[CANCER RESEARCH 42. 3741-3746, September 1982]
0008-5472/82/0042-0000$02.00
Seasonal and Geographical Changes of Spontaneous Skin Papillomas
in the Japanese Newt Cynops pyrrhogaster ^
Makoto Asashima, Shinji Komazaki, Chikako Satou, and Tsutomu Oinuma
Department of Biology. Yokohama City University, 22-2, Sefo. Kanazawa-ku. Yokohama 236. Japan ¡M.A., C S.. T. O.¡,and Second Department
Sa/fama Medical School. Kero-Hongo 38. Keroyama, Iruma-gun, Sa/fama 350-04. Japan [S. K.¡
ABSTRACT
The occurrence of spontaneous skin papillomas in Japanese
newts (Cynops pyrrhogaster) from Niigata prefecture in North
ern Japan was monitored over four seasons covering a period
of 2 years. Of 13,613 newts, 249 were found to possess
papillomas. The percentage of newts with papillomas was
highest in autumn, ranging from 1.93 to 5.45% of the total
average. These values were more than four times as high as
values obtained in the other three seasons (0.16 to 0.50%).
A total of 12,167 newts were collected from their natural
environment in 10 prefectures of Japan in the autumn of 1980
and 1981. Newts collected from the northern, seaside prefec
tures had higher papilloma rates (1.00 to 5.45%) than did
newts from the southern, Pacific Ocean prefectures of Japan
(0 to 0.27%). Male and female newts were affected equally
often by the epitheliomas.
Virus-like bodies, resembling herpes-type virus, were found
in the cytoplasm of the epitheliomas. These virus-like bodies
were not seen in the control epithelium of newts with normal
skin and without papilloma or in nonaffected regions of newts
with papilloma. It is suggested that tumor and virus are causally
related.
INTRODUCTION
Although the occurrence of tumors in amphibians has been
reported by many investigators, not much is known about the
total number and the variety of the tumors when compared with
data of other vertebrates. In anurans, tumors such as Lücke
renal adenocarcinoma in Rana (11, 12) and lymphosarcoma in
Xenopus (24) have been reported. In urodeles, the reports of
tumors is smaller still, because of the strong regenerating
ability of this group (26). In newts, skin papillomas (1, 3, 8,
21 ), melanoma (29), lymphosarcoma (9), nephroblastoma (30),
liver sarcoma (19), and mesenchymal cell neoplasm (4) have
been found, while lymphosarcoma (6), skin papilloma (23), and
melanoma (25) have been reported from Ambystoma.
Spontaneous skin papillomas have been reported recently in
the Japanese newt, Cynops pyrrhogaster. When these newts
with skin papillomas were kept under laboratory conditions,
the epitheliomas disappeared or regressed in some cases (1,
21), while in others the tumorous growth increased in size and
sometimes produced multicentric forms (1).
Little is known of the etiology of tumors in amphibians. Lücke
renal adenocarcinoma, which is observed in Rana and occurs
spontaneously, is dependent on the season (13, 15). In spring
and autumn, the frequency of this tumor is high. These fluctu1 Supported in part by a Grant-in-Aid
for Scientific Research from the Japa
nese Ministry of Education, Science, and Culture.
Received January 12, 1982; accepted May 12, 1982.
SEPTEMBER
1982
of Anatomy,
ations are thought to reflect the virus origin of this tumor (5,
17). Although some chemical substances induce tumorous
growths in urodeles, these animals are generally resistant to
tumorigenesis (2, 10, 28). In Japanese newts, Pfeiffer et al.
(21) have earlier reported that virus-like bodies could be the
causative agents of skin papillomas.
In this paper, we recorded the seasonal changes and geo
graphical variations of newt papilloma density. Furthermore, by
examining progressive skin papillomas under the electron mi
croscope, we hoped to generate evidence at the fine structure
level on whether viruses were involved in the genesis of this
amphibian disorder.
MATERIALS
AND METHODS
Seasonal Examination. Newts were collected from their natural
environment, i.e., shallow ponds and small rivers of Niigata prefecture,
located in the northwestern region of Honshu. The collection was
carried out in certain limited areas of this prefecture. The places
chosen for the collection were located between the sea and the small
mountains. Newts, encompassing all size and age groups, were col
lected and sexed by their external appearance. They were examined
to determine whether skin papillomas occurred on any part of the body.
Newts with obvious papilloma were separated, sexed, counted, and
taken back to the laboratory. The project was started in the autumn of
1979 and completed at the end of 1981. Collections were made in
each of the 4 seasons.
Geographical Distribution of Papilloma. Newts collected in October
and November 1980 from 7 prefectures of Japan (Iwate, Niigata,
Yamanashi, Chiba, Shizuoka, Kochi, and Kumamoto) were examined.
The occurrence of papillomas was noted and compared with papilloma
data from newts which were collected from Iwate, Niigata, Gifu, Shi
zuoka, Shimane, Kumamoto, and Nagasaki 1 year later. Percentages
of papilloma-bearing male and female newts were recorded.
We also measured the body weight of all newts collected in 1980
with a balancer (Ishida Co., Kyoto, Japan). The body weight values are
given as the average ±S.D. of all newts collected in each prefecture.
Electron Microscope Observation. For electron microscopy, the
specimen preparation was as described previously (1). In the attempt
to demonstrate viruses, only newts from Niigata prefecture with epithe
liomas of 4 to 5 mm were used. Normal skin regions of tumor-bearing
newts from Niigata were also examined. The normal skin of newts
without papilloma from Niigata and Kumamoto served as the control.
RESULTS
Seasonal Analysis. The results are confined to Niigata pre
fecture where in over a period of 2 years a total of 13,613
newts was collected in 4 seasons. Data reflecting seasonal
changes of papilloma occurrence are summarized in Table 1
and Chart 1.
In the spring of 1980 and 1981, 4436 newts (1870 + 2566,
respectively) were collected. Newts with obvious skin papilloma
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M. Asashima et al.
Table 1
Seasonal changes in numbers of newts with spontaneous skin papi/lomas collected in Niigata prefecture
1981
Male newts
Time
Total no.
collected
No. with
papillomas
from October 1979 to November
Female newts
% with
papillomas
Total no.
collected
No. with
papillemas
Total
% with
papillomas
Total no.
collected
No. with
papillomas
% with
papillomas
1979(Oct.
Autumn
-Nov.)Winter
1980(Jan.
-Feb.)Spring
1980(Apr.
-May)Summer
1980(July-Aug.)Autumn
1980(Oct.-Nov.)Winter
1981(Jan.
-Feb.)Spring
1981(Apr.
-May)Summer
1981(July-Aug.)Autumn
1981(Oct.-Nov.)45122612138471292371186260619669156312321232.000.440.410.712.400.540.160.336.26275171657655532250704602933511118114351.820.580.150.153.380.400.
•
•Total
D—O
O
Female
O Male
Chart 1. Seasonal changes in percentages
of newts with papillomas in Niigata prefecture.
1979
Season
(Month)
Autumn
(10,11)
were observed in 10 cases (6 + 4), representing 0.32 and
0.16%, respectively. With regard to the sex of these newts,
3075 (1213 + 1862) were male and 1361 (657 + 704) were
female. Among male newts, 8 (5 + 3) of 3057 newts, repre
senting 0.41 and 0.16%, respectively, had the papilloma on
the skin. For the female population, the corresponding data
gave 2 (1 + 1) of 1361 newts possessing the papilloma (0.15
and 0.14%, respectively).
In the summer of 1980 and 1981, a total of 2710 (1502 +
1208) newts was collected. Newts with epithelioma were ob
served in 13 (7 + 6) cases, representing 0.47 and 0.50%,
respectively. In both sexes, tumor occurrence was below
0.71%.
3742
Spring
(4,5)
Summer
Autumn
(7,8)
(10,11)
In the autumn of 1979, 1980, and 1981, the total figure of
collected newts was 5449 (726 + 1824 + 2899). Altogether,
221 newts (14 + 49 + 158) were observed to possess papil
lomas. This corresponds to 1.93, 2.70, and 5.45% of the
respective number of newts collected over the 3-year period.
In the autumn of 1981 alone, 1966 male newts were collected
of which 123, a percentage of 6.26%, had the epithelioma on
their skin. Scattered epitheliomas were found on the skin of
some newts such as the one shown in Fig. 1. In female newts,
58 (5 + 18 + 35) of 1740 had papillomas, representing 1.82,
3.38, and 3.75%, respectively, of the newts collected in 1979,
1980, and 1981.
In the winter of 1980 and 1981, the total number of newts
CANCER
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VOL. 42
Seasonal and Geographic
Changes of Newt Papilloma
collected was 1018 (397 + 621). Obvious skin papillomas
were observed in 5 cases (2 + 3), representing 0.50 and
0.48%, respectively. During the winter, fewer newts were col
lected than in the other 3 seasons because of the difficulties
attached to finding them under the snow. In spring and autumn,
on the other hand, it is easy to collect the newts because they
gather together for reproduction in the water.
As is apparent from Table 1 and Chart 1, there are obvious
seasonal changes in the occurrence of skin papilloma. The
highest peak of papilloma occurrence in newts was seen in
autumn, when the rate was more than 4 times as high as that
of the other 3 seasons. Throughout the year, there were no
obvious differences with regard to the sex of the papillomaaffected animals.
Geographical Analysis. In the autumn of 1980 and 1981,
the total number of newts collected from 10 prefectures in
Japan was 12,167. A summary of papilloma occurrence with
regard to geographical location is given in Table 2 and Chart
2. A total of 2281 newts (male, 1318; female, 963) was
collected from Chiba, Kochi, Kumamoto, and Nagasaki prefec
tures, but none of them was observed to suffer from epitheliomas. Eight-hundred five newts were collected from Shizuoka
Chart 3. The heaviest female newts occurred in Kumamoto
prefecture, while the lightest males were collected in Kochi
prefecture. Generally, female newts tended to be heavier than
males.
Electron Microscopic Observation. In comparison with the
normal epidermis, large amounts of lysosome-like organelles
were observed in the cytoplasm of tumorous epithelium. Fur
thermore, intercellular spaces were considerably wider.
In the cytoplasm of the large epitheliomas of the newts from
Niigata, virus-like bodies as shown in Fig. 2 were observed in
variable density. These virus-like structures were obviously
different from lysosomes and other organelles. Some of the
virus-like particles even exhibited a clear 6-edged form such
as that from the typical herpes virus. The size of these particles
ranged from 0.05 to 0.2 /im. Virus-like bodies could not be
in 1980, but once again no papillomas were observed among
the newts of this lot. In the same place, a single female newt
with a small papilloma was found in 1981. Newts collected in
1980 and 1981 from Iwate, Niigata, Yamanashi, Gifu, and
Shimane prefectures numbered 2857, 4723, 223, 306, and
599, respectively. Papillomas were seen in 46 (1.61%), 207
(4.38%), 2 (0.90%), 4 (1.31%), and 6 (1.0%) cases, respec
tively. Altogether, 266 (188 male and 78 female) newts of
12,167 had the epithelioma.
Differences in the body weight of newts collected from 7
prefectures in 1980 were recorded and are summarized in
A few reports dealing with the seasonality of spontaneously
occurring tumors in amphibians have been published previ
ously. McKinnell (13, 14) reported that the renal adenocarcinoma found in the leopard frog, Rana pipiens, showed an
obvious seasonal fluctuation. He examined the seasonal vari
ation of renal adenoma occurrence in a Minnesota population
from 1965 to 1968 and found that 5 and 4.4% of the frogs had
the tumor in spring and autumn, respectively. Tumor occur
rence in summer was low, amounting to only 0.14% of the total
population. Our study confirms McKinnell's observation of a
found in the cytoplasm of skin epithelium from control newts
(those without epithelioma from Niigata and Kumamoto prefec
tures) nor in the normal skin taken from papilloma-bearing
newts.
DISCUSSION
low summer incidence, but maximum occurrence of tumors
was restricted to the autumn period (1.93 to 5.45%). In spring,
summer, and winter, tumor-bearing newts (0.16 to 0.50% of
the population) were encountered much less frequently. The
discrepancy between Rana and Cynops data could be due to
a number of factors such as species-specific differences, virus,
tumor type, environmental dissimilarities, etc. Full agreement,
however, existed with regard to the high percentage of tumor
occurrence in autumn and the low percentage in summer. The
seasonal changes of Lückeadenoma in Rana was attributed
by Mizell ef al. (17, 18) to 2 separate, temperature-dependent
disease forms: (a) summer ("virus-free") and (b) winter ("viruscontaining") type. At this stage, we do not know whether 2
kinds of tumor, ;'.e., "virus-free" and "virus-containing"
papil
1
Fig. 1. Scattered papillomas seen on the entire body of the newt.
SEPTEMBER 1982
lomas exist in Cynops pyrrhogaster. What we do know is that
virus-like bodies were not seen in any of the healthy animals
that served as controls or in normal skin areas of tumor-bearing
specimens. Other possible factors involved in producing sea
sonal changes of amphibian tumors may be selective prédation
(17), natural regression (1, 21), temperature effects in combi
nation (22, 27) with virus type, and ecological phenomena such
as hibernation, regeneration, and metamorphosis.
With regard to the geographical distribution of Lückeade
noma in Rana, there were also obvious differences. McKinnell
ef al. (14, 16) collected tumor-bearing frogs from some coun
ties in Minnesota and reported on the obvious geographical
variation of the frogs with tumors. In our study, we found a high
percentage of spontaneous skin papillomas in northern and
northwestern prefectures of Japan (Table 2; Chart 2). These
3743
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M. Asashima et al.
Table 2
Percentages of newts with papillomas in 10 prefectures
newtsNo.
collected during the autumn of 1980 and 1981
newts%
with
in sam
with
with
in
papillo
with
in sam
with
with
papillomas1315311231300020000Female
papillomas2.262.382.406.261.042.130000.630000No.
PrefecturesIwate19801981Niigata19801981Yamanashi1980Qrfu1981Chiba1980Shizuoka19801981Shimane1981Kochi1980Kumamoto19801981Nagasaki1981No.
ple5756301292196696141221512170315173413298213Male
sample925727532933127165190293203284216229120208No.
mas12618351100140000%
papillomas1.300.833.383.750.790.61000.491.410000No.
ple1500135718242899223306411805373599389642418421TotalN
papillomas2521491582400160000%
papillomas1.671.552.705.450.901.31000
\fw\
1n
Newt body weight ( g )
4.0
5.0
6.0
7.0
3.0
8.0
9.0
1
M.Li
1980 198ÃŽ7
Iwa«*/'/? - §
(527)
1-0-
U
temale (925)
Magata
(Ã-' 1980 1981
1V^
(34—0—
male (1292)
/^
(532)Yamanashimale
female
J^
/m^Ã-
14.6O—
96)female
(
(127)
}i|
1980
0
r
Yairanash.-^/
,
/7
)
^198°
•pc
Chiba1980
£ L/
Chiba
1
/,'
1981
Shizuoka^
male
C4.3—0—
(221)
female (190)
Shuuoka
(40—
mate
(512)female
(293)Kochimate
fiI
3/l\ |
(50-0-
•—
F
¿ÉïlÃ-iiPrefecturesIwatemale
(173)
3-0—
(3
ternate (216)
Kumamoto
mate
(39
(413)
female (229)''—o—
Chart 2. Percentages
prefectures in Japan.
of male and female newts with skin papillomas in 10
data agree with observations on newts with papillomas which
were collected in Kyoto (21), Fukui (20), and Niigata (8). No
newts with papillomas were found in the southern parts of
Japan, e.g., Kumamoto, Nagasaki, and Kochi.
Concerning the body weights of newts, it is well known that
female newts are generally heavier than males. An obvious
relationship between the body weight and the geographical
fluctuations of the newts with papillomas, however, was not
3744
Chart 3. Body weight of newts collected in 7 prefectures in 1980. Numbers
in parentheses, number of newts examined; numbers in brackets, mean body
weights ±S.D.
apparent (Chart 3).
Possible causes of the tumors may be viruses, chemical
substances, or genetic properties. In the case of the Lücke
adenoma, the herpes-type virus was isolated and identified
(17, 18). It is, therefore, probable that at least one of the
causative agents of skin papilloma in Japanese newts is a virus.
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RESEARCH
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VOL. 42
Seasonal and Geographic Changes of Newt Papilloma
We demonstrated
the existence
of virus-like
bodies in the
cytoplasm of the progressive skin papillomas. The size of these
virus-like bodies was between 0.05 and 0.2 firn, which is very
nearly the same size as the Lückeadenocarcinoma virus in
Rana (5, 7, 17). The fact that our virus-like structures were not
observed at all in the normal skin of newts supports the theory
of a viral genesis of the papilloma, but the true test would
obviously have to come from the successful transmission of the
tumor, and experiments along these lines are being conducted
presently in this laboratory.
ACKNOWLEDGMENTS
We wish to thank Dr. T. Ishikawa of the Cancer Institute (Tokyo) for his critical
advice throughout this work and to Dr. B. Meyer-Rochow for proof reading the
manuscript of this paper. We also thank H. Takano, T. Hamazaki, H. Tsutsui, and
K. Matsumoto for their help in the field. We are also indebted to the pet-shop
owners Inoue, Ooba. and Sakaguchi for their instructions on how to best transport
and keep live newts.
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M. Asashima et al.
Fig. 2. Electron micrograph of virus-like bodies in the cytoplasm of a large newt epithelioma. x 35,000. Inset, same particles at higher magnification,
Specimen was fixed in 2.5% glutaraldehyde and embedded in Epon. and sections were stained with 2% uranyl acetate and lead citrate.
3746
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x 70,000.
VOL.
42
Seasonal and Geographical Changes of Spontaneous Skin
Papillomas in the Japanese Newt Cynops pyrrhogaster
Makoto Asashima, Shinji Komazaki, Chikako Satou, et al.
Cancer Res 1982;42:3741-3746.
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