Environ Health Prev Med (2011) 16:224–231
DOI 10.1007/s12199-010-0190-x

REGULAR ARTICLE

Long-term administration of the fungus toxin, sterigmatocystin,
induces intestinal metaplasia and increases the proliferative
activity of PCNA, p53, and MDM2 in the gastric mucosa
of aged Mongolian gerbils
Masahiro Kusunoki • Junichi Misumi • Tatsuo Shimada •Kazuo Aoki • Noritaka Matsuo • Hideaki Sumiyoshi •Takeshi Yamaguchi • Hidekatsu Yoshioka
Received: 4 August 2010 / Accepted: 27 October 2010 / Published online: 13 January 2011Ó The Japanese Society for Hygiene 2010

Abstract
Objective The causal agents of gastric cancer could include
fungus toxins. Sterigmatocystin (ST), a fungus toxin, is arisk factor of gastric cancer. We investigated the effects of
ST on the stomach tissues of Mongolian gerbils.
Methods Seventy-five-week-old male Mongolian gerbils
received ST ad libitum at a concentration of 0 ppb (nontreated, n = 11), 100 ppb (n = 7), or 1,000 ppb (n = 13)dissolved in drinking water for a period of 24 weeks. Afteradministration, we tested the histopathological changes andimmunostaining for proliferating cell nuclear antigen(PCNA), p53, and MDM2 expression.
Results We investigated the histopathological changes
and determined the incidence of histopathological changes
in animals with various gastric diseases after ST administration at a dose of 0 ppb (non-treated control), 100, or
1,000 ppb as follows: firstly, indices for gastritis were 18.2,
100, and 100%, those for erosion events were 9.1, 100, and
M. Kusunoki (&)  N. Matsuo  H. Sumiyoshi  H. Yoshioka
Department of Matrix Medicine, Faculty of Medicine,
Oita University, Idaigaoka 1-1, Hasama-machi,
Yufu, Oita 879-5593, Japane-mail: e21501@oita-u.ac.jp
J. Misumi  T. Shimada
Professor Emeritus, Oita University, Hasama-machi,
Yufu, Oita 879-5593, JapanK. AokiDepartment of Public Health and Hygiene,
University of the Ryukyus Faculty of Medicine,
207 Uehara, Nishihara-cho, Okinawa 903-0215, JapanT. Yamaguchi
Department of Functional Anatomy,
Faculty of Medicine, Kanazawa University,
13-1 Takaramachi, Kanazawa 920-1192, Japan

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92.3%, and those for polyps were 0, 71.4, and 61.5%,
respectively. These incidences in the ST-administeredgroups (100 or 1,000 ppb) showed significant increases
compared with those in the non-treated control group. And,lastly, indices for intestinal metaplasia were 0, 100, and15.4%, respectively. Furthermore, immunostaining for
PCNA, p53, and MDM2 expression showed significantly
greater rates in the ST-administered groups (100 or
1,000 ppb) than in the non-treated control group.
Conclusion The histopathological and immunohistopathological findings of this study indicate that ST exerts amarked influence on gastric mucus and gland cells, showing dominant gastritis, erosion events, polyps, and intestinal metaplasia in these animals.
Keywords Sterigmatocystin  Intestinal metaplasia Proliferating cell nuclear antigen (PCNA)  p53  MDM2

Introduction
Previous studies in humans have clarified that Helicobacter
pylori infection induces the infiltration of inflammatory
cells into the gastric mucosa [1], which leads to thedevelopment of chronic atrophic gastritis, gastric ulcers,
duodenal ulcers [2], and gastric cancer [3]. The prevalenceof H. pylori infection before 1949 was over 70% in Japan[4], and H. pylori infection is the major cause of histological gastritis and carcinogenesis in humans [1–3].
However, the death rate from gastric cancer in thesepatients is unexpectedly low compared with that from othercancers, such as lung cancer in Japan [5]. Furthermore, theprevalence rate of gastric cancer has decreased each year[6]. In addition, half of the patients with early gastriccancer are negative for H. pylori antibody [7].

Environ Health Prev Med (2011) 16:224–231

We have speculated that, besides H. pylori, there must
be other causal agents that produce gastric cancer. The
causal agents of gastric cancer could include fungus toxins,
which are universally distributed in Asian countries. The
genus Aspergillus, which is indigenous to human habitats,is known to grow in corn, barley, wheat, peanuts, andwalnuts and to produce various mycotoxins [8]. Specifically, the mycotoxin sterigmatocystin (ST), a substanceproduced by A. versicolor, the chemical structure of which
has been elucidated, is a precursor of aflatoxin B1. It is also
widely distributed in soil, agricultural crops, and grain
crops [9]. Xie et al. [10] have shown that Aspergillusinoculated corn flour causes typical hyperplasia of theglandular stomach in mice. In addition, an analysis of dietand gastric juice samples from patients with chronic gastritis in rural regions with high gastric cancer mortality
showed that they were frequently contaminated with
A. versicolor [10].
Truong Minh et al. [11] reported that gastric cancer
mortality rates for men and women increase with age. And
the deterioration in immune function with aging is thought
to make a major contribution to the increased morbidity
and mortality from infectious disease with advancing age[12]. Furthermore, elderly people frequently have symptoms of fullness and appetite loss due to impaired gastricmotor activity [13].
In our previous study, the administration of ST to
H. pylori-infected Mongolian gerbils induced a significantly higher incidence of gastric carcinogenesis comparedwith that in H. pylori-infected animals without ST treatment [14]. The above-mentioned study suggests that ST isa potential factor of carcinogenesis on the stomach in
Mongolian gerbils. However, to our knowledge, there havebeen no reports showing that ST alone induces stomach
cancer in the experimental animals with aging.
In the present study, we examined whether the fungus
toxin, sterigmatocystin, which is produced by fungi such as
A. versicolor and flavus, has a carcinogenetic effect on thegastric mucosa in aged Mongolian gerbils.

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the Guidelines for Animal Experimentation of the Facultyof Medicine, Oita University, Japan, and was approved by
its Ethics Committee.
Experimental protocol
Seventy-five-week-old Mongolian gerbils were randomly
assigned to the following groups: (1) non-treated control(n = 11), (2) ST 100 ppb administration (n = 7), and (3)ST 1,000 ppb administration (n = 13). ST (3a,12c-dihydro-8-hydroxy-6-methoxy-7H-furol[30 ,20 :4,5]furo[2,3-c]
xanthen-7-one) [10048-13-2] was purchased from Sigma
Chemical Company (St. Louis, MO, USA), and ST withover 98% purity was used. The LD50 value of ST has beenreported to be 166 mg per kg in rats with oral administration [15]. ST was administered for 24 weeks at the
specified concentrations in drinking water. In order for thisagent to dissolve well in water, ST was dissolved with
distilled water over 24 h and then adjusted to the specified
concentration. The ST water was made fresh every week,
stored at 10°C, and stirred again before administration.
Histological examination
The stomachs were opened along the greater curvature andgastric tissues, including the cardia, body, and antrum,
were fixed in 10% neutral formalin solution for more than
4 h dehydrated and then embedded in paraffin. Thereafter,
7-lm-thick sections were stained with hematoxylin andeosin (H&E) for morphological examination or with periodic acid Schiff’s (PAS) reaction and Alcian blue (pH 2.5)
to detect mucin-containing cells and observed.
All tissue preparations were observed by light microscopy (Nikon, Tokyo) at 509, 1009, and 4009 magnifications. Histopathologic findings of the glandular stomach
were categorized as follows: (1) active gastritis, (2) erosion
events, (3) hyperplastic polyps, and (4) intestinal metaplasia. These were classified as positive or negative asreported by Watanabe et al. [16] and Ma et al. [17].
Immunohistochemistry

Materials and methods
Animals
Seventy-five-week-old male specific pathogen-free Mongolian gerbils (NOs/Sea, Kyudo, Kumamoto, Japan),weighing a mean of 91.6 ± 12.36 g at the beginning of the
ST administration, were used in this study. The animals
were housed 4–5/cage in an animal room maintained at
23 ± 2°C with 55 ± 5% humidity under a 12/12-h light–
dark cycle, with free access to food (CE-2; CLEA Japan,
Inc., Tokyo) and drinking water. This study was based on

Gastric sections were examined by immunohistochemical
staining with mouse anti-proliferating cell nuclear antigen(PCNA) antibody (Nichirei, Inc., Japan), mouse anti-p53
antibody (Nichirei, Inc., Japan), and rabbit anti-MDM2
antibody (Ana Spec, Inc., USA), respectively. The paraffin
sections were autoclaved in TE buffer (pH 9.0) for 15 min
to retrieve the p53 antigenicity and for 20 min to retrieve
the MDM2 antigenicity, or were microwaved in 10 mM
citrate buffer (pH 6.0) for 15 min to retrieve PCNA antigen. The slide was subsequently incubated with the primary anti-PCNA for 1 h at room temperature, the primary

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Environ Health Prev Med (2011) 16:224–231

anti-p53 for overnight at 4°C, and the primary anti-MDM2
for 40 min at room temperature, together with theirrespective secondary antibodies (biotin-conjugated antiimmunoglobulin) at room temperature for 10 min, followed by reaction with the streptavidin peroxidase method
at room temperature for 5 min.
The PCNA labeling index (LI), p53 positive index (PI),and MDM2 LI expression were evaluated by lightmicroscopy [18, 19]. Three microscopic fields of the gastric body and pyloric region were photographed, and eachindex was calculated by counting about 1,000 positive andnormal nuclei in the selected fields.
Statistical analysis
The gastric sections that were positive for histopathologicalchanges were analyzed by the Chi-square test. Findings of
p53 PI and MDM2 were also compared between groups
using the Mann–Whitney U-test adjusted by the Bonferroni
method. Body weight at autopsy and PCNA LI werecompared by one-way analysis of variance (ANOVA)
followed by Tukey’s multiple comparison test.

Results
General findings
Ninety-nine-week-old Mongolian gerbils showed mean
body weights in the respective groups as follows: ST nontreated control group 106.1 g, ST 100 ppb-administeredgroup 111.5 g, and ST 1,000 ppb-administered group86.7 g (Table 1). The mean index of body weight wassignificantly lower in the ST 1,000 ppb-administered groupthan in the ST 100 ppb-administered group. Furthermore,
the feed consumption ranges during the experimental

period were as follows: ST non-treated-control group
5.5–8.1, ST 100 ppb-administered group 4.5–7.0, and ST
1,000 ppb-administered group 3.9–5.3 (g per animal perday), respectively. And the ST consumption ranges during
the administration period were ST 100 ppb-administeredgroup 0.007–0.015 and ST 1,000 ppb-administered-group0.088–0.132 (mg per kg per day), respectively. There were
no intergroup differences in either feed consumption orwater intake.
Histopathological findings
Figure 1 shows the histologically normal appearance of thegastric mucosa and glands from the ST non-treated control
group. There were no foci of inflammation in the gastricmucosa.
Macroscopically, in the gastric mucosa of some STadministratered Mongolian gerbils, polyps and edema were
found (Fig. 2).
Table 1 shows the incidences of the histopathologicalchanges in the Mongolian gerbils with various gastric diseases after ST administration at a dose of 0 ppb (non-treatedcontrol), 100, or 1,000 ppb. Firstly, gastritis was found in
18.2% of the non-treated control, 100% of 100 ppb, and100% of 1,000 ppb groups. Secondly, erosion events were
found in 9.1% of the non-treated control, 100% of 100 ppb,
and 92.3% of 1,000 ppb groups. Thirdly, hyperplastic polyps
were found in 0% of the non-treated control, 71.4% of
100 ppb, and 61.5% of 1,000 ppb groups. These incidenceswere significantly greater in both ST-administered groups
than those in the non-treated control group. Lastly, intestinal
metaplasia was found in 0% of the non-treated control, 100%of 100 ppb, and 15.4% of 1,000 ppb groups. The incidence
of intestinal metaplasia was also significantly greater in the
ST 100 ppb-administered group than that in either the ST
1,000 ppb-administered group or non-treated control group.

Table 1 Body weight and histopathological changes in the glandular stomach of Mongolian gerbils in the sterigmatocystin (ST)-administeredgroupsBody weight at
99 weeks (g)a

Activegastritisb

Erosioneventsb

Hyperplastic
polypsb

Intestinal
metaplasiab

11106.1 ± 19.5

2/11 (18.2)

1/11 (9.1)

0/11 (0.0)

0/11 (0.0)

7111.5c ± 13.0

7/7 (100.0)d

7/7 (100.0)d

5/7 (71.4)d86.7 ± 22.0

d

Group

n

Non-treated control
ST (100 ppb) administration
ST (1,000 ppb) administration

13

13/13 (100.0)

12/13 (92.3)

d8/13 (61.5)

d7/7 (100.0)e2/13 (15.4)

a

The results represent the mean ± SD. The data were analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s multiple
comparison testb

The results show the number of animals that were histopathologically positive/the number of animals examined (the percentage of positive
cases). The data were analyzed by the Chi-square testcde

P \ 0.05 compared with the ST (1,000 ppb) administration groupP \ 0.01 compared with the non-treated control groupP \ 0.01 compared with the non-treated control and ST (1,000 ppb) administration groups

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Fig. 1 Light microscopic views of the normal gastric mucosa of
Mongolian gerbils. a Normal stomach tissue is smooth and withoutfoci of inflammation in the gastric mucosa. b Findings in theluminal surface of the normal stomach body. The epithelium is wellpreserved and Alcian blue staining does not demonstrate acidic

227

mucin. c Periodic acid Schiff’s (PAS) reaction is negligible.
Characteristics of adenocarcinoma and mucus production are notconfirmed. Stains: hematoxylin and eosin (H&E) staining (a), pH
2.5 Alcian blue staining (b), PAS staining (c). Group: non-treatedcontrol group (a–c)

Figure 3 demonstrates the histopathological findings in
the ST-administered Mongolian gerbils. Figure 3a, b shows
intestinal metaplasia forming villus-like structures in themucosal and muscular layers of the pyloric antrum in the
ST-administered groups. Mucous cells, which were positively stained with Alcian blue staining, contained acidmucins. Figure 3c shows intensive erosions in the deep
regions of the antrum mucosa and Fig. 3d shows polyps
with many glands in the mucosa.
PCNA LI, p53 PI, and MDM2 LI expression
Analysis of the effects of dose-dependent ST on theexpression of PCNA showed that positive cells were moreabundant in the gastric mucosa of the ST 1,000 and
100 ppb administration groups than those in the non-treated control group (Fig. 4a), and extended into the middle ofthe basal layers of the mucosa. Figure 4b, c show PCNA
immunostaining of gastric mucosa from ST 1,000 and
100 ppb-administered Mongolian gerbils, respectively.
The PCNA LI, p53 PI, and MDM2 LI are shown in
Table 2. The PCNA LI, p53 PI, and MDM2 LI values in theST 1,000 and 100 ppb administration groups were significantly greater than those in the non-treated control group.
However, no p53 or MDM2 was detected in the normalpart of the gastric mucosa (Fig. 5a, b). The p53- andMDM2-positive cells were observed due to the presence of
faintly stained nuclei (Fig. 5c, d), indicating significantly
greater expression rates of tumor suppressor genes.

Discussion
The present study demonstrated that long-term administration of the mycotoxin ST induced a metaplastic effect,
including increases in the PCNA LI, p53 PI, and MDM2 LI

Fig. 2 Macroscopic image of the luminal surface of stomach fromsterigmatocystin (ST)-administered Mongolian gerbils. Edema(arrows) in the gastric mucosa is found and the gastric mucosal
surface is irregular in appearance. Group: ST 1,000 ppb administration groupexpression rates, in the gastric mucosa of Mongolian gerbils. We also found gastritis, erosion events, polyps, andintestinal metaplasia of the gastric mucosa in ST-administered Mongolian gerbils. Histopathological changes for
active gastritis and erosion events were found in all animalsof both ST administration groups. Active gastritis wasmarked in the ST 1,000 ppb administration group compared with that in the ST 100 ppb administration group.
And ulceration was demonstrated in some gerbils receiving
ST 1,000 ppb. Intestinal metaplasia was confirmed in all
animals of the ST 100 ppb administration group, but onlyin 15.4% of the ST 1,000 ppb administration group. The
difference in the incidence of intestinal metaplasia mightprovide a clue to the mechanism underlying the

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Fig. 3 Histopathological findings in the gastric mucosa of Mongolian
gerbils. a, b Intestinal metaplasia forming villus-like structures(arrows) in the mucosa of the pyloric antrum. c Erosion events in the
antrum mucosa. Not only the gastric epithelium but also the gastricglands were severely damaged. d Polyps consisting of several glandsin the mucosa. A large polyp like a cauliflower is found (arrows).
Stains: H&E staining (a, c), pH 2.5 Alcian blue staining (b), PAS
staining (d). Groups: ST 100 ppb administration group (b), ST
1,000 ppb administration group (a, c, d)development of intestinal metaplasia. In other words, theadministration of over 1,000 ppb of ST induced G1 arrestat the G1 check point of the cell cycle, but the administration of less than 100 ppb of ST produced a failure of G1
arrest [20]. Various histopathological changes were confirmed in most of the ST administered animals. These
histopathological changes suggested a precancerous condition [16]. In our results, we were able to capture imagesof these precancerous lesions.
Histopathological examinations reconfirmed thatH. pylori clearly produced metaplastic changes in thegastric mucosa of Mongolian gerbils, leading to atrophyand cancer of the stomach [21]. Ma et al. [22] reported thatST enhanced the development of H. pylori-induced gastricmucosal injury. Honda et al. [23] reported that H. pylori

infection was closely associated with the development ofintestinal metaplasia; however, Shousha et al. [24] suggested the effect of factors other than H. pylori infection on
the gastric mucosa. The present study indicated that otherfactors, such as ST, produced changes similar to thoseinduced by H. pylori in the gastric mucosa of aged Mongolian gerbils.
PCNA is an auxiliary protein of DNA polymerase delta
and is synthesized in the cell nucleus in the late G1 and S
phases of the cell cycle [25]. It is a very reliable indicatorof cell proliferative activity in tumorous and non-tumorous
tissues [26]. We evaluated PCNA expression in several
experimental groups. The PCNA LI rate was significantly
greater in both of the ST-administered groups compared tothat in the non-treated control group. These results indicate

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229

Fig. 4 Proliferating cell nuclear antigen (PCNA)-immunostaining
images of the gastric mucosa of Mongolian gerbils. a Non-treatedcontrol animals, whose nuclei show almost negative reaction for
PCNA antibody. b, c The gastric mucosa of ST-administered

Mongolian gerbils clearly has PCNA-positive nuclei. Stain: PCNA
immunohistochemistry (a–c). Groups: non-treated control group (a),ST 1,000 ppb administration group (b), and ST 100 ppb administration group (c)Fig. 5 p53 and MDM2 immunostaining images in the gastric mucosa
of Mongolian gerbils. a, b Non-treated control gerbils, whose nuclei
are negative on p53 and MDM2 immunostaining. c, d p53 and MDM2
expressions are observed in the gastric mucosa from ST-administeredgerbils. Faintly stained cells are seen in the mucous glands. The
arrows indicate positive nuclei. Stains: p53 immunohistochemistry(a, c), MDM2 immunohistochemistry (b, d). Groups: non-treatedcontrol group (a, b), ST 1,000 ppb administration group (c, d)

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Table 2 Proliferating cell nuclear antigen (PCNA) labeling index (LI), p53 positive index (PI), and MDM2 LI in the ST-administered groups
PCNA LI (%)a

P53 PI (%)b

MDM2 LI (%)b

Group

n

Non-treated control

1115.3 ± 6.7

0 ± 0.1

0±0

ST (100 ppb) administration

7

29.6c ± 12.7

4.0d ± 4.4

2.1d ± 2.7

ST (1,000 ppb) administration

13

29.7c ± 13.9

5.5d ± 4.7

1.7d ± 1.5a

The results represent the mean ± SD. The data were analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s multiple
comparison testb

The results represent the mean ± SD. The data were analyzed by Mann–Whitney’s U-test adjusted by the Bonferroni methodc

P \ 0.05 compared with the non-treated control groupdP \ 0.01 compared with the non-treated control groupthat ST changes the patterns of active cell proliferation in
the gastric glands.
The p53 gene is a tumor suppressor gene that is mostfrequently expressed at the time of transformation to amalignant tumor, including to those of gastric cancer [27].
In this study, p53 PI expression was significantly greater inthe gastric mucosa of both ST-administered groups compared to that in the non-treated control group. The p53
expression was identified in occasional epithelial cells thatwere concentrated in the neck region, as reported by previous studies [28]. The p53 gene expression has beenreported in precancerous lesions [29]. This suggests that ST
increases DNA damage in epithelial cells.
The MDM2 oncoprotein is a cellular inhibitor of the p53
tumor suppressor in that it can bind the transactivation
domain of p53 and downregulate its ability to activatetranscription. In certain cancers, MDM2 amplification is acommon event and contributes to the inactivation of p53[30, 31]. The interaction and relative ratios of p53 andMDM2 protein seem to play an important role in regulatingcell division [32]. In this study, MDM2 LI expression was
significantly greater in the gastric mucosa in both the ST
100 and 1,000 ppb-administered groups compared to thatin the non-treated control group. Furthermore, Xie et al.
[20] reported that, in an in vitro study to elucidate themechanism of ST-induced carcinogenesis in mouseembryonic fibroblasts, the ST-induced activation andoverexpression of MDM2 led to the suppression or inhibition of p53 gene function, and impairment of the DNA
repair function resulted in the failure of G1 arrest; thus, ST
induced the loss of genomic integrity, thereby, increasing
carcinogenicity. In addition to the regulation of p53,
MDM2 can also abrogate Rb-induced growth arrest andinteract with the S-phase-promoting transcription factor
E2F1/DP1 and activate it [20, 33–35]. In response to DNA
damage, the p53 regulated pathway involves several of its
downstream genes, including p21, cyclin-dependent kinases, Rb, and E2F. The effects of alteration of any of thesedownstream components may be similar to those of p53
inactivation, and, consequently, lead to failure of function

123of the entire pathway [36]. This indicates that ST promotes
the malignant transformation of cells.
We administered ST to aged Mongolian gerbils to
examine its carcinogenicity. The mean lifespan of Mongolian gerbils is reportedly about 3 years [37]. If a crude
extrapolation is be made, 7 weeks corresponds to a humanage of 3–4 years, 26 weeks to 14 years old, and 1 year to
25 years old [38]. Therefore, the Mongolian gerbils at thestart of this study were estimated to be in their fifties interms of human age.
The mortality rates of gastric cancer for men and women
increase greatly with age [11]. Furthermore, aging factors
are closely involved in the extension of atrophic gastritis[39]. Our results indicated that the environmental toxic
substance ST produced the changes seen in the gastricmucosa of aged Mongolian gerbils and suggested that ST
induced gastric carcinogenesis in the gastric mucosa. The
mechanism of these ST-induced histopathological changes
remains unclear, so further detailed studies on the basearrangement of genes with regard to their carcinogenic
effect are needed.
Acknowledgments We are grateful to Y. Makino, K. Kawahara,
T. Kishida, N. Eshima, Y. Yamaoka, R. Hamanaka, and S. Ishii from
Oita University Faculty of Medicine, Japan, and N. Ebine of Doshisha
University, Faculty of Health and Sports Science, Japan, for theirhelpful guidance in the preparation of this manuscript. We also thankA. Okamoto, M. Hikida, M. Yoshikawa, M. Tamori, Y. Miyanari,
Y. Takakura, S. Sonoda, Y. Sakamoto, T. Shinohara, H. Akashi, andall staff members of the Department of Matrix Medicine, Oita University Faculty of Medicine, Japan, for their technical assistance inthis study. This work was supported by Grants-in-Aid for Scientific
Research from the Ministry of Education, Culture, Sports, Scienceand Technology of Japan (17390176).

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