Effects of Oxygen Concentration on Carcinogenesis
Induced by Iransplacental Exposure to Urethan
JOSEPHA. DiPAOLO
(Rosiceli Park Memorial Institute, ¿fewYork State Department of Health, ¡iiiffalo,\ew York)
SUMMARY
The incidence of lung tumors was studied in hyperoxic and hypoxic pregnant A mice
given injections intravenously of 25 mg. of urethan, and in their offspring. Among
progeny born within 24 hours after their mothers received urethan, those born in an
environment of approximately 100 per cent or 10 per cent Oz developed a significantly
greater number of pulmonary tumors than did controls born in room air. The average
number of tumors decreased significantly in all progeny from mothers given injections
earlier than 24 hours prepartum. Myeloid leukemia and extramedullary hematopoiesis
were also induced or potentiated by urethan. Mothers in all groups had more pulmo
nary tumors than their offspring had ; 48-hour exposure resulted in an increased average
number for mothers kept hyperoxic, as compared with those kept hypoxic or in room
air. Mothers in all three groups developed a high incidence of mammary carcinomas.
Tumors have been induced in rivo by injection environment (3). The present study further ex
plores the phenomenon of urethan-induced pul
of polycyclic hydrocarbons and urethan into preg
nant mice. As early as 1940, Law (9) injected monary tumors, including observations made in
0.125 mg. of dibenz[a,/i]anthracene into the am- connection with intra-uterine exposure of embry
niotic fluid of mice 3-6 days prepartum, and into onic tissue to the action of urethan in 100 per cent
mice 24 hours old and 2 months old. Observations oxygen, 10 per cent oxygen, and room-air environ
made 6-8 months later revealed that 80 per cent ments. Also described are the effects of varying
of the mice exposed to carcinogen in utero and oxygen concentrations and urethan on the inci
100 per cent of those injected within a day after dence of lung adenomas and breast carcinomas
birth had multiple pulmonary nodules; only two in the mothers.
of 29 animals given injections at the age of 2
MATERIALS AND METHODS
months had tumors. Embryonic lung was thus
Plexiglass chambers were designed so that oxy
proved responsive to the carcinogen and possibly
more susceptible than adult lung. A high frequency gen concentration could be maintained at 10 or
of pulmonary tumors in offspring of mice given 100 per cent while temperature, humidity, and
intravenous injections of urethan during preg carbon dioxide concentration were kept fairly con
stant. Medicinal oxygen of 99.4 per cent purity
nancy, especially when the chemical was adminis
tered during the last day before parturition, was and water-pumped 99.7 per cent nitrogen were
reported by Larsen (8) and Klein (7), establishing supplied from high-pressure tanks, and concen
the transplacental effect of the compound as well tration was regulated by Venturi valves. Rubber
as the fact of increased penetration just before tubing completed the flow line from the valves
to metal pipes on each side of a fan on one side
parturition.
It has previously been demonstrated that hy- of the chamber. The percentage of oxygen was
measured with a Beckman oxygen analyzer about
peroxia increases the incidence of pulmonary ade
1 inch from the bottom of the chamber on the side
nomas in mice given injections of urethan, as opposite the inlets.
compared with animals given injections of urethan
The A mice used in these experiments were
and kept in a compressed-air, hypoxic, or room-air
bred in this laboratory and are derived from a
colony obtained from Dr. Strong in 1958. They
Received for publication September 25, 1961.
299
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Cancer Research
are genetically highly susceptible to pulmonary
tumors and are very responsive to urethan. Virgin
females 8-10 weeks of age were mated with young
males and were examined at least once daily for
vaginal plugs. The majority of pregnant females
were treated during the final 48 or 24 hours of
pregnancy, although some were 72-96 hours prepartum.
Each animal was given one injection, in a lateral
tail vein, of 0.25 ml. of water containing 25 mg.
of urethan, and the injection was carried out
slowly, so that the dose was received in not less
than 60 seconds. Controls were given intravenous
injections of water, maintained as treated groups,
and then examined for the presence of tumors.
After injection, an animal to be exposed to a high
or low concentration of oxygen was placed in a
transparent plastic cage with Purina chow and
water, and was sealed in an exposure chamber.
Immediately after birth, the animals were placed
in metal cages and returned to the animal room.
Mice were grouped on the basis of the interval
between injection of the mother and birth of the
young.
Progeny were killed at the age of 6 months, and
the mothers at the age of 9 months. One ml. of
Fekete's modification of Tellyesniczky's fixative
was intratracheally injected into the lungs before
the chest cavity was opened. The lungs were then
excised, the lobes were separated, and the tumors
in each lobe were counted in saline solution under
a magnification of 12X- Tumors appearing on the
surface of each lobe and those within the lung
parenchyma, detected by compressing the tissue
between the jaws of a pair of smooth-tipped for
ceps, were included in the total. Questionable
masses, as well as at least one lung tumor from
each animal, were diagnosed histologically.
RESULTS
Contrary to experiments previously reported,
exposure caused some deleterious effects. Mothers
given intravenous injections of urethan and ex
posed 24 hours to 100 per cent or 10 per cent
oxygen had fewer litters (35 and 24 per cent,
respectively) and fewer viable mice per litter (av
eraging 4.1 and 2.4, respectively) than did mothers
kept in room air (42 per cent, with litters averag
ing 6.0 mice). The average lung tumor incidence
for progeny from untreated mothers was 10 per
cent for the 69 progeny of mothers exposed to
100 per cent oxygen and for the 53 progeny of
mothers exposed to 10 per cent oxygen, as com
pared with 6 per cent in 53 mice from mothers
kept in room air.
Table 1 presents the average number of lung
Vol. 22, April 1962
adenomas per progeny from mothers given injec
tions at different intervals prepartum. The number
of lung adenomas induced at 100 per cent Oj
following 24 hours' exposure was statistically sig
nificantly greater than the number obtained at
room air conditions. The same was also true
of animals exposed to 10 per cent oxygen, but
they did not have an average number of lung
tumors significantly different from that in animals
exposed at 100 per cent oxygen. The average
number of lung adenomas per group decreased
abruptly after the first 24-hour exposure period;
and the differences between the average numbers
in the various groups decreased until it was im
possible to distinguish between animals born in
the different environments.
A comparison of the number of lung tumors
per mother with the number of lung tumors per
litter, as well as the percentage of mothers having
large or small litters (the latter consisting of fewer
than four mice), indicates that size of litter does
not vary with number of lung tumors per mother.
Some mothers eventually developed as many as
33 lung adenomas each. The number of lung
adenomas did not vary greatly from mouse to
mouse within a litter; the average number per
litter, however, did vary. This seems unrelated
to a dosage-response phenomenon, because the
number of lung adenomas per mother, as shown
by a scatter diagram, was not closely related to
the number of lung adenomas in litters.
A study of number of adenomas, on the basis
of cumulative percentage (Chart 1), allows com
parison of the average number of tumors per
litter. For example, 14 per cent of litters born
in 100 per cent oxygen environment averaged
fewer than 4.5 adenomas per litter, whereas 43
per cent of litters born in 10 per cent oxygen
environment and 63 per cent of litters born in
room air had fewer than 4.5 tumors. The litters
from animals exposed to 100 per cent oxygen
during the first 24 hours had the highest cumula
tive proportion at all points. Mice from the 10
per cent oxygen environment were intermediate,
and mice from the room-air environment had
the lowest cumulative percentages.
Since significant differences in the number of
lung adenomas have been found only in mice
born during the first 24 hours after injection,
changes in the number of lung adenomas per
litter for room air may be considered representa
tive of the situations in the other environments.
Mice born during the first 24 hours following in
jection had a larger number of lung adenomas,
on the average, than did animals born later. The
highest average number of lung adenomas per
Downloaded from cancerres.aacrjournals.org on July 31, 2017. © 1962 American Association for Cancer Research.
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Cancer Research
302
litter for any of the litters born 48-96 hours
after urethan injection was 2.25, whereas 75 per
cent of the litters born 24 hours subsequent to
injection exceeded this number.
Consideration of the cumulative percentage of
the average number of tumors in each mother
that gave birth during the first 24 hours subsequent
to urethan injection reveals a different incidence,
depending upon the group (Chart 2). Mothers
exposed to 10 per cent oxygen had fewer lung
adenomas than did those exposed to 100 per cent
oxygen. Mothers exposed only to room air subse
quent to urethan injection were intermediate in
Vol. 22, April 1962
carcinoma found in a control group of 32 females
that had been bred only once. Since this strain
of mice always develops a high incidence of mam
mary carcinomas after multiple breeding and at
a greater age, it cannot be ascertained whether the
urethan accelerated or simply induced the forma
tion of mammary carcinomas in the mice. In sever
al instances, mice with two or three mammary
carcinomas were studied by Dr. T. S. Hauschka,1
who found that each tumor was cytologically
distinct from the other carcinomas in the same
individual host.
Extramedullary hematopoiesis occurred in the
livers and spleens of eighteen progeny and twelve
mothers about equally divided among the urethan
groups, although none occurred in the controls.
Myeloid leukemia was found in five progeny from
mothers exposed to room air and hyperoxia; leu-
— MICEBORNIN 100% OXYGENENVIRONMENT
-«MICEBORNIN 10% OXYGENENVIRONMENT
•MICEBORNIN ROOMAIR ENVIRONMENT
15
7,
,3.5
,95
24,
AVERAGENUMBEROF LUNG ADENOMAS/ LITTER
•
MICE FROM100% OXYGENENVIRONMENT
•>•••<•
MICE FROM10% OXYGEN ENVIRONMENT
— MICE FROMROOMAIR ENVIRONMENT
CHART1.—Kffectof ¿4-hourexposure to different oxygen
Concentrationson progeny regrouped according to litter.
incidence of lung adenomas. Curves for the roomair and 10 per cent oxygen groups cross finally,
so that at 100 per cent the mothers from the
10 per cent group had an average number of
adenomas greater than the mothers exposed to
room air.
If the average number of lung adenomas per
mother exposed for 48 hours is considered, the
number of lung adenomas in the hyperoxia group
(18.2 + 1.2) is significantly different from the
other groups (8.6 ±1.7 for room air and 8.7 + 0.9
for 10 per cent oxygen), at the 1 per cent level
of statistical significance. This observation is in
agreement with one made on 6- to 8-week-old
A mice in which intraperitoneal injection of 1
mg urethan/gm body weight resulted in a statisti
cally significant difference at the 1 per cent level
of significance between the hyperoxia group and
others, including a compressed air chamber group
(20.5 per cent oxygen).
A high incidence of mammary tumors subse
quently developed in mothers treated with urethan
(Table 1), as compared with a single mammary
0
1.5
4.5
7.5
10.5
13.5
16.5
195
225
25.5
285
30.0
AVERAGENUMBEROF LUNG ADENOMAS/MOTHER
CHART2.—Effect of different oxygen concentrations on
induction of lung adenomas in strain A mice injected 24 hours
prepartum.
kemia was found in only one mother from the
room-air group. Increased mitosis was recognized
in the livers of six mothers exposed to hyperoxia.
DISCUSSION
The results of combinations of oxygen and ure
than on the progeny are strikingly different from
those on adult mice. The average numbers of
lung adenomas per mother in this study and per
adult mouse given 1.0 mg urethan/gm body weight
(3) are within the same range for identical ex
posure periods in hyperoxia, hypoxia, or room air.
The number of lung adenomas was less in
progeny than in adult mice, even at the 24-hour
interval, where the most significant increases and
differences were found. Either all stages of embry1Personal communication.
Downloaded from cancerres.aacrjournals.org on July 31, 2017. © 1962 American Association for Cancer Research.
DiPAOLO—C'arcinogenesis and Oxygen Concentration
onic lung tissue are less susceptible to this par
ticular combination than adult lung tissue, or a
partial placental barrier inhibits equal distribution
of urethan.
Hyperoxia or hypoxia alone did not significantly
increase the lung tumor incidence. Hyperoxia with
urethan must produce a change at the tissue level,
as demonstrated by the increased carcinogenic
effects. This is suggestive of the effect of high
oxygen concentration on mutation rates. The car
cinogenic effect of the combination with 10 per
cent oxygen suggests a relatively more severe
hypoxia in embryonic tissue than can be obtained
in adult lung tissue. Progeny from hypoxic mothers
came from smaller litters and fewer surviving
litters.
Warburg's theory (15) suggests that anaerobic
conditions select toward the survival of malignant
cells because of their glycolytic metabolic mecha
nisms. Mammalian carcinogenesis experiments in
volving decreased oxygen are necessarily limited,
because mice survive only a few hours in 5 per
cent oxygen, and local anoxia cannot be obtained
for any extended time without gangrene resulting.
Heston and Pratt (5) found that strain A mice
injected with dibenz[a,A]anthracene and exposed
for 48 hours to 8 per cent oxygen had a decreased
number of tumors induced by the carcinogen.
Mori-Chavez (10, 11), studying the effect of high
altitudes on neoplastic growth, found decreased
incidence of spontaneous leukemia in mice kept
at high altitude, as compared with mice at sea
level, whereas the number of tumors per lung
and the average size significantly increased in
strain A mice treated with a single dose of urethan
at high altitude in the mountains of Peru as
compared with those maintained at sea level.
The number of pulmonary adenomas induced in
progeny from mothers given injections earlier than
2-1hours prepartum was relatively small compared
with that in animals from mothers given injections
approximately 24 hours prepartum. Both Larson
(8) and Klein (7) have also found that offspring
of mothers given injections more than 2 days
prepartum had fewer tumors than did mice in
jected during the last 24 hours. The factors re
sponsible are obviously physiological and physical.
Urethan is rapidly eliminated from the body (1,
18), and the C14of urethan-C14 is uniformly dis
tributed in the various organs and tissues of the
normal body. Increased transplacental permeabili
ty may occur with physiological changes before
and during the process of birth and may account
for the response of lung cells to urethan.
Besides prenatal respiratory movements, lung
circulation competency is also extremely impor
303
tant. Since fetal lungs are not concerned directly
with respiration, only circulation involving growth
would be expected. Histological examination by
DiPaolo2 of embryonic lung tissue showed that
lungs were similar at 0-24 hours prepartum. The
alveolar epithelium was flat, whereas the bronchiolar epithelium was cuboidal, blood was in the
capillaries, and mitotic figures were rare. Younger
lung tissue, as from embryos 48 hours prepartum,
was completely collapsed, with tall epithelium
and numerous mitotic figures.
Urethan has been described as a multipotential
carcinogen because of the various neoplasms it
can initiate or potentiate. Tannenbaum (14) has
found that repeated painting of urethan on the
interscapular region of mouse skin augmented
the formation of mammary carcinomas in mice.
One intravenous injection of urethan late in preg
nancy appeared to increase the incidence of mam
mary tumors in all groups. The average age at
the appearance of the first tumor was 35 weeks.
Since none of the tumors were noticed when the
young were weaned from the mothers, urethan
was not proved to be an initiator of carcinogenesis.
Kawamoto et al. (6) and Berenblum and Trainin
(2) found urethan to be a promoting agent in
leukemogenesis. Fiore-Donati et al. (4) and Pietra
et al. (12) reported that a single subcutaneous injec
tion of urethan solution into newborn Swiss mice
less than 24 hours old resulted in malignant lymphomas in approximately 22 per cent of the ani
mals. The leukemia found in the present study
is interesting and would be of greater significance
if other animals having increased hematopoiesis
could be classified. Even though embryonic lung
tissue was perhaps less sensitive than adult lung
tissue to urethan, development of leukemia does
demonstrate the generally increased sensitivity
of treated embryos as an entity.
ACKNOWLEDGMENTS
The author is indebted to Dr. John E. Dowd for statistical
advice and to Mr. George Olshevsky for technical assistance.
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Effects of Oxygen Concentration on Carcinogenesis Induced by
Transplacental Exposure to Urethan
Joseph A. DiPaolo
Cancer Res 1962;22:299-304.
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