ICANCER
RESEARCH
27, 1192-1195, July 1967]
Life Term Studies on the Effects of Arsenic, Germanium,
Vanadium on Spontaneous Tumors in Mice1
MASAYOSHI
KANISAWA
Department of Physiology,
Vermont 05S01
AND HENRY A. SCHROEDER2
Dartmouth Medical School, Hanover, New Hampshire,
SUMMARY
Mice fed a diet low in many trace elements and raised in an
environment where contamination from extraneous trace ele
ments was controlled were given small amounts (5 jig/ml) of
arsenite, germanate, stannous, or vanadyl ions in drinking water
for their life times. Animals receiving arsenite had a significantly
decreased incidence of all tumors, and of tumors of the lung,
compared to those receiving tin, vanadium, and to controls.
Fewer spontaneous tumors were also found in animals given
germanium. Significant differences in the incidences of malig
nant tumors did not appear. None of the elements was carcino
genic in the doses given.
INTRODUCTION
During studies on the effects of small doses of various trace ele
ments on the growth and survival of mice (9), we have observed
that the incidences of visible spontaneous tumors were apparently
affected by the ingestion of cadmium, lead, and nickel (8). Male
mice receiving cadmium or lead salts (5 /ig/ml) in drinking water
for their lifetimes exhibited significantly fewer tumors, especially
in lung; female mice given nickel (5 jug/ml) in water also had
fewer tumors than did control animals and those receiving chro
mium (III) or titanium (IV) salts, and all were in lung. The pres
ent report considers the incidences of spontaneous tumors in
mice of both sexes given trivalent arsenic, divalent tin,
tetravalent germanium, and tetravalent vanadium salts for their
lifetimes.
MATERIALS AND METHODS
The experimental conditions of a relatively metal-free en
vironment has been reported in detail (9). Six hundred one ran
dom-bred white Swiss mice of the Charles River strain (CD),
born in our laboratory from pregnant females, were exposed from
the time of weaning (20-22 days of age) until death to doubly
deionized drinking water containing the essential trace metals
manganese (10 Mg/ml), chromium (1 Mg/ml), cobalt (1 Mg/ml),
copper (5 Mg/ml). zinc (50 MgMl), and molybdenum (1
1Supported by grants-in-aid from the National Heart Institute,
USPHS, HE 05076, U. S. Army Contract DA 2595, Germanium
Research Committee, and the American Cancer Society.
2Requests for reprints should be sent to Dr. H. A. Schroeder,
9 Belmont Avenue, Brattleboro, Vermont 00301.
Received November 21, 1966; accepted February 28, 196
1192
Tin, and
and the Brallleboro Memorial Hospital,
Brattleboro,
as the acetate, citrate, or molybdate. To groups of 23-71 of each
sex randomly selected from litters were given the additional ele
ments at 5 Mgmetal/ml of water: sodium arsenite, sodium germa
nate, stannous chloride (stabilized with ascorbic acid), or vanadyl
sulfate. The diet was composed of seed rye flour (60r0), dried
skim milk (30%), corn oil (9%), and sodium chloride (1%) to
which 100 mg/kg ferrous sulfate and a vitamin mixture (9) were
added. The diet contained 0.46 Mgarsenic/gm (3), 0.32 Mg germanium/gm (4), 0.28 /ig tin/gm (7) and 3.2Mg vanadium/gm (6).
Extensive precautions were taken to avoid extraneous metallic
contamination (9).
Dead animals were weighed and 542 were dissected. Gross
anatomical lesions were recorded, and tissues of 433 considered
abnormal were fixed in Benin's solution, sectioned, and stained
with hematoxylin and eosin. Microscopic examinations were
made on heart, lung, kidney, liver, spleen, and on other organs
considered grossly abnormal, as well as on visible tumors. No
detailed search for microscopic tumors was conducted by serial
sections; therefore, the data here presented represent only tu
mors visible under a magnifying lens. Sixty-three animals dying
during a weekend with gross autolytic changes or cannibalized
were not included except for determination of life spans. Nu
merical data were treated by chi-square analysis and by Student's
t test.
The following criteria were used in examining the sections:
"Pretumorous" lesions, found in lung or liver, were growths of
atypical parenchymal cells lacking proper stroma, replacing nor
mal epithelial cells in bronchus, alveolus, or hepatic sinusoids.
Benign tumors were autonomous growths of well-differentiated
cells which might be slightly atypical, but which did not metastasize or invade. Malignant tumors were autonomous, de
structive growths of highly atypical cells which sometimes metastasized to other organs or in the organ of origin.
RESULTS
The data from examination of the microscopic sections were
analyzed according to the presence of clusters of cells in lung
and liver appearing "pretumorous," of circumscribed benign
adenomata, or carcinomata, sarcomata, lymphomata, leukemias,
and of other tumors benign and malignant in lung, liver, mam
mary glands, and other locations. As no significant differences
appeared between incidences of tumors in the two sexes, they
were grouped together.
In Table 1 are shown the numbers of benign and malignant
tumors found in the various groups related to the number of
CANCER RESEARCH VOL. 27
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Effects of Trace Elements on Spontaneous Mouse Tumors
TABLE 1
Number of Deaths and Number of Tumors Found in Mice (Both Sexes) in Each Three-Month
Period
Period
ending(months)91215182124273033Total%
(.I)0310
(5)21
(3)89
(6)1155
(2)7
(2)13
(1)2
(2)1
U)11"
(1)7
U)5
(2)3
(1)25«
(2)6
(1)4
(1)3
(1)i
(3)1
U)4
(2)6
(2)1
U)15
U)222
(6)%144201005010.7GermaniumDeaths(No.)910153433216121314TumorsNo.3
(7)%304721151419.1TinDeaths(No.)5941824166228622Tumors(No.)114
(.15)%10817324920645010032.4ArsenicDeaths(No.)6224282415221035Tumors(No.)4
(6)%1125222525505010025.6VanadiumDeaths(No.)
(9)%1001424605032.0
totalsAutopsies
of
notdoneControlDeaths(No.)101218314339142117028TumorsNo.11
" Numbers in parentheses represent number of malignant tumors.
6 Differs from control number P < 0.0005.
c Differs from control number P < 0.025.
deaths in each three-month period. Mice receiving trivalent
arsenic showed significantly lower incidences of tumors than did
the controls, although a decreased incidence of malignant tumors
was not statistically significant. Mice receiving tin and vanadium
did not exhibit significantly decreased or increased incidences of
tumors based on the to tal number of animals autopsied. Somewhat
fewer tumors were found in those given germanium.
In Table 2 are shown deaths and tumors in mice dying between
15 and 24 months of age, the highest death rates being in that
period. Although the percentages of deaths were quite similar in
the five groups, as were the percentages of tumors, there were sig
nificantly fewer tumors in the arsenic and germanium groups
than in the controls, based on total number of animals autopsied.
Organ distribution of tumors and their types are given in
Table 3. There were significantly fewer adenomata of the lung
in arsenic-fed mice and also fewer adenomata and carcinomata of
the lung combined (P < 0.0001). Differences in other groups and
in types of tumors were not significant.
Another comparison was made on the incidences of tumors and
"pretumorous" lesions in the sections examined, excluding those
mice on which sections were not taken (Table 4). Mice receiving
arsenic and germanium had significantly fewer lesions than did
the controls. They also had fewer tumors. Significant differences
were not found for malignant tumors.
The life spans of all mice receiving metals, which will be re
ported elsewhere (5), can be calculated from Table 1 for those
autopsied. Large numbers of early deaths of animals in any
group did not occur. Significant differences in survival of all mice
at any three-month period were not observed for the tin and
vanadium group. There were fewer males given arsenic ali ve at 18
months of age (P < 0.025) and fewer males given germanium
(P < 0.005) compared to controls. At 21 months of age, there
were fewer females given arsenic (P < 0.05). Other differences
were not significant.
JULY 1967
TABLE 2
Death Rates and Incidences of Spontaneous Tumors in Aulopsied
Mice Receiving Various Trace Elements during
15-24 Months of Age
of
of
all
deaths
MetalControlArsenicGermaniumTinVanadiumNo.
deaths11367885834%all
deaths66.565.167.167.472.3No.
tumors398°15»1411%
tumors70.972.760.063.673.3%
withtumors34.511.917.124.232.4
" Differs from control number P < 0.001.
6 Differs from control number P < 0.01.
Note: Between 12 and 21 months of age there were 76 deaths
with 5 tumors in the arsenic group and 92 deaths with 34 tumors
in the control group (P < 0.0001).
There were 140 of 198 control mice alive after 15 months of
age (70.6%) of which 25 had tumors of the lung at death. Of the
arsenic-fed mice, 74 of 108 survived 15 months (68.6%), of which
number 3 subsequently had pulmonary tumors (P < 0.01).
There was no evidence of an earlier than usual appearance of
tumors in any of the other three groups.
Bod}' weights of the mice given arsenic and germanium are
presented in Table 5. At one year and eighteen months, males
fed arsenic or germanium were smaller than their controls, as
were females fed germanium at eighteen months. The greatest
decline in weight amounted to approximately 17.0-19.3% of that
expected. Similar changes were not found in the tin- and vana
dium-fed animals, which weighed approximately the same as their
controls at all ages.
1193
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Masayoshi Kanisawa and Henry A. Schroeder
TABLE 3
Types of Tumors in Various Organs of Mice Receiving Arsenic, Germanium, Tin, or Vanadium
LesionAdenoma,
benignLungLiverMammary
glandSubtotalMalignant
tumorsLungLiverMammary
glandLeukemiaLymphomaHypernephromaOtherSubtotalMiscellaneousLymphoma,
benignOther,
benignSubtotalTotalPretumorous
lesionsLungLiverControlNo.20722960522001538115534%52.727.320.0100ArsenicNo.2»013110300160221131%27.354.518.2100Ge
" Differs from control P < 0.005.
Note : Differences of percentages between other groups are not significant.
TABLE 4
Tumors and Pretumorous Lesions Found in Sections Examined
from Mice Receiving Arsenic, Germanium, Tin, and Vanadium
TABLE 5
Weight of Mice Given Germanium and Arsenic
of
S.E.)7137.1
(gm ±
S.E.)5438.4
(gm ±
S.E.)5438.8
±
age60901201501803605406090120150180360540Control(gm
lesionsNo.6215°32*2516150%43.818.028.343.142.134.6All
tumorsNo.5511°25«2215128%39.013.222.138.039.429.5Malignant
tumorsNo.15676640%10.67.26.210.415.89.2
of
ElementControlsArsenicGermaniumTinVanadiumTotalsNo.
mice141831135838433All
Number
malesNumber
of
0.3345.2
±
0.7546.7
±
0.3648.4
±
0.2648.5
±
0.6556.7
±
5.9357.0
±
1.955428.5
±
°Differs from control number,
6 Differs from control number,
' Differs from control number,
Other differences from control
P < 0.0005.
P < 0.025.
F < 0.005.
not significant.
DISCUSSION
Arsenic, especially in its trivalent form, has been considered a
carcinogen for many years (2). This idea, which appears to have
had little experimental backing, has been refuted (1). The pres
ent experiments, carried on for the lifetimes of mice, indicate that
at the doses given, trivalent arsenic as sodium arsenite is not
only noncarcinogenic, but actually appears to suppress spon
taneous tumors of the lung.
1194
1.0542.1
±
1.3644.1
±
1.1046.7
±
1.2648.4
±
1.4348.6
±
1.71°47.8
±
2.66=6430.0
±
0.7143.8
±
0.7146.6
db
0.7649.9
±
0.8750.3
±
0.8852.0
±
1.00"46.0
±
1.77°5429.8
±
of fe
malesDays
1.1534.8
±
0.7935.3
±
0.6235.2
±
0.9240.8
±
1.2539.5
±
0.5339.5
±
1.0346.1
±
1.6743.9
±
0.7544.5
±
1.9945.5
±
1.1547.2
±
0.9945.1
±
1.1253.6
±
1.9152.1
±
0.7253.7
±
1.5547.2
±
2.3946.1
±
1.5555.5
±
±1.51°Arsenic
±4.83
±1.60Germanium
" Differs from control value, P < 0.005.
6 Differs from control value, P < 0.05.
c Differs from control value, P < 0.025.
CANCER RESEARCH VOL. 27
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Effects of Trace Elements on Spontaneous Mouse Tumors
Germanium, as sodium germanate, did not appear to be carci
nogenic. There was some indication that mice receiving this ele
ment had fewer tumors (Table 1) without reference to type. There
was no evidence in this study that the ingestion of stannous tin
or tetravalent vanadium influenced the incidence of tumors. The
diet, however, was unavoidably high in vanadium, although
fairly low in the other metals studied, making this group of ques
tionable significance.
According to chemical analyses, the metals given accumulated
in the tissues of mice, especially arsenic, germanium, and tin.
These data will be reported elsewhere (5). Although it is difficult
to measure the intake of water accurately, owing to the habits of
mice in wasting it, we have calculated that they ingested roughly
7 ml/100 gm body weight/day, which amount would supply ap
proximately 35 /ig metal/100 gm/day, or about 13 nig/100 gm/
year. The ingestion of 6 gm food/100 gm/day would supply an
additional 2.76 ¿igarsenic, 1.92 ng germanium, 1.68 ng tin, and
19.2 /ig vanadium per 100 gm body weight per day. The yearly
totals from both food and water, on these bases, would amount to
13.8 mg arsenic, 13.5 mg germanium, 13.4 mg tin, and 18.9 mg
vanadium per 100 gm body weight. These intakes are 17-29
times those measured for a 70-kg man in the cases of arsenic (3),
germanium (4), and vanadium (6), and 6.4 times that of tin (7).
Experience with random-bred mice of this strain in three
groups comprising over 400 control animals has revealed tumor
incidences of 32% (8) to 40%. We do not believe that the animals
given arsenic represented a strain which was resistant to tumors,
for they were randomly selected littermates distributed among
the five groups. Because of the large number of animals, the con
trol groups and those given arsenic and germanium were run con
currently; those on vanadium and tin were started a year earlier
and accompanied by other controls (5).
REFERENCES
1. Frost, D. V. Arsenicale in Biology—Retrospect and Prospect.
Federation Proc., 26: 194-208, 1967.
2. Report of Royal Commission on Arsenical Poisoning (2 vols).
London: His Majesty's Stationary Office, 1903.
3. Schroeder, H. A., and Salassa, J. J. Abnormal Trace Metals in
Man: Arsenic. J. Chron. Dis., 19: 85-106, 1966.
4. Schroeder, H. A., and Balassa, J. J. Abnormal Trace Metals in
Man: Germanium. J. Chron. Dis., SO: 211-224, 1967.
5. Schroeder, H. A., and Balassa, J. J. Arsenic, Germanium, Tin
and Vanadium in Mice. Effects on Growth Survival and Tissue
Levels. J. Nutr., in press.
6. Schroeder, H. A., Balassa, J. J., and Tipton, I. H. Abnormal
Trace Metals in Man: Vanadium. J. Chron. Dis., 16: 1047-1071,
1963.
7. Schroeder, H. A., Balassa, J. J., and Tipton, I. H. Abnormal
Trace Metals in Man: Tin. J. Chron. Dis., 17: 483-502, 1964.
8. Schroeder, H. A., Balassa, J. J., and Vinton, W. H., Jr. Chro
mium, Lead, Cadmium, Nickel, and Titanium in Mice: Effect
on Mortality, Tumors and Tissue Levels. J. Nutrition, 8S: 239250, 1964.
9. Schroeder, H. A., Vinton, W. H., Jr., and Balassa, J. J. Effect
of Chromium, Cadmium and Other Trace Metals on the Growth
and Survival of Mice. J. Nutrition, 80: 39-47, 1963.
JULY 1967
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1195
Life Term Studies on the Effects of Arsenic, Germanium, Tin,
and Vanadium on Spontaneous Tumors in Mice
Masayoshi Kanisawa and Henry A. Schroeder
Cancer Res 1967;27:1192-1195.
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