Urethan and Leukemogenesis in Mice*
S. KAWAMOTO,
N. IDA,A. KIRSCHBAUM,|ANDG. TAYLOR
(Department of Anatomy, Baylor University Collegeof Medicine, and Department of Pediatrics,
The University of Texas SI. D. Anderson Hospital and Tumor Institute, Houston, Texas)
Urethan
perimental
suppresses hemopoietic activity in ex
animals (13) and is "radiomimetic"
in
other respects. It induces pulmonary tumors in
rodents (10, 11); when administered
orally to
mice, papillomas of the forestomach may appear
(1). Urethan initiates skin carcinogenesis in mice,
although when administered alone skin tumors do
not appear (14). Hemangiomas of the mouse liver
have been induced by this chemical (6).
The objective of this investigation was to test
the influence of this radiomimetic agent on mouse
leukemogenesis.
X-rays are independently
leukemogenic, or may act either additionally or synergistically with other leukemogenic agents (e.g.,
methylcholanthrene,
estrogenic hormone) to aug
ment the induction of mouse leukemia (3, 9). If
urethan by itself is not leukemogenic, but aug
ments or promotes leukemogenesis, then it could
be classified as a "co-leukemogen,"
following the
terminology of Berenblum in designating agents as
"co-carcinogens"
(2). Urethan was administered
as follows: (a) independently,
(6) together with
x-rays, (c) with estrogenic hormone, (¿)with both
of these agents, and (e) with methylcholanthrene,
to test its influence on leukemogenesis in pure
strain C57BL, BALB/c, and DBA/2 mice, and
C57BL-C3H F! hybrids. All these stocks are con
sidered to be "low-leukemia" from the standpoint
90
SO
WHOLE BODY JO .
X-R(80rxll) 18
WHOLE BODY .„
X-R(40rill)
•J2.-35.3H
70
-»•URETHAN "''
60
WHOLE BODY 5
X-R(40r xll) 52
SO
URETHAN
ONLY
„
. „
*'B *
0%
40
30
20
of spontaneous occurrence (Chart 9).
In addition, experiments are being reported in
volving the effect on mouse leukemogenesis
of
shielding bone marrow or thymus during x-radiation under urethan anesthesia. In preliminary ex
periments it had been found (7) that thigh-shield
ing of C57BL mice under urethan anesthesia did
not protect against the induction of leukemia, this
observation
leading to the present studies on
augmentation
of mouse leukemogenesis by ure
than. Either thigh-shielding
or thymus-shielding
under barbiturate anesthesia (5) is protective.
* This investigation lias been supported by grants from
the National Cancer Institute, Public Health Service, and
the American Cancer Society.
t Deceased.
Received for publication February 10, 1958.
EXPERIMENTAL
X-rays combined with urethan: leukemogenic ef
fects.—Urethan was administered together with a
dose of x-rays of low leukemogenic activity to the
following groups of C57BL mice (Chart 1), begin
ning at 6 weeks of age. C57BL mice are susceptible
to the leukemogenic action of x-rays (4).
10
200
300
400
AGE AT DEATH
500
(DAYS)
CHART 1.—Effect of urethan on induction of leukemia
by x-rays in intact (male and female) C57BL mice. Treatment
with urethan and x-rays begun at 6 weeks of age.
1. 40 r of x-rays1 whole-body under urethan
anesthesia every 4th day, for 11 times. The dose of
urethan (ethyl carbamate) was 1 mg/gm of body
weight, administered
intraperitoneally
in 10 per
cent aqueous solution.
2. Same x-ray treatment—no urethan.
3. Urethan only—unirradiated.
1Physical factors: 140 kvP, 5 ma., 2.0 mm. Al. added
filter, 30 cm. target mouse distance. Output, 58.8 r/min.
725
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1958 American Association for Cancer Research.
Vol. 18, July, 1958
Cancer Research
726
4. 80 r of x-rays whole-body every 4th day 11
times—no urethan.
In a similar manner, the effect of urethan on ir
radiation-induced
leukemogenesis was tested with
BALB/c and DAB/2 mice (Charts 2 and 3). For
each strain, groups of mice given the indicated
treatments (beginning at 6 weeks of age) were ob
served. Both of these strains were known to be
susceptible to the induction of leukemia by x-rays
(8).
1. 90 r of x-rays whole-body under urethan
anesthesia every 4th day, for 4 times.
2. Same treatment with x-rays—no urethan.
3. Urethan only—unirradiated.
50-1%
WHOLE BODY
57'28.9%
(S90r_x4)
+ uIRETHAN 31
40-
URETHAN
ONLY
•0%
20
-*-(90r
WHOLEx BODY
4)
IO
300
400
AGE AT DEATH (DAYS)
CHABT2.—Effect of urethan on induction of leukemia
by x-rays in intact (male and female) BALB/c mice. Treatment
begun at 6 weeks of age.
tary effect of urethan on the combined (additive)
leukemogenic action of x-rays and estrogenic hor
mone was studied in C57BL-C3H
FI hybrids
(Chart 5). The incidence of leukemia in untreated
controls is indicated in Chart 9. The following
groups of FI hybrid mice were studied :
1. 40 r of x-rays whole-body every 4th day, 11
times—no anesthesia.
2. Same treatment
with x-rays under urethan
anesthesia. One thigh was shielded, a treatment
which under nembutal anesthesia protects against
leukemogenesis.
3. 25 //g. of estradici dipropionate weekly.
4. Same treatment with estradici dipropionate
plus 40 r of x-rays to the whole body every 4th
day, 11 times.
5. Estradiol dipropionate,
plus urethan anes
thesia 11 times at 4-day intervals when 40 r of
x-rays were given with the left thigh shielded.
Urethan supplemented
the combined leukemo
genic effects of x-rays and estrogenic hormone
(Chart 4). Whole-body exposure of 90 r at 4-day
intervals for 4 times was not as active in inducing
so
200
30
2O
10
In all three strains, although urethan per se was
not leukemogenic, it significantly augmented the
induction of leukemia by x-rays (Charts 1-3).
Estrogenic hormone combined urith urethan: leuke
mogenic effects.—To determine whether urethan
might act synergistically with estrogenic hormone
in inducing leukemia, the following groups of
C57BL mice were studied (Chart 4).
1. 25 Micrograms of estradici dipropionate
in
oil (CIBA)2 weekly, given subcutaneously.
2. Same treatment plus urethan every 4th day,
11 times.
3. Urethan only, no estradici.
In this strain, compared with others, estrogenic
hormone proved to be only mildly leukemogenic;
urethan alone induced no leukemia. In mice re
ceiving both agents, the incidence of leukemia was
almost 60 per cent, a synergistic effect (Chart 4).
Estrogenic hormone combined with both urethan
and x-rays: leukemogenic effects.—Thesupplemen2Ciba Pharmaceutical Products, Summit, N.J. Estradiol
dipropionate was kindly supplied throuh the courtesy of Dr.
Robert Gaunt, Director of Endocrine Research.
----
200
WHOLE BODY .4
<4*90r)+
-57- -45.2%
URETHAN
WHOLE BODYJL
.,30.
(4I90O
33
URETHAN
0
ONLY
Jo"
300
4OO
AGE AT DEATH (DAYS)
500
CHART3.—Effect of urethan on induction of leukemia by
x-rays in DBA/2 mice (male). Treatment begun at 6 weeks
of age.
leukemia as 40 r X 4 with one thigh shielded under
urethan anesthesia.
Combined
treatment
with
40 r X 4 (whole-body,
no urethan) and estro
genic hormone yielded 48 per cent leukemia,
whereas the addition of urethan resulted in an
earlier appearance of leukemia and a higher inci
dence (60 per cent), even though thigh shielding
was employed during x-radiation.
A similar experiment was conducted on C57BL
mice (Chart 6) with a larger dose of x-rays,
40 r X 11, rather than 40 r X 4. The incidence of
leukemia in mice that were thigh-shielded under
uethan anesthesia was 37.5 per cent. If mice were,
in addition, estrogen-treated,
the incidence was in
creased to 57.2 percent with earlier onset. If thighshielded mice were irradiated under amytal (rather
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1958 American Association for Cancer Research.
KAWAMOTOet al.—Urethan and Leukemogenesis
than urethan) anesthesia and treated with estro
gen, the leukemia incidence was only 15.8 per cent
(three out of nineteen), not significantly different
from the incidence with estrogen only (Chart 5).
Although the incidence of leukemia was 59.4 per
cent in mice receiving estrogen plus urethan, the
addition of urethan accelerated onset (Chart 6).
Influence of thigh and thymus shielding during xradiation upon potentiation of leukemogenesis by
urethan (Charts 6 and 7).—When C57BL mice
were thigh-shielded
during x-radiation under so
dium amytal anesthesia, the leukemogenic effects
of 80 r every 4 days X 11 were remarkably re
ESTROGEN+ 11.594«
URETHAN
37 594*
40
ESTROGEN
JO'
URETHAN
•A-°*
incidence of leukemia in both thigh- and thymusshielded mice receiving estrogenic hormone was
low when amytal anesthesia was employed during
irradiation.
Effect of urethan on induction of leukemia by
methylcholanthrene (Chart 8).—DBA/2 mice were
given either urethan or skin paintings of methyl
cholanthrene
in benzene (0.25 per cent solution)
alone. Either eighteen or nine skin paintings were
given, a different skin site being used for each of
the paintings (3 times a week). In a third group
for each dose of methylcholanthrene,
urethan and
methylcholanthrene
treatments
were combined.
Onset of leukemia was significantly accelerated,
and the total incidence was greater when urethan
was given in addition to the methylcholanthrene.
As in the other strains, urethan by itself did not
induce leukemia.
66X
60
30
727
THIGH SHIELDED
(40 r 14) + URETHAN
* ESTROGEN
WHOLE BODY X-R
(«On 4) + ESTROGEN
M
20
ESTROGEN
4O
THIGH
f/
100
to
200
300
400
AGE AT DEATH (DAYS)
;Jg"286X
SHIELDED
(40 r I 4)+ URETHAN
WHOLE BODY X-R
(90 r »4)
90
IO
^•478»
lt_
52
_9_
(O
500
CHART 4.—Synergistic effect of urethan and estrogeuic
hormone on induction of leukemia in castrated C57BL mice.
Treatment was begun at 6 weeks of age. Negative mice on
estrogen either died after 300 days of age or are still living
beyond this age.
duced. If the same treatment
was given under
urethan anesthesia, the incidence of leukemia was
higher (39.1 compared with 18.2 per cent) and on
set earlier (Chart 7). Although leukemogenesis was
only moderately inhibited by thigh shielding un
der urethan anesthesia, thymus shielding was more
effective (Chart 7).
The difference between thymus
and thigh
shielding is further demonstrated
by the results
illustrated
in Chart 6. When mice were thighshielded under urethan anesthesia and given estro
genic hormone, the incidence of leukemia was
57.2 per cent. However, when the mice were
thymus-shielded
during x-radiation and similarly
given urethan and estrogenic hormone, the leuke
mia incidence was only 26.1 per cent. Since this
incidence is lower than when estrogenic hormone
and urethan were given without irradiation, it is
concluded that in C57BL mice extra-thymic
ir
radiation inhibits estrogen-urethan-induced
leuke
mogenesis.
Contrary to the results of Toch et al. (15), the
10
ZOO
30O
400
500
600
AGE
TOO
AT DEATH
BOO
(DAYS)
CHART5.—Effect of urethan on induction of mouse leu
kemia by x-rays and estrogenic hormone in castrated C57BLC3H FI hybrids. Treatment was begun at 6 weeks of age.
Controls.—The incidence of leukemia in pure
strain controls is shown in Chart 9. All pure strains
have been inbred in this laboratory, the DBA/2's
since 1945 when the original breeder pair was ob
tained from the Jackson Memorial Laboratory.
C57BL, CB (BALB/c without mammary tumor
agent), and C3H breeding stocks were obtained in
1952 from Dr. John Bittner, University of Minne
sota, and have since been maintained by brothersister matings. The controls were all breeding
stock from which the test mice were derived. Data
on 110 C57BL-C3H FI hybrids are shown in Chart
9.
DISCUSSION
Urethan remarkably
augments the leukemo
genic activity of x-rays, estrogenic hormone, and
methylcholanthrene
in mice, although by itself it
does not seem to be leukemogenic in low-leukemia
strains, at least in the doses which augment leuke
mogenesis. The influence of urethan on "viralleukemogenesis"
is not known. Presently, high-
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1958 American Association for Cancer Research.
Cancer Research
728
leukemia C58 and AKR mice are being treated
with urethan beginning at weaning age, to deter
mine whether the onset of the disease is hastened,
and low-leukemia mice are being treated with
urethan prior to the administration of cell-free
leukemic tissue extracts.
Vol. 18, July, 1958
It is remarkable that an agent without demon
strable leukemogenic activity so strongly aug
ments leukemogenesis induced by other agents.
There are probably other "radiomimetic" agents
which produce similar enhancing effects upon the
induction of leukemia. The problem of leukemo
genesis may be complicated by the promoting ac
tion of agents which might escape detection as
leukemogens if tested independently.
-IBMCA +
14
URETHAN
II
18MCA jj-34.4%
URETHAN
9 MCAj^j -15.7%
JO"'366X
----
200
100
300
AGE AT DEATH
400
(DAYS)
ESTROGEN +URETHAN
||-59.4X
t II) 6
„
23 'z6-1*
THIGH SHIELDED X-RI40r«ll)
+ AMYTAL + ESTROGEN
3 ._
»
-¡flS.SX"
THIGH SHIELDED
Ja
+ URETHAN
X-R(40r*ll)
URETHAN
ONLY
*
« £-8.7*
„._»
52"
THYMUS SHIELDED X-R(40rxll)
+ AMYTAL OESTROGEN
' - >J
A
,4«. *
2,-l4.3X
-WHOLE BODY
X-H(80r«ll)
Ti-55.5%
NOANESTHESIA '
-THIGH SHIELDED
X-R(80rill)
+URETHAN
SO
40
•-THI6HSHIELDED
Or.ll)
THYMUS SHIELDED
X-R(80rill)
tAMYTAL
CO
800
700
B.
200
C.
4
g^-18.2%
300
400
500 600
700
800
900
20
IS
10
a
A-39.1%
<J
•THYMUSSHIELDED3
X-R(SOrjll)
a-ll.5%
•»•URETHAN
SO
600
900
- OX
CHART6.—Additiveeffects of x-rays, estrogen, and urethan
on leukemogenesis in C57BL mice (male). Treatment was
begun at 6 weeks of age. In the groups indicated by asterisks,
most of the negative mice are still living and are all beyond
250 days of age.
60
500
300
400
A6E AT DEATH (DAYS)
200
.«
'°*
CHART8.—Effect of urethan on induction of leukemia
by methylcholanthrene in DBA/2 male mice. Treatment was
begun at 6 weeks of age.
THIGH SHIELDED X-R(40f» II) J2_ „,.
+URETHAN
+ESTROGEN
21 >57Z*
THYMUS SHIELDED X-R(40r
-r URETHAN
+ ESTROGEN
MO
SOO
URETHAN
ONLY
200
300
400
500
600
IO
D.
•ir-»'»
600
700
800
900
AGE AT DEATH (DAYS)
10
200
300
400
AGE AT DEATH (DAYS)
500
CHART7.—Effect of partial-body shielding on induction
of leukemia by x-rays in intact male and female CS7BL
mice unanesthetized or anesthetized during irradiation by
either urethan or amytal. Treatment was begun at 6 weeks
of age. In the thymus-shielded group, most of the negative
mice are still living and are all beyond 300 days of age.
CHART9.—A. Incidence of spontaneous leukemia in un
treated C57BL mice; B. Incidence of spontaneous leukemia
in untreated BALB/c mice. C. Incidence of spontaneous
leukemia in untreated DBA/2 male mice; D. Incidence of
spontaneous leukemia in untreated C57BL-C8H FI hybrids.
These charts represent the observed occurrence of spontaneous
leukemia in the untreated mice of the strains used for testing
the leukemogenic action of urethan in combination with leu
kemogenic agents.
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1958 American Association for Cancer Research.
KAWAMOTO
et al.—Urethan and Leukemogenesis
The fact that a single agent such as urethan
enhances the leukemogenic action of either polycyclic hydrocarbons, estrogenic hormone, or ioniz
ing radiations makes it seem likely that there is a
stage in the leukemogenic process common to all,
where a single agent may exert its influence.
In DBA/2 and BALB/c mice as few as four
anesthetic doses of urethan added to a dose of xrays of relatively low potency (4 X 90 r) increased
the incidence of leukemia 3| times and accelerated
its onset (Charts 2 and 3). The minimum effective
dose of urethan is being determined.
Certain strains of mice are relatively refractory
to the leukemogenic action of methylcholanthrene,
and in these the combined action of urethan and
the polycyclic hydrocarbons is being tested. The
influence of orotic acid (and similar agents), found
by Rogers (12) to inhibit the carcinogenic action of
urethan on the lung, is now being tested in this
laboratory for possible antileukemogenic action.
Urethan to a considerable degree nullifies the
protection afforded by thigh (marrow) shielding
against x-ray-induced leukemogenesis (Chart 7).
Estrogenic hormone has a similar effect (15). On
the other hand, as in the case of estrogenic hor
mone (15), shielding of the thymus during x-radiation under urethan anesthesia inhibits the induc
tion of leukemia (Charts 6 and 7). Experiments in
progress demonstrate that thigh shielding of
C57BL mice does not protect against the lethal
effects of x-rays if the mice are urethan-anesthetized.
SUMMARY
1. Urethan augmented the induction of leuke
mia in mice by x-rays, estrogenic hormone, or
methylcholanthrene.
2. Urethan was not leukemogenic for low-leuke
mia strains of mice when administered alone in
doses which remarkably augmented the leuke
mogenic activity of other agents.
3. The protective effects upon leukemia induc
tion of shielding bone marrow were nullified if
mice were anesthetized with urethan during x-radiation.
729
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Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1958 American Association for Cancer Research.
Urethan and Leukemogenesis in Mice
S. Kawamoto, N. Ida, A. Kirschbaum, et al.
Cancer Res 1958;18:725-729.
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