TUMOR GROWTH IN MICE ONE-FIFTH SATURATED WITH
DEUTERIUM OXIDE (HEAVY WATER) 1
HENRY G. BARBOUR
AND
EDGAR ALLEN
with the collaboration of
w.
U. GARDNER, L. C. STRONG, JAMES B. HAMILTON, ARTHUR KIRSCHBAUM.
PAUL H. BARBOUR. JR.
AND
(From the Laboratory of Pharmacology and Toxicology and the Laboratory of Anatomy, Yale
University School of Medicine, New Haven, Conn.)
For the mouse, as one of us (1) has shown, a condition of one-fifth saturation with deuterium oxide, while compatible with a state of apparent
health, is associated with definite physiological effects. These include, for
example, stimulation of the sympathetic nervous system as well as phenomena
attributable to reduced vapor pressure (2). The fact that autoxidation of
epinephrine (3), as well as the splitting of acetyl choline by esterase (4), can
be inhibited by deuterium oxide in vitro illustrates its stabilizing influence
upon certain important hormones.
The effects of deuterium oxide on the growth of mammals appear unknown
except for a few scanty data of our own, though the inhibitory action of heavy
water on the development of amphibian eggs (Ussing, 5) has been demonstrated. On tumor growths in mammals no studies have been made in which
the body has been over 0.3 per cent deuterium-saturated. Negative results
obtained with mouse sarcoma and carcinoma in animals maintained three or
four weeks at such a level have been described by Woglom and Weber (6).
Rea and Yuster (7) employed even lower concentrations, 0.11 per cent deuterium oxide, merely added to sarcoma inoculations in rats or injected around
tumors. Negative results with such concentrations are scarcely surprising,
as nearly, if not all, satisfactorily established pharmacological effects of
deuterium oxide, whether on high or low forms of life, require a concentration
of at least several per cent. In the process of transplanting mouse sarcoma
and rat carcinoma Sugiura and Chesley (8) immersed bits of these tumors
for twenty-four hours in 14,40 and 94 per cent deuterium oxide but with negative results." Hitherto no one has studied tumors in mice saturated with
deuterium to a degree which could be expected to exert any influence.
We shall report here the results of 9 tumor transplantations in mice saturated one-fifth or more with deuterium oxide. Young male mice of the
Strong A strain were used, varying in weight from 12 to 23 gm, at the time
of tumor inoculation. Seven of the transplants were from carcinoma of the
1 The expenses of this work were defrayed in part from a grant from the Research Funds of
the Yale University School of Medicine. Dr. W. U. Gardner made available the tumors used,
Dr. L. C. Strong supplied the special inbred strain of mice, Dr. James B. Hamilton injected the
carcinomata. Dr. Arthur Kirschbaum transplanted the lymphosarcomata and made careful studies
of the blood. Paul H. Barbour, Ir., made the observations on tumor size, body weight, and water
consumption of the mice.
2/n vitro, concentrations of deuterium oxide incompatible with healthy mammalian life have
been found to inhibit the growth of mouse carcinoma and chicken sarcoma (Fischer, A.: Protoplasma 26: 51, 1936).
440
TUMOR GROWTH IN MICE ONE-FIFTH D20-SATURATED
441
mammary gland and the other two from lymphosarcoma. Both these tumors
are said to be transplantable with nearly 100 per cent success.
All the deuterium mice were brought to the level of one-fifth saturation by
the substitution of 40 per cent deuterium oxide for their ordinary drinking
water, for several days. This concentration was continued throughout experimental periods of sixteen to nineteen days following tumor inoculation
except in the case of two of the carcinomatous mice in which the concentration
imbibed was raised to 50 per cent on the sixth day before, and to 60 per cent
one and two days, respectively, before tumor inoculation. The degree of
saturation of the body water with deuterium was determined by the fallingdrop method for specific gravity applied to insensibly lost water caught in
CO2 cooled condensers (9). The results showed that the body water of mice
receiving 40 per cent deuterium oxide was maintained at a level approximating
1.021, and of those receiving 60 per cent at 1.032, indicating respectively
one-fifth and one-third saturation of the body water with deuterium oxide.
The tumors were implanted in pieces weighing a very few milligrams, in
the region of the axilla. Such tumors could be palpated sometimes as early
as the fourth day after injection. The conventional size index (mm. length
X breadth X thickness) was employed. On the average, dividing this index
by two gives a rough approximation of the tumor weight in grams; in the
earlier days the index/weight ratio is much smaller, and toward the end much
greater than two.
The mice drank, usually in pairs, from fountains, and the water consumption was measured daily. The weights of the animals were also recorded
daily. The fountains are specially constructed 1O-c.c. calibrated tubes with
ground glass valves at the tapered lower end. They are kept closed at the
top and never allow leakage of more than 0.1 c.c, of water in twenty-four hours.
Effects of 40 Per Cent Deuterium Oxide on Normal Mice: One of us (10)
has shown that 40 per cent deuterium oxide imbibed by normal mice over
considerable periods of time produces certain effects including changes in the
weight curve, stimulation of the sympathetic nervous system, and changes in
the water balance. Three adult mice drinking 40 per cent deuterium oxide
for over seven weeks showed the following net losses in weight: 0.8, 2.8, and
2.5 gm. The last-mentioned animal, when continued on the same water had,
by the twelfth week, regained his original weight; during the seventeenth and
eighteenth weeks the weight was nearly 1.0 gm. above this level (development of tolerance?) and the mouse was then killed while in excellent health.
The sympathetic effects on mice include especially a metabolic increase,
often amounting to 20 per cent or more (10). Daily determinations show
that this effect continues for a week or two, thereafter diminishing gradually.
The one-fifth saturation level, which is maintained when all water drinking
is confined to 40 per cent deuterium oxide, is associated with such. other
sympathetic phenomena as a general pilomotor effect and a disposition to
prolonged exophthalmos on slight provocation, both of which effects, like the
metabolic stimulation, are easily inhibited by ergotoxine (10, 11).
When mice are being raised either in two days (by 99 per cent D 2 0 ) or
in five days (by 40 per cent D 2 0 ) to the level of one-fifth saturation, it has
been found that the insensible water loss fails to rise as rapidly as the metabo-
442
HENRY G. BARBOUR AND EDGAR ALLEN
lism and may even be reduced (2). This indicates water retention, which
may well be ascribed to the fact that at body temperature the vapor pressure
of deuterium oxide is 11 per cent lower than that of ordinary water. The
water accumulation affects the body weight curve, and, with higher concentrations of deuterium, is sometimes associated even with oliguria (12).
Effects of Deuterium Oxide on Tumor Growth: The effects of deuterium
oxide on the growth of mouse carcinoma in a typical pair of mice are illustrated in Fig. 1. It will be seen that significant growth of the tumors became
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GROWTH OF MAMMARY CARCINOMA IN MICE ONE-FIFTH SATURATED WITH DEUTERIUM OXIDE
Ordinates: size index of tumors. Abscissae: days (tumors transplanted on zero day). The
solid lines represent tumors in mice receiving 40 per cent D,O instead of H,O j the broken lines
tumors in mice receiving H,O.
evident in the control mice on the fifth day, but did not appear in the deuterium mice until the ninth day. The growth curve of these carcinomata is
sinusoidal, the more linear portion continuing as a rule for two or more weeks.
As shown in the figure, the mean size of the tumors in the deuterium mice was
only 30 per cent of that of the control tumors at the end of eighteen days.
During this period all the tumor mice which had received deuterium showed
some net gain of weight except those receiving 60 per cent, which showed a
slight loss. They did not, however, gain weight as rapidly as the controls.
Fig. i gives a typical weight curve from two mice (Nos. 20 and 40) which
0.03 gram as against
0.26 gm,
showed a mean daily weight increase of
in the controls. This tendency to increase in weight less rapidly is in accord
with observations on water drinking. It will be seen from the curves (Fig.
2) that the water consumption of the pair of mice taking 40 per cent deuterium oxide, instead of ordinary water, was considerably less than for the
+
+
443
TUMOR GROWTH IN MICE ONE-FIFTH D20-SATURATED
controls. The mean daily imbibition of the two deuterium mice was 2.13 as
against 3.30 c.c. for the mice receiving ordinary water.
The results of all 4 carcinoma experiments on 7 mice and one lymphosarcoma experiment in 2 mice 8 are summarized in Table 1. The largest
tumor in any of the deuterium mice was seen in mouse No.2, being 51 per
cent of the size of the tumor in the control mouse. The two lymphosarcomata
were also about half as large as their controls on the sixteenth day after
injection, at which time one of the mice died.
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FIG. 2.
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16
BODY-WEIGHT AND WATER CONSUMPTION OF MICE BEARING TRANSPLANTS OF
MAMMARY CARCINOMA
Ordinates: c.c, water consumed per pair of mice j grams body weight of individual mice.
Abscissae: days before and after carcinoma inoculations. The solid lines represent mice drinking
D,O, 40 per cent; broken lines mice drinking H,O. The two upper lines show the water consumption in two pairs of mice. The four lower lines show the body weights of mice plotted individually. The net weights of mice at the end of the experimental period, after allowance for the
estimated weight of tumors, are indicated by the circles at the right of the chart.
As regards body weight, all of the deuterium mice showed some gain except
those receiving 60 per cent, in which there was an average daily loss of 0.39
gm. The gains in body weight correspond in a general way to the size of
the tumor. In Fig. 2 are indicated by circles the net weights of the four mice
at the end of the experiments, calculated by subtracting one-half the index
size from the gross weight. With the exception of one control mouse, the
growth shown was accounted for essentially by the tumors.
The water consumption by tumor mice is shown in the last two lines of
8 Neither of these mice differed significantly from their controls with respect to blood counts,
including differential counts, at any time during the experiment.
444
HENRY G. BARBOUR AND EDGAR ALLEN
TABLE
I
7, 1
5, 9
13, 14
Mouse number ..............
20, 40
2
Tumor ..................... Carcinoma Carcinoma Carcinoma Carcinoma Lymphosarcoma
40
60
40
40
Per cent of 0 20 imbibed .....
40
19
16
18
13
Days after injection ..........
18
1
4
2
Number of H 20 controls ......
4
2
Mean tumor size 0 20 mice
.03
.48
.51
.13
...
.30
Mean tumor size H 20 mice
0 20 mice: Mean daily change
-.39
in body weight (gm.) .......
+.07
+.17
+.03
+.03
H 20 mice: Mean daily change
in body weight (gm.) .......
+.09
+.23
+.47
+.17
+.26
Mean daily imbibition of 0 20
2.62
2.53
3.49
2.50
(c.c.) .....................
2.13
Mean daily imbibition of H 2O
5.59
5.65
3.74
(c.c.) .....................
4.45
3.30
Table 1. It will be seen that in each experiment the deuterium mice drank
only two-thirds or less the amount of water taken by their controls.
The final outcome of the various experiments was as follows. All of the
carcinoma controls survived at least thirty-four days, while the 5 carcinoma
mice drinking 40 per cent heavy water died, in the order given in the Table,
on the 30th, 25th, 29th, 16th, and 32d days. The last of these died in spite
of replacement, on the 2Zd day, of heavy water by ordinary water. The two
carcinoma mice drinking 60 per cent heavy water died in 13 and 34 days
respectively, but the latter was withdrawn from the heavy water on the 22d
day. The control lymphosarcoma mice died in 19 and 21 days respectively
as against 16 and 21 days for the deuterium mice.
It is obvious, therefore, that deuterium oxide contributed some unfavorable
element to the outcome of each experiment in spite of the very large reduction
in the growth of the tumors. This unfavorable factor does not appear to
have been nutritional, as there was no depression of the body weight curves
except with the one-third-saturated mice.
Gross examination of the tumors after death has revealed no essential
difference in their nature between the controls and the mice receiving deuterium oxide.
DISCUSSION
Speculation as to the cause of the retarded tumor growth under the influence of deuterium oxide suggests three general possibilities: (1) it may be a
specific toxic effect, (2) it may be part of a general inhibition of growth; (3)
it may be a result of catabolism.
( 1) A specific toxic effect of deuterium oxide on the tumor might be
exerted either directly or by means of some indirect mechanism acting through
the circulation. Further, a specific toxic effect on the tumor might be due to
interference with some enzyme mechanism responsible for tumor growth apart
from the enzyme systems concerned with body growth in general, though no
such enzyme system peculiar to tumor cells is known. It is also conceivable
that the tumor cells might be damaged by the osmotic effects of heavy water
(Brooks, 13) owing to a weaker resistance in this respect than that of
ordinary cells.
TUMOR GROWTH IN MICE ONE-FIFTH D~O-SATURATED
445
(2) The second general possibility is that the inhibition of the growth
of the tumors is merely one manifestation of a general inhibition of growth
throughout the body. While some evidence has been mentioned for inhibition
of growth in general, we have also to consider that water drinking is decidedly
diminished in the case of. all mice given 40 per cent heavy water as compared
with ordinary water. This is undoubtedly associated with a decreased food
intake. Such factors, however, as decrease in food and water intake do not
suffice to explain why the tumor growth tends to suffer more than the weight
of the body in general. No studies on young, rapidly growing mice have been
made with deuterium oxide, and the slight loss in weight in normal adult mice
referred to early in the paper is not substantiated by our results with the
tumor mice which, at least up to the eighteenth or nineteenth day, showed no
significant weight loss even when allowance is made for the size of the tumor.
It may be expected, however, that deuterium will retard the rapid growth
of young mice, and this may place it in the category with those "carcinogenic" agents for which Haddow and his collaborators (14) have shown a
capacity to retard the growth of rat tumors as well as the growth of young
rats. Haddow suggests the possibility that a period of inhibited growth of
all cells affords for a few of them an unusual opportunity to grow and multiply
later, thus explaining how growth inhibitors may become carcinogenic agents.
As to whether tumors will tend to grow more rapidly on withdrawal of
deuterium oxide, or whether this substance can, from any point of view, be
considered a carcinogenic agent, evidence is still wanting.
(3) The retarding influence of deuterium oxide may perhaps be entirely
accounted for by its previously described calorigenic effect. A metabolic rate
from 10 to 20 per cent or more above the normal would certainly indicate a
catabolism sufficiently augmented to inhibit both body growth and tumor
growth appreciably.
CONCLUSIONS
1. The growth of carcinoma in 7 mice and lymphosarcoma in 2 mice was
one-half as rapid, or less, in mice drinking 40 per cent deuterium oxide as in
mice drinking ordinary water.
2. Drinking 40 per cent deuterium oxide did not decrease the weight of
young adult mice injected with carcinoma or lymphosarcoma. Mice receiving ordinary water grew faster but much of the difference in weight could be
accounted for by their larger tumors.
3. Mice receiving deuterium oxide drank less than their controls receiving
ordinary water by about one-third or more.
4. Survival of the tumor-bearing mice was shortened by deuterium oxide.
5. Carcinoma mice drinking 60 per cent heavy water failed to maintain
their body weight, and died sooner than those receiving 40 per cent.
6. It is not yet certain whether deuterium oxide exerts a specific action on
the tumors in question by virtue of its osmotic properties or its capacity to
interfere with enzyme systems, whether the tumors are influenced by a growthinhibiting property, or merely by the established catabolic effect of deuterium
oxide.
446
HENRY G. BARBOUR AND EDGAR ALLEN
REFERENCES
1. BARBOUR, H. G.: Yale ]. Bio!. & Med. 9: 551, 1937.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
BARBOUR, H. G., AND RICE, L. E.: J. Pharm. & Exper. Therap. 1938. In press.
SALOMON, K., AND BARBOUR, H. G.: To be published.
DICKERSON, V.: Am. ]. Physio!., 1938 (Proc.). In press.
USSING, H. H.: Skandinav. Arch. f. Physio!. 72: 192,1935.
WOGLOM, W. H., AND WEBER, L. A.: J. A. M. A. 102: 1289,1934.
REA, C. E., AND YUSTER, S.: Proc. Soc. Exper. Bio!. & Med. 31: 1058, 1934.
SUGIURA, K., AND CHESLEY, L. C.: Proc. Soc. Exper. Bio!. & Med. 31: 659, 1934.
BARBOUR, H. G., AND COCHRAN, F.: Science 82: 179,1935.
BARBOUR, H. G., ~ND RICE, L. E.: J. Pharm. & Exper. Therap. 1938. In press.
BARBOUR, H. G., AND HERRMANN, ]. B.: J. Pharm. & Exper. Therap. 1938. In press.
BARBOUR, H. G., AND TRACE, J.: J. Pharm. & Exper. Therap. 58: 460, 1936.
BROOKS, S. C.: Science 86: 497, 1937.
HADDOW, A., SCOTT, C. M., AND SCOTT,]. D.: Proc. Roy. Soc. 122, Ser. B: 477, 1937.
HADDOW, A., AND ROBINSON, A. M.: Proc. Roy. Soc., 122, Ser. B: 422, 1937.
See also Lees, J. C.: Quart.]. Exper. Physio!. 27: 161,171, 181,1937.