for incidences and multiplicities of colonic tumors in DDD-initiated groups.
Adenomas
Adenocarcinomas
Total tumors
Treatment
(DDD-initiated groups)
Dose
(ppm)
No. of
rats
Incidence (%)
Multiplicity
Incidence (%)
Multiplicity
Incidence (%)
Multiplicity
Re-80
Re-80
Am-580
Am-580
Am-55P
ACA
All-trans-retinoic acid
All-trans-retinoic acid
Basal diet
1
0.4
20
5
20
100
10
2
—
18
18
18
18
18
18
17
18
18
2 (11.1)
6 (33.3)
5 (27.8)
7 (38.9)
2 (11.1)
4 (22.2)
7 (41.2)
4 (22.2)
4 (22.2)
0.11 ± 0.32
0.39 ± 0.61
0.28 ± 0.46
0.44 ± 0.62
0.17 ± 0.51
0.22 ± 0.43
0.65 ± 1.00
0.33 ± 0.69
0.44 ± 0.98
7 (38.9)
11 (61.1)
5 (27.8)
9 (50.0)
4 (22.2)
7 (38.9)
8 (47.1)
4 (22.2)
7 (38.9)
0.50 ± 0.71
0.72 ± 0.67
0.50 ± 0.99
0.67 ± 0.77
0.17 ± 0.38
0.50 ± 0.79
0.59 ± 0.71
0.44 ± 0.98
0.67 ± 1.14
8 (44.4)
14 (77.8)
7 (38.9)
12 (66.7)
5 (27.8)
9 (50.0)
15 (88.2)∗
7 (38.9)
9 (50.0)
0.61 ± 0.78
1.11 ± 0.76
0.78 ± 1.31
1.11 ± 1.02
0.33 ± 0.59
0.72 ± 0.89∗
1.24 ± 1.20
0.78 ± 1.35
1.11 ± 1.45
∗
p < 0.05 vs basal diet group.
chromosomal change, and activation of cytoplasmic signal
transduction pathways related to cell growth (Cerutti, 1994).
For this reason, it is to be expected that ACA would inhibit
all stages of tumor development including initiation, promotion and progression. In fact, with treatment in the initiation
and/or early promotion stages, ACA exhibited strong chemopreventive effects on 4-nitroquinoline 1-oxide-induced oral
carcinogenesis (Ohnishi et al., 1996) and on azoxymethane
(AOM)-induction of colonic aberrant crypt foci (Tanaka et al.,
1997b) in rat models at doses of 100-500 ppm in the diet. In
the present 2-stage rat carcinogenesis model, however, ACA
administered in only the promotion stage at the dose of
100 ppm did not show any inhibitory effects on tumor growth
in any organs. In an AOM-induced rat colon carcinogenesis model, ACA suppressed the growth of adenocarcinomas
by treatment during either initiation or promotion stages at
500 ppm in the diet, but there was only a weak effect in
the promotion stage at 100 ppm (Tanaka et al., 1997a). In
the rat hepatocarcinogenesis model induced by a choline–
deficient/L-amino acid-defined (CDAA) diet, ACA at doses
of 0.005–0.05% in diet reduced the number of GST-P positive foci and 8-hydroxyguanine level as the index of oxidative
damage to DNA, but it did not influence 2-thiobarbitric acidreacting substance levels as an index of the magnitude of
oxidative injury to subcellular components other than DNA
(Kobayashi et al., 1998). On the other hand, ACA did not inhibit GST-P positive foci development in the post-initiation
stage of diethylnitrosamine (DEN)-initiated hepatocarcinogenesis (Kobayashi et al., 1998). Therefore, chemopreventive effects of ACA may depend on the dose level, organ site,
stage of carcinogenesis, and initiators used.
Retinoid is a generic name for compounds that have
retinoic acid-specific biological activities due to binding to
retinoic nuclear receptors (Kagechika, 1994). Nevertheless,
retinoids also exert strong chronic toxicity and teratogenicity
(Kagechika, 1989a; Bollag and Holdener, 1992; Tallman and
Wiernik, 1992; Kagechika, 1994; Elmazer et al., 1997) so that
the balance with efficacy is of great importance in development of novel retinoids as anti-tumor drugs (Lippman et al.,
1995). Re-80, Am-580 and Am-55P, synthesized as novel
retinoic agonists (Kagechika, 1994), also have demonstrated
chemopreventive activity in various models in vitro and in
vivo (Jetten et al., 1987; Kagechika et al., 1989a; KuleszMartin et al., 1995; Lee et al., 1995b; Cho et al., 1997). These
compounds show biological activities similar to retinoic acid
in various models (Jetten et al., 1987; Kagechika et al., 1988,
1989a,1989b; Kagechika, 1994; Lee et al., 1995a, 1995b;
Lippman et al., 1995; Brooks et al., 1996; Gianni et al., 1993,
1996). Some retinoids specifically regulate the differentiation
and/or proliferation of cell lines such as HL-60 cells by receptor binding and might be expected to show anti-tumor effects
as inducers of cell-differentiation (Moon, 1989; Tallman and
Wiernik, 1992; Lippman et al., 1995).
All-trans-retinoic acid, an oxidative metabolite of vitamin
A alcohol (retinol), acts as an agonist for RARs but not for
RXRs (Allenby et al., 1993; Lippman et al., 1995). It inhibits the growth of human colon carcinoma HT29 cells, and
an RAR-alpha selective antagonist suppresses this inhibition
(Nicke, 1999). Thus, RAR-alpha might be concerned in the
anti-proliferative effects of retinoids. We surmised that the
chemopreventive effects of retinoid were mainly dependent
on activation of RAR-alpha. We expected to gain dissociation
Table 4.—Data for incidences and multiplicities of tumors of other organs in DDD-initiated groups.
Liver
Kidney
Treatment
(DDD-initiated groups)
Dose
(ppm)
No. of
rats
Adenoma (%)
Carcinoma (%)
Total tumor (%)
RCT a (%)
(NB b %)
S.intestine
adenocacinoma (%)
Zymbal’s gland
tumor (%)
Re-80
Re-80
Am-580
Am-580
Am-55P
ACA
All-trans-retinoic acid
All-trans-retinoic acid
Basal diet
1
0.4
20
5
20
100
10
2
—
18
18
18
18
18
18
17
18
18
2 (11.1)
4 (22.2)
1 (5.6)
1 (5.6)
3 (16.7)
3 (16.7)
1 (5.9)
0
3 (16.7)
1 (5.6)
0
0
0
2 (11.1)
1 (5.6)
2 (11.8)
2 (11.1)
0
3 (16.7)
4 (22.2)
1 (5.6)
1 (5.6)
5 (27.8)
4 (22.2)
3 (17.6)
2 (11.1)
3 (16.7)
0
0
2 (11.1)
7 (38.9)
2 (11.1)
3 (16.7)
6 (35.3)
5 (27.8)
3 (16.7)
7 (38.9)
8 (44.4)
2 (11.1)∗
7 (38.9)
3 (16.7)
2 (11.1)∗
5 (29.4)
6 (33.3)
8 (44.4)
1 (5.6)
0
2 (11.1)
0
0
1 (5.6)
1 (5.9)
1 (5.6)
2 (11.1)
3 (16.7)
3 (16.7)
2 (11.1)
2 (11.1)
0
6 (33.3)
1 (5.9)
4 (22.2)
3 (16.7)
a
b
∗
RCT: Renal cell tumor.
NB: Nephroblastoma.
p < 0.05 vs basal diet group.
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256
ORITA ET AL.
between efficiency and toxicity by using RAR-alpha agonists.
Therefore, we examined the novel retinoic acids, which are
RAR-alpha selective and/or dominant agonists, and all-transretinoic acid in this study. Re-80, an RAR-pan-agonist, shows
about 40-fold the differentiation-inducing activity of retinoic
acid in HL-60 cells (Kagechika et al., 1989a). Am-55P is an
RAR-alpha-dominant agonist, and Am-580 is an RAR-alpha
selective agonist (Elmazer et al., 1997). Re-80 and Am-580
function not only as regulators of cell growth and differentiation, but also as inhibitors of angiogenesis needed for the
development of solid tumors (Oikawa et al., 1993).
In the present study, all-trans-retinoic acid delayed mammary tumor development at high dose, but the novel synthetic
retinoids showed only limited inhibitory effects. There were
no data of the absorption of these compounds in this study,
but Re-80 and Am-580 caused the deformation of the forefoot or foot. Therefore, we inferred that the animals were
sufficiently exposed to Re-80 or Am-580. It remained to be
seen whether Am-55P was enough to expose to rats.
Retinoids have been shown to suppress the induction of
mammary tumors by a variety of carcinogens in rat models
(Moon, 1989; Tallman and Wiernik, 1992), and Re-80 and
Am-580 also have anti-proliferative effects on normal mammary epithelium or human breast carcinoma cells (Lee et al.,
1995b; Cho et al., 1997). Nevertheless, there were no differences in incidences, multiplicities and volumes of mammary
tumors with the novel retinoids-treated groups in the present
study. On the contrary, high-dose treatment with all-transretinoic acid increased the colonic tumor incidence. Experimental data regarding the potential chemopreventive effects
of retinoids in colon carcinogenesis have revealed conflicting
results (Nicke et al., 1999). There are no available data for the
molecular mechanisms of effects of retinoids on chemically
induced colon carcinogenesis. The sensitivity to chemopreventive effects of retinoids varies between any organs and
tumors (Laura et al., 2000). Detailed knowledge of the celltype-specific expression patterns for each retinoid receptor
subtype might reveal the reasons for different responses to
retinoid treatment.
Am-580 and Re-80 have caused bone deformation at doses
of 20 and 1 ppm, respectively, whether the rats were treated
with carcinogens or not. These compounds also decreased
the body weight gain and the survival rates. Oral administration of Am-580 in pregnant mice was recently reported to
induce various RAR-alpha-mediated malformations in pups
(Elmazer et al., 1997, 2001). Clinically, limited therapeutic success has been achieved with retinoids because longterm and high-dose treatment leads to undesirable side effects (Bollag and Holdener, 1992). The side effects observed
in preclinical and clinical trials might be partly due to the
activity of RAR-alpha.
Under the conditions of the present study, the physiological disturbance induced by the novel synthetic retinoids exceeded any benefit. Specifically, the doses of Re-80 and Am580 might be too excessive. In addition, the mechanism of
anti-tumor effects of retinoids is generally regarded through
modulation of cell proliferation and differentiation, but it has
been reported that retinoids are more effective when administered shortly after carcinogens (Moon, 1989). The treatment
in only the promotion stage might be unsuitable for evaluation of anti-tumor effects in this model. For these reasons, it
TOXICOLOGIC PATHOLOGY
seemed that there was no difference between the result of each
retinoic compound in this study, although it was the purpose
to examine the influence on anti-tumor effect by selectivity
to RARs, especially on RAR-alpha.
In conclusion, ACA and the novel synthetic retinoids Re80, Am-580 and Am-55P did not show obvious chemopreventive effects in this study, except weak inhibition by
Am-55P of colon carcinogenesis and nephroblastoma development by Am-580 and ACA. It seems that physiological disturbance exceeded any benefit under the present experimental condition. As a result, these compounds did not
demonstrate the effect sufficiently in this multi-organ carcinogenesis model.
ACKNOWLEDGMENTS
This research was supported in part by grants-in-aid for
cancer research from the following organizations: the Ministry of Education, Science, Sports and Culture of Japan; the
Ministry of Health and Welfare of Japan; the Ministry of
Health and Welfare for the Second Term Comprehensive 10
Year Strategy for Cancer Control of Japan; and the Society
for Promotion of Pathology of Nagoya, Japan.
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