Arginase Activity and Nitrogen Content in Epidermal
Carcinogenesis in Mice
EUGENE ROBERTS AND SAM FRANKEL
(From the Department of Anatomy, Washington Unicerrity Sc/toolof Medicine, St. Louis, Missouri)
This
is one of a series of investigations
the pur
pose of which is to characterize
the nitrogen
metabolism of mouse epidermis in various phases
of growth and in carcinogenesis. It was reported
previously that mouse epidermis has high concen
trations of urea and preformed ammonia (1) and
considerable arginase activity (92). The purpose of
this communication
is to report data on arginase
activity in the epidermis of mice undergoing car
cinogenesis as a result of treatment with methyl
cholanthrene
in benzene and in control mice.
These include animals treated with benzene alone
or with a dilute solution of croton oil in benzene, a
non-carcinogenic
irritant. Finally correlative data
on total nitrogen, trichloroacetic
acid-soluble ni
trogen (TCA-soluble N), urea, and preformed am
fnonia contents will be presented.
Reports in the literature give the general im
pression that the arginase activity of malignant
tissues
is higher
than
that
of normal
and this has led to the suggestion
creased
arginase
accelerated
activity
rates
that
synthesis
weighing
and
with
cell
as part of
lism of the arginine-rich
roles suggested for arginase
S Aided
the Charles
by
grants
from
F. Kettering
the
Health
bottle
Service
in an ice bath
immediately
after
re
determined in aliquots of the freshly prepared homo
genate in the presence and absence of added Mn++ and
in samples incubated
manner
for activities
in 0.05 M MnCl2 for 5 hours at
previously
measured
described
(2).
in the presence
Only
the
of excess
Mn@@ before and after incubation will be reported be
cause of the possibility of previous contamination of the
epidermis with traces of activating ions. The results are
expressed both on a fresh weight and on a total nitrogen
basis.
When
the results
were calculated
in terms
of dry
weight of tissue the relative values differed little from
those
calculated
in terms
of nitrogen.
nation of total and TCA-soluble
ly from
the dermis
N were removed rapid
at 50°C. on a constant
temperature
hot plate, weighed individually on a torsion balance, and
then immediately placed in the calibrated tubes of small
in
with the metabo
Public
rapidly
distilled water. An aliquot of each homogenate was em
ployed for the determination
of total nitrogen by a
semi-micro Kjeldahl procedure. Arginase activity was
homogenizers
manganate
containing
water
redistilled
wiuch had been placed previously
boiling water. The tubes were removed
cooled, the volume
adjusted
so that
fresh tissue was approximately
50 mg.
tissue was homogenized.
One ml. of the
from
per
in gently
after 10 minutes,
the quantity
of
per ml. and the
homogenate
was
employed for the determination
of total nitrogen. To
92ml. of the homogenate was added 0.5 ml. of 192.5per
cent TCA with stirring and the mixture was allowed to
histones.
None of the
in the cellular economy
U.S.
was removed
genized in a ground-glass homogenizer in ice-cold re
vestigation
at the present time. It has been sug
gested that arginase may participate
in the trans
fer of the amidine group of arginine to glycine to
form guanidoacetic
acid in the kidney (7). This
enzyme may also be concerned
free of dermis
The samples of epidermis employed for the determi
ginine (6) and arginase
activity
(92) suggest the
possibility
that the ornithine
cycle may also be
This is under
almost
METHODS
moval, weighed rapidly on a torsion balance, and homo
results
The presence of large quantities of urea and pre
formed ammonia (1), and the detection of free ar
in mouse epidermis.
Epidermis
AND
by scraping at room temperature (8) for the determina
tion of arginase activity. The tissue was chilled in a
the in
the ornithine
cycle. However,
the function
of ar
ginase in the extrahepatic
tissues is not known.
operative
MATERIALS
500 C. in the
division (3, 4). Liver tumors, however, have much
less arginase activity
than the normal parent tis
sue (5). Probably
the chief function of arginase in
the liver is to form urea from arginine
veloped (92)was employed in the present study.
tissues,
may be associated
of protein
has received suitable quantitative
expression
be
cause most of the methods for estimation
of ar
ginase activity
in crude tissue preparations
have
been inadequate
in one or more respects. An im
proved
procedure
which has recently
been de
stand for 20 minutes. Nitrogen determinations
were per
formed on aliquots of the filtrate. The filtrate gave no
visible precipitate when more TCA was added or when
picric acid, metaphosphoric
acid, or a mixture of sul
and
Foundation.
9231
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Cancer Research
92392
furic and phosphoric
ammonium
sulfate
acids were added. Saturation
also had
no effect.
It
with
is recognized
that all known procedures for the precipitation of pro
teins from such complex mixtures give only comparative
results.
Nevertheless,
when
the conditions
are carefully
controlled valuable information can be gained about the
relative qualities of nitrogen contained in higher and
nitrogen
(see Table
weight
of normal
3) found
The results show clearly that the maximal po
tential arginase activity of the transplantable
squamous
cell carcinomata
is much
series. The relative
determination
of urea and ammonia have been de
scribed previously (1).
Adult female Swiss mice were painted with methyl
that for epidermis
increase
than
for any of the other
tissues
beuzene alone. With one exception the experimental
mice were sacrificed .5 days after the last application of
the test material, and the control mice 5 days after the
backs were shaved. The solutions were applied thrice
weekly. The tumors studied were obtained from 92lines
of transplantable
squamous cell carcinomata originally
derived from primary tumors produced by the applica
tion of methylcholanthrene
to the skin of a mouse. The
of liver, Tumor II exhibited
activity of any tissue studied
small
young
tumors
from
in activity
receiving
smallest.
to very
than
on incuba
tion at 50°C. with Mn@ was greater for Tumor II
cholanthrene in reagent grade benzene (0.6 gm. in
88 gm.), with 0.1 per cent croton oil in benzene, or with
were limited
greater
that found in the other tissues of the carcinogenic
lower molecular weight fractions. The methods for the
analyses
in an equal fresh
epidermis.
The
examined
while
3 paintings
was the
significance
of this
physiological
latter finding is not yet clear. With the exception
od of determination
Influence
taming
the highest arginase
to date by the meth
employed
of Benzene
(92).
Alone
and
Benzene
Con
0.1 Per Cent Croton Oil on Arginase
tivity—Six
and
1 1 paintings
produced approximately
arginase
activity
with
benzene
Ac
alone
a two fold increase in the
of epidermis
(Table
92). When
which the connective tissue capsule and the small
amount of central necrotic material were removed.
benzene containing 0. I per cent croton oil was ap
plied 6 times over a period of 92weeks the arginase
activity was normal. The hyperplasia which the
RESULTS
latter solution produces, which is somewhat
great
er than that resulting from benzene alone, regresses
Arginase
Activity
ogen-treated
epidermis
heating
in Normal
Epidermis,
Epidermis,
and
Tumors—Normal
had only slight arginase
with
Mn@
(Table
Carcin
activity
1). However,
prior to
after
activating
the preparations
to the maximal extent
under the conditions
employed the mean value at
tamed was 9 times greater than that found prior
to activation. The arginase activity rose markedly
after 3 paintings with methylcholanthrene
in ben
zene so that even before activation the mean ac
after cessation of treatment and does not result in
tumor formation.' When the croton oil solution
was applied
daily for 6 days and the animals
These
results
indicate
that
stimulation
tivity was greater than that of the normal epi
dermis after activation.
The completely
activated
homogenate
the arginase activity of the tissue. Epidermis
3 times as active
as the comparably
treated normal tissue. After 11
paintings
with the carcinogen
the arginase
level
was considerably
lower than after 3 paintings
but
was still slightly
higher than normal.
However,
when 924applications
were made over a period of
8 weeks the arginase activity was reduced to slight
ly lower values than were found in the normal con
trols. Maximally
activated
homogenates
of Tu
mors I and II were approximately
6 and 18 times
more active than normal
epidermis
on a fresh
weight basis and approximately
11 and 33 times
agent
of the epidermis
the
growth
was approximately
does not necessarily
genesis—The
determined
weights
of epidermis
in which
approximately
painted,
and removed.
the tissue
receiving
paintings
approximately
observations
(8) or
in an increase
in
which
dermis
per mouse
were
on samples obtained by heat from mice
than when wet weights were employed because a
given wet weight of tumor tissue contains only
weight
result
plications of benzene containing the carcinogen.
Total Nitrogen, Tissue Weights, and TCA -soluble
Nitrogen in Normal Epidermis and in Carcino
cholanthrene
of the dry
to
by a non-carcinogenic
had received 1 1 paintings with benzene had greater
arginase
activity
and less hyperplasia
than that
shown by tissues receiving the same number of ap
more active when expressed in terms of total nitro
gen. These values were also considerably
higher
than those found after 3 paintings.
The arginase
activity of the tumors was relatively greater when
the results were expressed
on the nitrogen
basis
one-half
were
killed on the day following the last treatment an
even greater hyperplasia was found on histological
examination than in the previous 92groups. How
ever, in the latter tissue the arginase activity was
lower than that found in the normal epidermis.
equal
areas
were shaved,
It should be noted that in
924paintings
with the methyl
solution there are projections
into the dermis.
An undetermined
of epi
portion
of this material was left behind in the dermis when
the separation
was made. The area of epidermis
used may also have been slightly smaller in the 924
1 Berenbium,
material.
personal
communication,
and
unpublished
in this laboratory.
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1949 American Association for Cancer Research.
TABLE
1
ARGINASE AcTIVITY IN EPIDERMAL
CARC1NOGENESIS
(Results are expressed in micrograms of urea liberated in the presence
of Mn@
under standardassayconditions)
ARGINASE
ACTIVITY
Before activation
Mierograma
Nvaaaa or
TISSUE
Nt'MBSS OF
per mg
wt.
total
3*
73
8
40
(1—10)11
(30—110)
3 paintings with MC5
10
30
(24—46)
11 paintings with MC
5
15
10
(5—18)
24 paintings with MC
10
38
(0—4)
Tumor It
10
10
24
(10-68)
Tumor JI@
10
10
(8—71)
@
in
per nig.
total N
27
586
(19—37)
615
(421—830)
83
1,540
(57—121)
38
(31—59)
20
(16—25)
165
(64—257)
479
179
(97—329)
43
(0—88)
995
(431—2,700)
89
S Methylcholanthrene
N
(448—842)
2
Micrograms
per mg.
fresi, wt.
MICE
37
activation
Micrograms
per mg.
SAMPLES
Normal
fresh
After
Micrograms
1,579
(1,120-2,190)
675
(536—1,000)
421
(328—495)
6,690
(2,980—9,360)
19,400
(160—721)
(330—2,500)
(6,620—27,700)
benzene
6th transplantation
t 68th transplantation
§Average value
IIRangeofvalues
TABLE 2
INFLUENCE
OF PURE BENZENE
AND BENZENE
CONTAINING
0.1 PER CENT CROTONOIL ON ARGINASEACTIvITY
(Results
are expressed
in micrograms
of urea liberated
in the presence
of Mn@ under standard assay conditions)
.
AROINASE
Before activation
Micrograms
Micrograms
NUMBER
TISSUE
OF
NusiaSS
SAMPLES
OF
per
MICE
mg.
per
fresh wt.
ACTIVITY
Micrograms
mg
per
total N
After activation
Micrograms
mg.
per
fresh wt.
mg.
total N
3
73
27
586
Normal
8
40
(1—10)
7
(30—110)
155
(10—37)
58
(421—830)
1,244
6 paintings with benzene
6
20
(6—11)
(121—192)
13
11 paintings with benzene
S
15
(5—21)
6 paintings with croton oil in benzene
6
11
(2—7)
6 paintings with croton oil in benzene
6
15
(4—9)
group
of
animals
was
painted
on
6 successive
days
and
the
epidermis
was
analyzed
28
day
after
598
(233—949)
18
(76—160)
the
(795—1,385)
(11—43)
112
on
1,167
(40—81)
112
(46—195)
6
(1,090—1,400)
63
(107—199)
5
S This
(48-65)
165
the
last
347
(12—27)
treatment.
All
other
(226—486)
experimental
groups
were painted thrice weekly and killed 5 days after the last painting.
TABLE
TOTAL NITROGEN
AND TCA-SOLUBLE
3
NITROGEN
WEIGHT
OF
TISSUE
SAMPLES
Normal
7
3 paintings with MC5
6 paintings
24 paintings
with MC
with
MC
Tumor It
S Methyleholanthrene
in
benzene
OF
NUMBER
MICE
30
12
29
5
15
10
32
10
10
OF
CARCINOGENESIS
TOTAL
EPIDERMIS
NUMBER
IN EPIDERMAL
NITROGEN
Mg. per
Per cent of
mouse
56
fresh
wt.
4.56
(48—62)
(3.98—5.05)
127
5.39
(98—195)
92
(71—98)
(5.19—6.08)
4.76
(4.64—5.00)
59
(47—68)
4.66
(4.32—5.05)
2.28
(1.97—2.61)
TCA-SOLUBLE
Per cent of
fresh
wt.
0.79
(0.68—0.84)
0.74
NITROGEN
Per cent of
total
N
17.4
(15.6—20.0)
13.7
(0.70-0.79)
0.82
(11.6—17.5)
17.2
(0.69-0.92)
(14.8—19.9)
0.95
(0.84—1.14)
0.23
(0.17—0.28)
20.3
(18.1—22.1)
10.1
(7.7—11.7)
t 6@ndtransplantation
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Cancer Research
9234
The weights of the epidermal sample per mouse
showed
an increase
to 1927 mg. per mouse
after
conditions.
paintings
3
However,
with
in the
benzene
paintings from the normal mean value of 56 mg.
(Table 3). The values dropped to 992mg. and 59
there
mg.,
ratio of the two remaining
respectively,
after
methylcholanthrene.
pattern
of changes
6 and
924 paintings
with
decreased
in the total
f@
croton
oil
nitrogen
and a
increase in the TCA-soluble
N, the
in the normal
range.
—Three applications
of methylcholanthrene
in
benzene produced a decrease of urea nitrogen from
77 mg. per cent to 44 mg. per cent (Table 5). No
further
significant
decrease took place in tissues
receiving
6 and 924 paintings
of the carcinogen.
There
of the much higher water content of the tissue. No
comparison
of the weights
of the transplanted
tumors and the epidermal samples is possible. The
mean percentage
of the total nitrogen found in the
fraction
receiving
Urea and Ammonia in Epidermal Carcinogenesis
The total nitrogen showed a
similar to that found in the
weights, increasing markedly after 3 paintings and
decreasing thereafter. In contrast, the TCA-sol
uble N decreased after 3 paintings and increased
thereafter.
Both the total nitrogen and TCA
soluble N were lower in the tumor studied because
TCA-soluble
was an increase
corresponding
tissues
containing
was also a fall to 48 mg. per cent in the
epidermis
of mice which
lanthrene
contained
had received
in a mixture
methylcho
of benzene and
lanolin (0.6 gm. methylcholanthrene
in 88 grams
of a thixture containing
equal weights of lanolin
from 17.4 in the
and benzene) . It has been shown that when lanolin
alone is the solvent for methylcholanthrene
the
carcinogenic action is prevented, although the tis
untreated
controls to 13.7 after 3 paintings
of the
carcinogen,
returned
to 17.92 per cent after 6 ap
TABLE 4
INFLUENCE
OF PURE BENZENE
AND BENZENE
ON TOTAL NITRoGEN
CONTAINING
AND TCA-SOLUBLE
WEIGHT
OF
NUMBER
SAMPLES
TISSUES
OF
Mg.
MICE
TCA-SOLUBLE
NITROGEN
Per cent of
fresh wt.
per
mouse
56
Normal
CONTENT
TOTAL
OF
EPiDERMIS
NUMBER
0.1 PER CENT CROTON OIL
NITROGEN
4.56
7
30
(48—62)
67
(3.98—5.05)
with henzene
6
22
(50—77)
43
(3 .89—4
.58)
6 paintings
with benzene
5
20
(37—49)
(3.17—4.65)
6 paintings
with croton oil in benzene
3
12
(46—73)
plications,
sue
and rose to 920.3per cent after 924appli
cations. These changes were in the opposite direc
tions
from the changes
in arginase
activity,
weights, and total nitrogen.
In the tumor studied
the percentage
value lower than
of TCA-soluble
that
found
N was
10.1, a
in any of the samples
of epidermis. This is consistent
with findings
which showed that there is a marked decrease in
the free amino
acids
in the
tumors
the decrease in urea and ammonia
(6) and with
to be reported
in a subsequent
section of this paper.
Influence of Benzene Alone and Benzene
Contain
0.79
17.4
(15.6—20.0)
1.06
24.8
(0.94—1.23)
(23.9—26.8)
4.19
5.54
(5.40—5
.63)
is sensitized
to
Per cent of
total N
(0.68—0.84)
4.19
3 paintings
60
NITROGEN
Per cent of
fresh wt.
0.92
24.3
(083—1.02)
1.01
(17.8—27.5)
18.2
(1.00—1.01)
(17.7—18.7)
further
applications
of the
carcinogen contained in pure benzene (9). Methyl
cholanthrene
contained in a mixture of benzene
and lanolin also does not exert its customary ac
tion (10). The sebaceous glands are not destroyed
and little if any hyperplasia occurs. It is, there
fore, apparent that the decrease in urea observed
in the animals receiving methylcholanthrene
in
benzene does not necessarily depend on the de
struction of the sebaceous glands, which occurs
after 3 paintings, or on the production of the in
tense hyperplasia which is usually induced by this
ing 0.1 Per Cent Croton Oil on Total Nitrogen, Tis
sue Weights, and TCA-soluble N—Three applica
tions of benzene alone produced a slight increase in
treatment.
the average
weight
treatments
there was a slight decrease
epidermal urea (1) comparable to that produced
by methylcholanthrene
in benzene in the present
study. It can, therefore, be concluded that agents
other than carcinogens can induce a marked de
of the epidermis,
while after
6
from the
normal values (Table 4) . There was a decrease in
the total nitrogen content
of these tissues and a
concomitant
increase in the TCA-soluble
N.
These changes resulted in a significant increase in
the percentage
not precipitated
of the total N found in the fraction
by TCA under our experimental
croton
The application
oil in benzene,
was previously
crease
reported
in epidermal
of a dilute
solution
a non-carcinogenic
urea.
to produce
of
agent,
a decrease
No significant
in
changes
in the ammonia content of the treated tissues were
observed with the possible exception of the de
creased
ammonia
level in the tissue
treated
with
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1949 American Association for Cancer Research.
ROBERTS
AND FRA@EL—Epidermal
Carcinogenesis
9235
in Mice
methylcholanthrene
contained in the mixture of
benzene and lanolin.
The mean values of the urea and ammonia con
tents of the tumors studied were significantly
lower than those shown either by the hyperplastic
or normal tissues. The samples obtained from the
57th transplantation
gave closely similar mean
values to those obtained from the 692nd transplan
changes in a number of other constituents studied
to date (16) were not like those shown by the ar
the TCA-soluble
arginase
activity
tation of Tumor I. The values for Tumor II were
slightly higher than those for Tumor I, but were,
nevertheless,
lower than those for any of the
samples of epidermis
studied.
paintings of methylcholanthrene
and the much
greater increases in the carcinomata were accom
panied by decreases in the proportion of TCA
soluble N. It is thus apparent that there is no ob
DISCUSSION
The pattern of changes in arginase activity re
ported in the present study differs from that
vious
ginase
activity.
The increased arginase activity after painting
with benzene was accompanied by an increase over
normal of the percentage of the total N found in
fraction,
while the
found in epidermis
correlation
between
these
elevation
receiving
variables
in
3
which
would hold for all of the tissues examined. How
ever, in the carcinogenic series changes in arginase
TABLE S
UREA AND AMMONIA
IN EPIDERMAL
NUMBER
TISSUE
OF
CARCINOGENESIS
NUMBER
DETERMINATIONS
OF
MICE
Normal epidermis
20
92
3 paintings
with MC* in benzene
17
54
6 paintings
with MC in benzene
S
15
6 paintings
with MC in benzene+lanolin
S
25
24 paintings with MC in benzene
12
Tumor It
10
Uaxt-N
AMMONIA-N
Mg. per 100 gms. fresh tissue
77
24
(55-98)
(16-31)
44
20
(32—66)
(16—28)
42
(34—46)
48
(35—63)
40
28
(21—31)
17
(14—18)
25
44
(26-67)
21
(20-31)
7
10
(15—28)
(4—9)
22
10
10
(19-26)
28
11
TumorII@
10
10
(20-46)
(9-15)
previously
for cytochrome
c (11), cyto
chrome oxidase (192), succinic dehydrogenase (192),
and adenylpyrophosphatase
(13). None of the
other enzymes showed any significant changes
from normal after 3 paintings with methylcholan
threne, while arginase increased three fold after
this treatment.
When the results were expressed
on a wet weight basis, Tumor I had 6 times as
much arginase activity as normal epidermis, while
the succinic dehydrogenase
and adenylpyrophos
phatase
activities
increased
two
and
three
fold,
respectively, and the cytochrome oxidase activity
and the cytochrome c content were below the nor
mal levels. In addition, pure benzene produced a
doubling of the arginase activity while not exert
ing any
influence
on the other
There is some parallelism
by arginase
activity
for cytoplasmic
pared material
changes
altogether
(7—10)
t 6@ndtransplantation
I 5th transplantation
. Methyicholanthrene
t .57thtransplantation
reported
9
Tumor I@
enzymes
studied.
in the changes exhibited
and those
previously
reported
ribonucleic acid in similarly pre
(14). On the other hand, the
in desoxyribonucleic
different
(15) and
acid
the
content
patterns
were
of
activity
were consistently
of opposite
accounted
of epidermis
lanthrene
correlated
sign in the percentage
for by the TCA-soluble
receiving
3 paintings
with changes
of the total
N
N. The samples
with methylcho
were more like the tumors with respect
to arginase activity and TCA-soluble
N than were
any of the other samples of epidermis
examined.
The greatly increased epidermal
weight and nitro
gen content after 3 paintings
suggest that the rate
of protein synthesis
is greater in this tissue than
after a greater number of applications
of the car
cinogen.
The changes in urea level in carcinogenesis
not
parallel
to the
changes
Even if arginase activity
in arginase
were
activity.
were entirely responsible
for the urea content of epidermis,
factors such as
substrate
concentration
and cell permeability
would play a role in the regulation
of the tissue
content
of urea. Also if the ornithine
be shown to exist in epidermis,
arginase
cycle should
would only
be one of a number of enzyme systems upon which
the formation of urea would depend, and it is en
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9236
Cancer Research
tirely possible that some of the other reactions
would constitute
the rate-limiting
steps in the
process. In this connection it is interesting to note
that liver tumors have been shown to retain some
of the arginase activity of normal liver but not the
ability to convert ammonia to urea (17) . The quan
titative changes in urea in carcinogenesis resemble
those reported for calcium (18) rather than those
found in the nitrogen fractions reported in the
present paper. On the other hand, the pattern of
changes in ammonia in carcinogenesis was similar
to that found for the TCA-soluble N. It is inter
5. Six and 11 paintings
duced a two fold increase
esting
decrease
that
the mean values
for urea and ammonia
in tumors were closely similar to those previously
found in mouse liver and in embryonic epidermis
(1). The changesin urea level in carcinogenesis
were negatively
correlated
with alterations
in
water content, since there was an increase in water
content over normal in the hyperplastic
tissues
and a further increase (8) in tumors at the same
time that progressive decreases in urea content
were recorded.
Although the free arginine content fell from rela
tively high levels in normal and hyperplastic epi
dermis to values below the limits of detection in
the tumors (6), the tumor tissue as a whole had the
same content
of arginine
as normal
epidermis
(19).
This indicates that the rapidly growing carcinoma
is able
to
obtain
arginine
at
a sufficiently
than
that
mis in the present
of normal
study,
or hyperplastic
epider
might be a factor
limiting
the quantity of arginine available for protein syn
thesis and thereby limiting the growth of the
tumor.
SUMMARY
1. Determinations
4. In Tumors I and II the mean arginase ac
tivities were approximately
6 and 18 times, re
spectively, that of the normal epidermis on a wet
weight basis and 11 and 33 times on a total nitro
gen basis.
The
percentage
in total
nitrogen
tissues
treated
with pure benzene or benzene
receiving
6 and
7. The
level
of urea
decreased
8. The
erence
pared
results
studies
C.
for
5 hours
in 0.05
M
that
found
with
made
on
special
similarly
1949.
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ref
pre
heating
at
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The
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3. The arginase activity, total nitrogen, and the
wet weight of the epidermis per mouse all increased
to a maximum after 3 paintings with benzene con
taming methylcholanthrene
and decreased after
subsequent applications
of the carcinogen. The
maximal mean value for arginase activity was 3
times
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Arginase Activity and Nitrogen Content in Epidermal
Carcinogenesis in Mice
Eugene Roberts and Sam Frankel
Cancer Res 1949;9:231-237.
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