The Lipide Composition of Epidermis and Dermis of Mice
Undergoing Carcinogenesis by Methylcholanthrene*
C. CARRUTHERS
ANDB. DAVIS
(Biochemistry Research Department, Rosweu Park Memorial Institute, Buffalo, N.Y.)
SUMMARY
The lipide composition of epidermis and dermis under the influence of the hair
growth cycle and following the topical application of methylcholanthrene was inves
tigated. The percentage of cholesterol and phospholipide was lowest in anagen and
telogen epidermis, was nearly doubled in early and late hyperplastic epidermis, and
rose to even higher values in squamous-cell carcinomas. The cholesterol ester content
and the iodine number of the triglycéridefatty acids were of the same order of mag
nitude in these tissues, but the percentage of total lipide and triglycérideswas much
lower in the carcinomas than in hyperplastic, anagen, or telogen epidermis. Early
hyperplastic male epidermis contained a much higher percentage of cholesterol esters
and cholesterol than did similarly treated female epidermis. Fast acting sterols of the
epidermis were limited essentially to the cholesterol ester fraction.
No appreciable change was found in the lipide composition of the dermis of early
and late hyperplastic epidermis, as compared with anagen and telogen dermis. The
lipide composition of epidermis was considerably different from dermis in having a
higher content of phospholipide, cholesterol, and cholesterol esters but less neutral
lipide and triglycérides.
Very little is known concerning the role of the
various lipides in carcinogenesis or of their impor
tance in the hair growth cycle. Both processes are
related to cell growth in that changes in the hair
growth cycle are related to normal growth, where
as alterations in the epidermis induced by methyl
cholanthrene are associated in part with the gene
sis of skin cancer. Chemical changes in the skin as
related to hair follicular activity (3, 8, 11) are
mainly obtained from histochemical procedures
(17), whereas some information is available on the
chemistry of epidermis undergoing malignant
transformation (6). The adipose tissue of the skin
of mice doubles in thickness during periods of
dynamic hair follicular activity (anagen) over that
during the quiescent stage (telogen) of these struc
tures (9), yet no difference was found in the lipide
composition of the skin in the two phases of the
hair growth cycle (7). In this report the lipide com* Aided in part
by Grant
C-3257(C3)
position of epidermis and dermis of mice under
going carcinogenesis by methylcholanthrene
is
given.
MATERIALS AND METHODS
Adult Swiss mice (female, except where indicat
ed), 4-6 months of age, were used throughout. The
back of each mouse was shaved with an electric
clipper, and after a 3- to 4-day wait a 0.3 per cent
solution of methylcholanthrene (MC) in benzene
was applied topically to the shaved area with a
camel's hair brush No. 4 thrice weekly on alternate
days. Early hyperplastic epidermis (and dermis)
and late hyperplastic epidermis (and dermis) con
sisted of skin treated 3 times during 1 week and 18
times during 6 weeks, respectively, with MC. The
mice were sacrificed by cervical dislocation 5 days
after the last application of MC. The fat below the
panniculus carnosus was carefully removed, after
which the epidermis was separated from the der
mis at 50°C. by the procedure of Baumberger et
al, (2). The specimens of dermis contained the
adipose layer and panniculus carnosus and thus
did not vary as a result of scraping away indefinite
from the National
Cancer Institute, TJ.S.P.H.S. A preliminary report of this work
has been made (Proc. Am. Assoc. Cancer Researck,3:lOO, 1960).
Received
for publication
August 3, 1960.
82
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CARRTJTHERS
ANDDAVIS—-LipideComposition of Skin in Carcinogenesis
and uncontrollable amounts of fat. The epidermis
of 50-100 mice and the dermis from six to ten ani
mals were employed for each analysis. MC-induced papillomas and squamous-cell carcinomas
were pooled for analysis. Necrotic material and
connective tissue were carefully removed from
each carcinoma, a small portion of which was saved
for histological examination. The various tissues
were stored at —25°C. under redistilled ethanol
until ready for lipide extraction.
The lipides were extracted from the different
tissues at room temperature by the procedure of
83
the 12th-day after plucking of hair or the growing
phase of the hair follicles) and telogen dermis (re
sulting from the 22d day after plucking of hair or
the quiescent phase of the hair follicles) are not
appreciably different from the values obtained
from the dermis of mice painted 3 and 18 times
with MC (Table 1). Also, the levels of cholesterol
esters, cholesterol, and triglycéridesand the iodine
number of the triglycéridefatty acids are nearly
the same for anagen and telogen dermis and for
dermis following three and eighteen applications of
MC (Table 2).
TABLE1
THECOMPOSITION
OFLIPIDEFROMDERMISOFMICEUNDERVARIOUS
CONDITIONS*
dermisAnagen
Source of
(5)f
Telogen (5)|
3 Applications MC (5)î
18 Applications MC (5)jWater
cent)64.8
(per
lipide
cent)16.4
(per
+ 3.1
62.6±3.4
66.0 + 1.2
64.3+0.7Total
+ 1.6
+ 2.1
19.0 ±1.5
6.3±1.5
17.9 + 1.4
8.4+1.4
18.9 + 3.2Phospholipide
7.0±0.6Neutral
cent)7.8
(per
lipide
cent)92.2
(per
+ 2.0
93.7 + 1.5
91.8±1.5
91.7±0.5
* Anagen and telogen values from (7).
t Anagen and telogen—skin removed from mice on 12th and 22d-23d day of hair growth cycle,
respectively.
t MC applied thrice weekly on alternate days, skin removed 5 days after last MC application.
No. of samples analyzed indicated in parentheses.
TABLE 2
THE COMPOSITION
OFLIPIDEFROMDERMISOFMICEUNDERVARIOUS
CONDITIONS*
dermisAnagen
Source of
esters
cent)0.27+0.060.44+0.090.15+0.050.40+0.2Cholesterol
(per
cent)0.75+0.20.5
(per
cent)99
(per
(9)fTelogen
t3
(6)
(5)î18
Applications MC
Applications MC (5)tCholesterol
value
Wijs71+1363+
±0.1699.1+0.299.2+0.298
+0.140.6
886+
992
+0.161.4
+ 15
+0.5Triglycérides+0.8Iodine
* Anagen and telogen values from (7).
tÃ-See footnotes, Table 1.
No. of samples analyzed indicated in parentheses.
Hanahan (12) as given in (7). The silicic acid
chromatography method of Hirsch and Ahrens
(13) was employed to fractionate the acetonesoluble fraction of the lipides into cholesterol
esters, triglycérides,and cholesterol. Fast-acting
sterols were estimated on some samples by the pro
cedure of Moore and Baumann (18), cholesterol
according to the method of Abell et al. (1), and
triglycéridesby weight. The iodine number of the
triglycéridefatty acids was determined by the
micro-absorption Wijs technic after Luddy et
al. (16).
RESULTS
The water, total lipide, phospholipide, and neu
tral lipide content of anagen dermis (resulting from
There was a gradual increase in the water con
tent of epidermis in the following sequence : anagen-telogen, early and late hyperplasia. The water
level was highest in the carcinomas (Table 3). On
the other hand, the amount of total lipide was very
low in the carcinomas and papilloma, intermediate
in hyperplastic epidermis, and highest in anagen
and telogen epidermis. The percentage of phospho
lipide was much higher in the carcinomas than in
the normal or hyperplastic epidermis. The choles
terol ester content of anagen, telogen, early hyper
plastic (male) epidermis, and the carcinomas was
about the same, whereas that of early and late hy
perplastic epidermis of female mice was considera
bly less (Table 4). Female mice were employed for
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84
Vol. 21, January 1961
Cancer Research
most of the experiments because of the aggressive
and fighting nature of male Swiss mice. The per
cent cholesterol was lowest in anagen and telogen
epidermis, intermediate in hyperplastic epidermis
(female), and highest in the carcinomas. Ehrlich
ascites tumor cells were analyzed to determine
whether another type of tumor cell had high cho
lesterol content. These cells had not only a high
percentage of cholesterol but also of cholesterol
esters and phospholipide. An interesting finding
was that early hyperplastic epidermis of male mice
highest in the carcinomas. There is no significant
difference in the cholesterol ester content or in the
iodine number of the triglycéridefatty acids in
epidermis undergoing carcinogenesis, but an ap
preciable decrease in the total lipide and percent
age of triglycéridestakes place. Kandutsch and
Baumann (14) and Brooks and Baumann (4)
found that the application of MC to mouse skin
lowers the A'-cholestanol content, whereas the
amount of cholesterol increases somewhat. The
diminution of A'-cholestanol in MC-treated epi-
TABLE3
THECOMPOSITION
OFLIPIDEFROMEPIDERMIS
OFMICEUNDERVARIOUS
CONDITIONS*
TissueEpidermis-anagen
(4)t
Epidermis-telogen (5)f
Epidermis from 8 applications MC (4)t
Epidermis from 18 applications MC (3)}
Papilloma (1)
Squamous-cell carcinomas (4)
Ehrlich ascites (2)Water
cent)54.1±2.7
(per
lipide
cent)19.9+0.7
(per
53.7+3.4
61.9+0.8
69.3 + 4.8
66.1
80.0+0.8Total
1.511.8
+
23.6 + 1.2
+ 1.2
10.0 + 2.8
27.9±4.4
12.7+0.3
20.010.7
4.0
34
3.3+0.2Phospholipide
52.5+0.8
cent)13.9
(per
64Neutral
lipide
cent)85.1
(per
+ 1.5
88.2 + 1.2
72.2 + 4.4
80.0+0.7
66
47 ±1.2
36
* Ânagenand telogen values from (7).
ft See footnotes, Table 1.
Number of samples analyzed indicated in parentheses.
TABLE 4
THE COMPOSITION
OFLIPIDEFROMEPIDERMISOFMICEUNDERVARIOUS
CONDITIONS*
TissueEpidermis-anagen
esters
(per
cent)3.1+0.5
cent)2.3
(per
(5)f
+0.6
Epidermis-telogen (4)f
1.8 +0.4
0.8 +0.1«
Epidermis from 3 applications MC (4)î
Epidermis from 3 applications MC (rf1) (3)î 2.6 ±0.5
Epidermis from 18 applications MC (5)|
0.96±0.09
Papilloma (1)
4.4
Squamous-cell carcinomas (3)
2.6 +0.3
Ehrlich ascites (2)Cholesterol
18.2Cholesterol
cent)95
(per
+0.6
96 ±0.4
2.1±0.4
5.4±1.0
94.1 ±1.4
83.6 + 4.1
13.8±2.3
93.2 + 1.0
5.8±0.9
65.6
30
72.7±2.4
25.4±2.5
25.3Triglycérides
56.5Iodine
value
\Vijs59±
3
62±4
73+ 2
62±2
57+ 4.5
60
68±10
118
* Anagen and telogen values from (7).
tÃ-See footnotes of Table 1.
Number of samples analyzed indicated in parentheses.
had a considerably greater content of cholesterol
esters and cholesterol than did similarly treated
epidermis from female mice.
DISCUSSION
The following sequence of changes occurs in the
lipide composition of epidermis undergoing malig
nant transformation where anagen and telogen
epidermis serve as the control tissues. The content
of water and the percentage of cholesterol and
phospholipide is lowest in anagen and telogen epi
dermis, intermediate in hyperplastic epidermis, and
dermis appears to be related to the disappearance
of the sebaceous glands, where this steroid is lo
cated (5). In the analysis reported here the fastacting sterols, present in the ester fraction only, are
some 18 per cent of the esters and 10 per cent of
the total steroids of anagen and 27 per cent of the
esters and 13 per cent of the total steroids of telo
gen epidermis (7). Hyperplastic epidermis and
squamous-cell carcinomas contain relatively small
amounts of fast-acting sterols, in agreement with
observations of Kandutsch and Baumann (14,15).
The presence of the fast-acting sterols in the ester
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CARRUTHERSAND DAVIS—Lipide Composition of Skin in Carcinogenesis
85
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C.; QUEVEDO,W. C., JR.; and WOERNLET,
D. L. Influence of Hair Growth Cycle on Cytochrome
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10. FRANTZ,I. D., JR.; DULIT, E.; and DAVIDSON,A. G.
The State of Esterification of the Sterols of Rat Skin.
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fraction is in agreement with the observation of
Kandutsch et al. (14) and Franta et cd. (10).
The increase in the cholesterol content of hyperplastic epidermis and carcinomas over that of
normal epidermis is in agreement with the results
of Kandutsch et al. (14) and Brooks et al. (4).
These investigators found that the level of A7-cholestanol in mouse skin is independent of sex, where
as the levels of cholesterol and cholesterol esters
are higher in early hyperplastic male epidermis
than in female epidermis (Table 4).
A comparison of the data in Tables 1 and 2 with
3 and 4 indicates the great difference in the lipide
composition between epidermis, which is largely
cellular, and dermis, which is quite acellular. Inde
pendent of the hair growth cycle epidermis has a
higher content of phospholipide, cholesterol, and
cholesterol esters, but less neutral lipide and tri
glycéridesthan does dermis. The lipide composi
tion of dermis is little affected by the application
of methylcholanthrene.
1.
2.
3.
4.
5.
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The Lipide Composition of Epidermis and Dermis of Mice
Undergoing Carcinogenesis by Methylcholanthrene
C. Carruthers and B. Davis
Cancer Res 1961;21:82-85.
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