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Factors Influencing the Incidence of Epidermal Methylcholanthrene Tumors in Mice Treated with Cortisone
FACTORS INFLUENCING THE INCIDENCE OF EPIDERMAL METHYLCHOLANTHRENE TUMORS IN MICE TREATED WITH CORTISONE IL INFLUENCE OF VARIATIONS IN THE TIMING OF CORTISONE ADMINISTRATION IN RELATION TO CARCINOGEN APPLICATION*
F. HERRMANN, M.D., R. W. SHERWIN, M.D., S. D. MORRILL, M.D., M. J. ROTHSTEIN, M.D. AND MARION B. SULZBERGER, M.D. OBJECTIVE
In the following we shall report on experiments concerning the question of whether or not the acceleration in the occurrence of epidermal methyicholanthrene tumors, observed by us previously in mice treated with cortisone (1—3), is
dependent on the timing of the administration of cortisone in relation to the carcinogen applications. Among several possibilities, it was, for example, conceivable that this acceleration is not caused by direct action of the steroid, but by
withdrawal of its use; in that case, a stronger acceleration was to be expected from cortisone administration preceding the course of methylcholanthrene
paintings, than from either concomitant or "overlapping" treatments with cortisone and methylcholanthrene. It was the attempt at clarifying this quest i'n and several related points which gave rise to the present study. The observations made in three of our pertinent experiments will be described in this presentation.' METHOD AND MATERIALS
Animals. Female albino mice (383) of the Swiss strain, from Carworth Farms, Inc., New
City, Rockland County, N. Y., three months of age; on unrestricted "Rockland mouse diet" and intake of water. Carcinogen. Methyloholanthrene, 0.6% in benzene. Cortisone. Cortone suspension,2 containing 2.5% cortisone acetate, 0.9% benzyl alcohol—
as preservative, and dispersing material (Tween 80 and Na-carboxy-methylcellulose-— * From the Department of Dermatology and Syphilology of the New York University Post-Graduate Medical School (Dr. Marion B. Sulzberger, Chairman) and the Skin and Cancer Unit of the University Hospital. Thi8 study was made possible by a grant of the United States Public Health Service (U.S.P.H.S.R.G. C 1379). All numerical results and presentations were supervised by Dr. R. R. Freudenthal, statistical consultant, Jackson Heights, N. Y. With the technical assistance of L. Mandol, B.A., L. Frank, B.A., J. Kallman, B.S., M. Cohen, B.S., M. Besen, B.A. and C. Guevara.
Presented at the Sixteenth Annual Meeting of the Society for Investigative Dermatology,
Inc., Atlantic City, N. J., June 5, 1955. 'Another group of pertinent experiments—cortisone administration beginning after, or shortly prior to termination of the methylcholanthrene applications—was started only recently; the results will be reported later. by Merck & Co., Inc., Rahway, N. J. 411
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THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
concentrations unknown) in physiologic saline solution, was diluted with saline solution to obtain a 0.17% solution of cortisone acetate; at each administration, 0.25 ml. of this dilution (containing approximately 20 mgm. cortisone acetate per kg. fasting weight) was injected subcutaneously. The right and left inguinal areas were injected on alternate days. Experimental Design.
Experiment I (Cortisone administration concomitant with applications of methylcholanthrene). The first experiment was designed as a pilot experiment assaying (1) the occurrence of skin tumors in response to only three methyicholanthrene paintings per week for a period of three weeks; and (2) any acceleration of this response after concomitant treatment with cortisone—in order to establish a base-line for appraisal of the results of variations in timing of the course of cortisone injections in relation to that of the carcinogen applications. The procedure of hair clipping at the beginning of the experiment, as well as the technic of painting were the same as described in our previous reports (1—3).
In all mice the shin of the interscapular area was painted with methyicholanthrene in benzene three times per week, for three consecutive weeks. The animals were divided in three groups: Group Ia—49 mice: Cortisone was injected on five consecutive days during each of the three weeks of painting. Control experiments were carried out in the two other groups as follows: Group Ib—48 mice—received injections of the (diluted) vehicle of cortisone (Merck & Co.)—instead of the cortisone injections. Group Ic—46 mice—did not receive any injections. Experimeni8 in which the period of cortisone administration did not coincide with that of the carcinogen applications:
Experiment II (Cortisone treatment prior to carcinogen applications). Group ha: Cortisone was injected in 40 mice on five consecutive days during each of three consecutive weeks. Following termination of the injections, i.e. from the first day of the fourth week of the experiment, methyleholanthrene was applied to the skin of the interscapular region three times a week, for a total of three consecutive weeks. Two other groups of 40 mice each were used for control experiments as follows: Group IIb—Instead of the cortisone injections, injections of the diluted vehicle employed
in Merck's Cortone suspension preceded the three weeks' period of methylcholanthrene applications. Group lie: Physiologic saline solution was injected instead of the cortisone suspension; otherwise the procedure was identical with that employed in groups ha and b.
Experiment III (Period of cortisone treatment overlapping first week of carcinogen applications). Group lila: In 40 mice, cortisone was administered by injection for a period of three weeks, in the manner outlined for our previous experiments. Painting with methyleholanthrene, however, was started at the beginning of the third week of treatment with cortisone and continued for two weeks after termination of the cortisone injections. Control groups: Group 11Th: Forty mice received injections of the (diluted) cortisone dispersing liquid, instead of the cortisone injections; otherwise, the procedure was the same as that in group
lila.
Group life: Forty mice received injections of physiologic saline solution, instead of the cortisone injections; otherwise the procedure did not differ from that employed in group
lila.
FACTORS IN TUMOR INCIDENCE IN MICE. II
413
RESULTS
Incidence of First Skin Tumors.
Experiment I (Concomitant treatment with cortisone and methylcholanthrene). Table I shows the incidence of first skin tumors and the number of mice which died without skin tumors, in each week of the experiment. The experiment was discontinued at the end of the 36th week. The average weight of the mice in each group before and after treatment is recorded below the table; a considerable drop is apparent in the cortisone group, but this weight was
regained two weeks after the termination of treatment.
The overall incidence of skin tumors was low, in particular when compared with the range of the incidence usually observed when methylcholanthrene is applied more frequently or for longer periods of time. Since distinctive rates in the occurrence of first skin tumors were apparent in the three groups during different periods of observation, our total 36 weeks of observation was subdivided into four periods, as indicated by the heavy vertical lines on Table I: (1) First two weeks: No tumor formation. (2) Third, fourth, and fifth week: Skin tumors appeared in eight mice of the cortisone group (Ta), in only one animal of control group Tb—and in two animals of control group Ic. During this period, therefore, the tumor incidence was significantly higher in the cortisone group than in both control groups.
This difference was made even more significant by the death rate of nontumor bearing animals, in as much as eight of the cortisone treated mice died within the first month of the experiment, whereas no death occurred in the control groups during the first two periods of observation. (3) Sixth through 16th week: During these 11 weeks, six of the mice injected with the suspending liquid of Merck's Cortone suspension developed first tumors (group Tb), whereas there was only one new tumor bearer in the cortisone group (Ta) and one in the control group in which injections were omitted (Ic). (4) Seventeenth through 36th week: During this last period, the incidence of first skin tumors was similar in all groups (eight new tumor bearers in group Ia, as well as in group Ic; six new tumors bearers in group Tb). Evidently, the results were considerably more distinctive regarding the time of appearance of first tumors than regarding the final proportion of tumor bearers per group.
Experiments in which the periods of cortisone administration and methyicholanthrene treatment did not coincide:
Table TI shows the weekly number of new tumor bearers and of the mice which died without skin tumors in each week during the course of experiments
II and III.
TABLE
I
T
48
46
Ibf
Jc
0 0
1
0 0
1
0 0
2
1 0
5 4
3
7
8
9 10
11
12
13
14
0
0
0 2 1 0
0 0 0
0 1 0 0 0
0 0 0 0
1 0
0 0
Period
0 0
18
0 0
19
0 0
20
0 0
21
1
1
22
24
25
26
27
28
29
30
31
0 1 0 0 0 1 0 0 0 0 0 0 1 0 0 0 0 0
23
1 1
32
34
35
36 Total
0
0 0 0
0 0 0 0 0 0
0 0
0
0 0 0 3 0 1 0 0 0 0 0 1 0 0 0 —4th Period
2 0 0 0
2 1
—
0 0 1 0
5
11
5
13
0 3 0 1 17 1 0 1 0 14
33
0 0 0 0 0 0 0 0 0 1 0 2 0 2 0 1 0 0 0 0 0 0 0 1 0 1 0 0 1 0 1 0 1 0 0 0 0 0 0 0
0 0
16 17
1 0 0 0 0 0 0 0 0 0 0 0 0 0 1 010 0 0 0 0 0 —* i— 3rd Period
1 0
0 0
15
At beginning of treatment period At end of treatment period
23.7
25.8
Average weight of mice in grams 1a Groups:
26.3 26.7
1W
27.6
26.0
Ic
* Ia: Subcutaneousinjections of cortisone acetate (20 mgm/Kg body weight—Cortone suspension,Merck & Co.) on five days per week, for three consecutive weeks. t Ib: Injectionsof suspendingliquid of cortisone insteadof the cortisone injectionsControl Groups SIc: No injections § T: Number of mice with (grossly visible) skin tumors. ¶ D: Numberof mice dead without (grossly visible) tumor development.
Penod
6
1 0 0 1 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0
5
0 0 0 0
2
4
T 00 00 20 00 00 D —1st-- -- 2nd
D1[
Dlf
T
49
Ia
Weeks:1
of Mice
Groups
No.
Experiment I: External treatment with methyicholanthrene 0.6% in benzene for three weeks. Concomitant injections of cortisone acetate. Occurrence of first skin tumors per week (and weekly death rate of mice without skin tumor)
FACTORS IN TUMOR INCIDENCE IN MICE. II
415
The average weights of the different groups of the mice, recorded at the end of the table,
showed a decline after the injection periods, especially in the cortisone-treated groups (ha and lila); but this weight was regained two weeks after termination of the injections.
The total period of observation was again subdivided into four periods in order to emphasize the pertinent differences in the incidence of first tumors observed in the individual groups.
Experiment II (Cortisone treatment prior to methyicholanthrene applications). There was no significant difference between the tumor incidence observed in the group in which the course of cortisone injections was followed by exposure to the carcinogen (ha) and the incidence observed in either one of the two control groups (JIb and TIc—injected with the plain vehicle of Merck's cortisone suspension and with saline solution, respectively—prior to the carcinogen applications). In all three groups, the incidence showed a "scattered" pattern similar to
that observed in the control group of experiment I, in which injections were omitted (group Ic). Experiment III (Period of cortisone treatment overlapping first week of methyicholanthrene applications). The occurrence of skin tumors was significantly accelerated in the group of animals in which the three weeks' period of cortisone administration overlapped the first week of painting with methyicholanthrene (Table II, group lila). During the four periods of observation, first tumors occurred as follows—in the three groups of this experiment (see Table II): (1) First three weeks: No tumor development. (2) Fourth and fifth week: Skin tumors developed in 12 mice of the cortisone group (lila), against two tumor bearers in the "vehicle" group (11Th) and one tumor bearer in the saline group (Tile). (3) Sixth through 16th week: First skin tumors appeared in six mice of the cortisone group, in three of the vehicle group, whereas no tumor appeared in the saline group. (4) Seventeenth through 36th week: Five additional animals showed tumors in the vehicie group, while there were one new tumor bearer among the cortisonetreated—and two among the saline mice.
It is evident, therefore, that the total number of first tumors observed in group lila, in which the cortisone injections were given two weeks prior to— and one week coinciding with the first week of painting with methylcholanthrene, was significantly higher than in the control groups. The number of tumor bearers of the vehicle group (Tub) somewhat exceeded that of the saline group (IhIc), but this difference was not significant statistically. Description of Tumors. Macroscopically, not more than one third of all the mice with skin tumors in experiment I showed tumors with the characteristics of malignancy, i.e. with nodular configuration, solid consistency, a broad base, and indurated surroundings. The other tumors were either papillomatous and pedunculated, or too small for grossidentification. In experiments II and III, two thirds of the total number of tumor bearers showed skin tumors with grossly distinctive features of malignancy.
37
hIc
111c4
37
1h1b3 37
lIla2 39
TO
40
11b0
00
0
T
D7
T° D7
4 0
0 0 0 0 0
TO D7
Period
0 0
Period
9 10
0 0
0 0 0 0
0 0
0
1
0 0 0 0
0 0
1 0
0 1 1 0 2 0 0 0 0 0
0
1
0 0
1 0
11
0 0
0 0
0 0
1
0
0 0
12
0
0 0
0
0
0 0
14
0
0 0 0
0 0
0
0 0
1 1
0 0
13
0 0
0 0
0 0
0 0
0 0
15
0 0
0 0
0 0
0 0
0 0
16
1 0
0
0
0 0
18
0
0
1
0
19
21
22
23
24
25
26 27
28
29
1 0 0
0 1 0
30
31
32
33
34
35
0 0
0
1
0 0
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0 0
0 0
0 0
0 0
0 0
0
0
0 0
1
0
1
0
1
0
1 0 1 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
1
0
0 0
0
0
0
0
0
0 1
0
0 0
0
0
0
1
0 0
0 0
0 1
0
0
0
0 0
0 0
36
5
19
6
5
7
4
4
8
Total
0 010 0 0
0 0 0 0
0 0
1
0
0 0 2 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0
20
0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 0 1 1 0 1 0 0 0 0
0
0
0
0
0
0
17
0
0
0
0
3rd Period
0
0
0
0
0
1
1
4th Period
0
1
0
0
1
0
00000000000 000010000000001000003 0 0 0 0 0 0 0 0 0 0 0 0 0 s
0 0 1 —1t—-' —2nd--÷
0
8
1 0 0 1 0 0 0 0 0 0
1
1
2
0
1
0 0
0
0
7
1 0 1 0 0 0 0 0 1
1
6
8
0
1
1 0
000 01
0
0 1 0 0
1
0
0
0
0
0 0
1
0 0 0 0
0 0
0 0 0 1 0 0
T 00
0
0
0 0
5
3
4
2
D7
D
TO D7
39
No. of Weeks:1 Miceb
ha1
Groups
TABLE II ExperimentII: Three weeks' period of cortisone treatment preceding three weeks' period of methyicholanthrene applications. ExperimentIII: Three weeks' period of cortisone treatment overlapping first of three weeks of methyicholanthrene applications. Occurrence of first skin tumors per week (andweekly death rate of mice without skin tumor)
I-a
Prior to period of injections After period of injSections
Groups:
25.3 22.7
ha lic 25.4 23.7
24.6
26.4 25.1
23.5
11Th
26.0
lila
Average weight of mice in grams
26.4
Ill,
25.6 24.6
Iflc
1 ha: Subcutaneousinjections of cortisone acetate (20 mgm/Kg body weight—Cortone Suspension,Merck & Co.) on five days per week for three consecutive weeks, followed by paintings with methylcholanthrene (0.6%) in benzene for another three weeks. lila: Subcutaneousinjections of cortisone acetate started two weeks prior to painting with methyicholanthrene and continued until end of first week of painting period of three weeks. JIb and Ilib: Control groups—injectionsof suspending liquid of cortisone, instead of cortisone injections. lic and hlic: Control groups—injectionsof physiologicsaline solution, instead of cortisone injections. 6 In most groups, one or more of the initially employed 40 mice died during the injection periods which preceded the applications of methylcholanthrene. • T: Number of mice with (grossly visible) skin tumors. D: Number of mice dead without (grossly visible) tumor development.
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THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
Microscopically, tumor specimens of 31 mice were examined thus far in experiment I; the characteristics of prickle cell carcinoma were found in at least one tumor per animal in 11; and those of sarcoma in one of the 31 mice, whereas benign tumors were found in the remaining 19 animals. These benign tumors were sebaceous adenomas and/or papillomas. Since in experiments II and III gross observation is still in progress and histologic specimens of skin tumors thus far have been procured only from animals which either were killed when there was tumor formation with grossly unmistakable evidence of malignancy—or had died during the later phase of the experiment, specimens with the microscopic features of malignancy were obtained in nearly all of these mice: Of 28 animals, 23 had at least one
tumor with the characteristics of prickle cell epithelioma, three had tumors suspect of prickle cell carcinoma, one was afflicted with sarcoma, and one died with sebaceous adenoma. DISCUSSION
Due to the chosen schedule of methyicholanthrene applications, the number of mice showing a tumor response was relatively small in most of our experimental groups (Tables I and II). As a consequence, the differences observed between the small numbers of tumor-bearers of certain groups present trends, rather than findings of statistical significance. The acceleration in the occurrence of tumors noted after the concomitant treatment with methyicholanthrene and cortisone (experiment I) is in line with the results of our previous experiments (1—3). As was indicated in our previous report (3), comparison of these findings with the seemingly conflicting observations made by other investigators (Boutwell and
Rusch (4)), Engelbrecht-Holm and Asboe-Hansen (5), Zachariae and AsbocHansen (6), Baserga and Shubik (7), Ghadially and Green (8)) brings out the importance of the time of treatment with cortisone in relation to the periods of exposure to the carcinogen and of tumor development; these investigators obtained a reduction in tumor formation by using the steroid not only during the period of tumor induction, but also for a long time thereafter, or even exclusively after the induction period.
The fact that the cortisone administration which was stopped prior to the course of applications of the carcinogen did not result in a significant change in the incident of tumors (experiment II, Table II) rules out the possibility that withdrawal of the steroid was responsible for the accelerated tumor incidence observed in our other cortisone experiments. The result of experiment II appears comparable with the unaltered tumor response observed in a somewhat similar experiment by Ghadially and Green, in which external cortisone applications were followed immediately by exposure to dimethylbenzanthracene and
subsequent paintings with croton oil (8).
The absence of acceleration in the tumor response in experiment II is in striking contrast with the significant acceleration noted in experiment III (Table II, group IIIa)—which reveals the crucial importance of the brief period of coincidence of cortisone administration and carcinogen applications for the occurrence of an accelerated response. Whereas it is unknown whether or not our cortisone injections during the two weeks prior to painting with methyicholanthrene
somehow contributed to the acceleration (experiment III), it is apparent that
FACTORS IN TUMOR INCIDENCE IN MICE. II
419
the injections during the first of the three weeks of painting were of outstanding significance, since certainly no stronger acceleration was observed in the experiment
in which the cortisone injections were combined throughout the three weeks' period of methyicholanthrene painting (experiment I, Table II). In fact, it appears conceivable that an effect comparable with the inhibitory influence of cortisone on growth formation noted by other investigators after prolonged or "late" administration of the steroid (4, 7, 8) may have set in some time during the second or third week after start of the carcinogen applications and thus interfered with further acceleration of the tumor response. The failure of cortisone to bring forth an acceleration in the tumor response, when employed prior to the carcinogen applications (experiment II), affords evidence that whatever factors may be responsible for the acceleration observed in our other experiments, the effect quickly subsides after cessation of the administration of the steroid. Pertinent in
this connection—and outstanding among the factors possibly responsible, appear the peculiar microscopic findings we previously encountered at the pilo-sebaceous organ of animals subjected to simultaneous administration of cortisone and paintings with methylcholanthrene (2, 9). These microscopic findings, in particular a relative inertia in the regrowth of hair during cortisone administration (as compared with follicular hyperactivity in the control animals treated with methylcholanthrene only), did not outlast the period of steroid treatment. Another indication that cortisone is capable of inactivating the hair follicles of rodents in a rapidly reversible manner is Castor and Baker's observation that (male) rats showed complete cessation of hair growth and a decrease in the size of the sebaceous glands during prolonged external applications of cortisone; but a sudden regrowth and return to normal after the 180th day of painting, even though the treatment was continued (10). Our pertinent specific findings regarding the question of an inter-relationship between the effect of cortisone on the hair-follicular cycle and the accelerated tumor response in our experiments will be described in a subsequent report.
The somewhat accelerated tumor response noted in our control groups Tb, 3rd period, and Tub—in which the diluted vehicle of cortisone was injected— in relation to the response obtained in the other control groups (Ic and Tile Tables I and II), was unexpected and is not readily explained. In view of the fact that no similar difference was apparent iii the corresponding groups of experiment II, in which the injections preceded our course of carcinogen applications, the trend observed in the two other experiments may be not a chance occurrence—even though the differences are not statistically significant—but may perhaps be related to the combined treatment with the vehicle and methylcholanthrene. Recent findings described by K. Setälä, H. Setäla and P. Hoisti (11) might well shed light on the mechanism underlying the trend in question. These authors observed a distinct co-carcinogenic effect of a series of surface active compounds, in particular of non-ionic
detergents including Span 20 (sorbitan monolaurate) and Tween 60 (polyoxyethylene sorbitan monostearate). Tween 60 was considerably more effective than Span 20. When applied to the skin of mice for several weeks, these agents produced not only a spectacular tumor promoting effect in animals subjected to a single painting with dimethyl-benzan-
thracene, but also characteristic histologic alterations of the epidermis ("thickening") and cutis in animals not subjected to any treatment with a carcinogen. Although under our experimental conditions presumably only traces of the Tween incorporated in Merck's
420
T}IE JOURNAL OF INVESTIGATIVE DERMATOLOGY
cortisone suspending liquid reached the site of tumor formation, our observation appears to be in line with the findings of Setala and collaborators, and may thus find a rationale. SUMMARY
(1) Significant acceleration in the occurrence of epidermal tumors was evident in mice painted with methyicholanthrene (0.6 %) in benzene three times a week for three consecutive weeks and concomitantly injected with cortisone (20 mgm/ Kg body weight) five times per week during the three weeks of methyicholanthrene application. (2) When cortisone was administered during the first three weeks of the experi-
ment and methyicholanthrene applied during the following three weeks, no significant difference was observed between the tumor response in the animals treated with cortisone and that in the control animals. This finding negates the possibility that the acceleration obtained with cortisone in our earlier experiments was caused by withdrawal of the steroid. (3) The tumor response was significantly accelerated when the last of the three weeks of cortisone administration coincided with the first week of painting with methyicholanthrene. (4) A slight trend toward acceleration in the tumor response was noted in the control groups in which the cortisone dispersing liquid was injected instead of cortisone—and in which the injections were not discontinued prior to treatment with methylcholanthrene. REFERENCES 1. SULZBERGEE, M. B., HEREMANN, F., PICCAGLI, H. AND FRANK, L.: Incidence of epi-
dermal methylcholanthrene tumors in mice after administration of cortisone. Proc. Soc. Exper. Biol. & Med., 82: 673, 1953. 2. PIcCAGLI, H. W., HEERMANN, F., FEAN, L., ROTHSTEIN, M. J., MORRILL, S. D. AND SULZBERGrn, M. B.: On the development of epidermal methylcholanthrene tumors in
mice receiving cortisone. J. Invest. Dermat., 22: 317, 1954. 3. HERUMANN, F., SHERWIN, R. W., MORRILL, S. D., R0TESTEIN, M. J. AND SULZBERGER,
M. B.: Factors influencing the incidence of epidermal methylcholanthrene tumors in mice treated with cortisone. I. External applications of methylcholanthrene combined with cortisone administration and biopsy trauma. J. Invest. Dermat., 25: 403, 1955. 4. BOUTWELL, R. K. AND RUSCH, H. P.: The effect of cortisone on the development of tumors. Proc. Am. Assoc. Cancer Research, 1: 5, 1953. 5. ENGELBBETH-HOLM, J. AND ASBOE-HANSEN, G.: Effect of cortisone on skin carcinogenesis in mice. Acta path. et microbiol. Scandinav., 32: 560, 1953. 6. ZACHABIAR, L. AND ASBOE-HANSEN, G.: Regression of experimental skin tumors in
mice following local injections of 17-hydroxycorticosterone-21-acetate. Cancer Research, 14: 488, 1954. 7. BASERGA, H. AND SHUBIK, P.: The action of cortisone on transplanted and induced tumors in mice. Cancer Research, 14: 12, 1954. 8. GHADIALLY, F. N. AND GREEN, H. N.: The effect of cortisone on chemical carcinogenesis in the mouse skin. Brit. J. Cancer, VIII: 291, 1954. 9. HERRMANN, F., MOBRILL, S. D., SHERWIN, H. W., ROTRSTEIN, M. J. AND SULZBERGEB,
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FACTORS IN TUMOR INCIDENCE IN MICE. ii
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THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
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30. Pearse, A. C. E.: p. 910, Histacheini.stry Thearetscal and Applied, 2nd ed., Churchill, London, 1960.
31. Daniels, F., Jr., Brophy, D. and Lobitz, W. C.: Histochemical responses of human skin fol-
lowing ultraviolet irradiation. J. Invest. Derm.,37: 351, 1961.
32. Bitensky, L.: The demonstration of lysosomes by the controlled temperature freezing section method. Quart. J. Micr. Sci., 103: 205, 1952.
33. Diengdoh, J. V.: The demonstration of lysosomes in mouse skin. Quart. J. Micr. Sci., 105: 73, 1964.
34. Jarret, A., Spearman, R. I. C. and Hardy, J. A.:
Tohoku, J. Exp. Med., 65: Supplement V, 10, 1957.
43. Bitcnsky, L.: Lysosomes in normal and pathological cells, pp. 362—375, Lysasames Eds., de Reuck, A. V. S. and Cameron, M. Churchill, London, 1953.
44. Janoff, A. and Zweifach, B. W.: Production of inflammatory changes in the microcirculation by cationic proteins extracted from lysosomes. J. Exp. Med., 120: 747, 1964.
45. Herion, J. C., Spitznagel, J. K., Walker, R. I. and Zeya, H. I.: Pyrogenicity of granulocyte lysosomes. Amer. J. Physiol., 211: 693, 1966.
46. Baden, H. P. and Pearlman, C.: The effect of ultraviolet light on protein and nucleic acid synthesis in the epidermis. J. Invest. Derm.,
Histochemistry of keratinization. Brit. J. 43: 71, 1964. Derm., 71: 277, 1959. 35. De Duve, C. and Wattiaux, R.: Functions of 47. Bullough, W. S. and Laurence, E. B.: Mitotic control by internal secretion: the role of lysosomes. Ann. Rev. Physiol., 28: 435, 1966. the chalone-adrenalin complex. Exp. Cell. 36. Waravdekar, V. S., Saclaw, L. D., Jones, W. A. and Kuhns, J. C.: Skin changes induced by
Res., 33: 176, 1964.
F. HERRMANN, M.D., R. W. SHERWIN, M.D., S. D. MORRILL, M.D., M. J. ROTHSTEIN, M.D. AND MARION B. SULZBERGER, M.D. OBJECTIVE
In the following we shall report on experiments concerning the question of whether or not the acceleration in the occurrence of epidermal methyicholanthrene tumors, observed by us previously in mice treated with cortisone (1—3), is
dependent on the timing of the administration of cortisone in relation to the carcinogen applications. Among several possibilities, it was, for example, conceivable that this acceleration is not caused by direct action of the steroid, but by
withdrawal of its use; in that case, a stronger acceleration was to be expected from cortisone administration preceding the course of methylcholanthrene
paintings, than from either concomitant or "overlapping" treatments with cortisone and methylcholanthrene. It was the attempt at clarifying this quest i'n and several related points which gave rise to the present study. The observations made in three of our pertinent experiments will be described in this presentation.' METHOD AND MATERIALS
Animals. Female albino mice (383) of the Swiss strain, from Carworth Farms, Inc., New
City, Rockland County, N. Y., three months of age; on unrestricted "Rockland mouse diet" and intake of water. Carcinogen. Methyloholanthrene, 0.6% in benzene. Cortisone. Cortone suspension,2 containing 2.5% cortisone acetate, 0.9% benzyl alcohol—
as preservative, and dispersing material (Tween 80 and Na-carboxy-methylcellulose-— * From the Department of Dermatology and Syphilology of the New York University Post-Graduate Medical School (Dr. Marion B. Sulzberger, Chairman) and the Skin and Cancer Unit of the University Hospital. Thi8 study was made possible by a grant of the United States Public Health Service (U.S.P.H.S.R.G. C 1379). All numerical results and presentations were supervised by Dr. R. R. Freudenthal, statistical consultant, Jackson Heights, N. Y. With the technical assistance of L. Mandol, B.A., L. Frank, B.A., J. Kallman, B.S., M. Cohen, B.S., M. Besen, B.A. and C. Guevara.
Presented at the Sixteenth Annual Meeting of the Society for Investigative Dermatology,
Inc., Atlantic City, N. J., June 5, 1955. 'Another group of pertinent experiments—cortisone administration beginning after, or shortly prior to termination of the methylcholanthrene applications—was started only recently; the results will be reported later. by Merck & Co., Inc., Rahway, N. J. 411
412
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
concentrations unknown) in physiologic saline solution, was diluted with saline solution to obtain a 0.17% solution of cortisone acetate; at each administration, 0.25 ml. of this dilution (containing approximately 20 mgm. cortisone acetate per kg. fasting weight) was injected subcutaneously. The right and left inguinal areas were injected on alternate days. Experimental Design.
Experiment I (Cortisone administration concomitant with applications of methylcholanthrene). The first experiment was designed as a pilot experiment assaying (1) the occurrence of skin tumors in response to only three methyicholanthrene paintings per week for a period of three weeks; and (2) any acceleration of this response after concomitant treatment with cortisone—in order to establish a base-line for appraisal of the results of variations in timing of the course of cortisone injections in relation to that of the carcinogen applications. The procedure of hair clipping at the beginning of the experiment, as well as the technic of painting were the same as described in our previous reports (1—3).
In all mice the shin of the interscapular area was painted with methyicholanthrene in benzene three times per week, for three consecutive weeks. The animals were divided in three groups: Group Ia—49 mice: Cortisone was injected on five consecutive days during each of the three weeks of painting. Control experiments were carried out in the two other groups as follows: Group Ib—48 mice—received injections of the (diluted) vehicle of cortisone (Merck & Co.)—instead of the cortisone injections. Group Ic—46 mice—did not receive any injections. Experimeni8 in which the period of cortisone administration did not coincide with that of the carcinogen applications:
Experiment II (Cortisone treatment prior to carcinogen applications). Group ha: Cortisone was injected in 40 mice on five consecutive days during each of three consecutive weeks. Following termination of the injections, i.e. from the first day of the fourth week of the experiment, methyleholanthrene was applied to the skin of the interscapular region three times a week, for a total of three consecutive weeks. Two other groups of 40 mice each were used for control experiments as follows: Group IIb—Instead of the cortisone injections, injections of the diluted vehicle employed
in Merck's Cortone suspension preceded the three weeks' period of methylcholanthrene applications. Group lie: Physiologic saline solution was injected instead of the cortisone suspension; otherwise the procedure was identical with that employed in groups ha and b.
Experiment III (Period of cortisone treatment overlapping first week of carcinogen applications). Group lila: In 40 mice, cortisone was administered by injection for a period of three weeks, in the manner outlined for our previous experiments. Painting with methyleholanthrene, however, was started at the beginning of the third week of treatment with cortisone and continued for two weeks after termination of the cortisone injections. Control groups: Group 11Th: Forty mice received injections of the (diluted) cortisone dispersing liquid, instead of the cortisone injections; otherwise, the procedure was the same as that in group
lila.
Group life: Forty mice received injections of physiologic saline solution, instead of the cortisone injections; otherwise the procedure did not differ from that employed in group
lila.
FACTORS IN TUMOR INCIDENCE IN MICE. II
413
RESULTS
Incidence of First Skin Tumors.
Experiment I (Concomitant treatment with cortisone and methylcholanthrene). Table I shows the incidence of first skin tumors and the number of mice which died without skin tumors, in each week of the experiment. The experiment was discontinued at the end of the 36th week. The average weight of the mice in each group before and after treatment is recorded below the table; a considerable drop is apparent in the cortisone group, but this weight was
regained two weeks after the termination of treatment.
The overall incidence of skin tumors was low, in particular when compared with the range of the incidence usually observed when methylcholanthrene is applied more frequently or for longer periods of time. Since distinctive rates in the occurrence of first skin tumors were apparent in the three groups during different periods of observation, our total 36 weeks of observation was subdivided into four periods, as indicated by the heavy vertical lines on Table I: (1) First two weeks: No tumor formation. (2) Third, fourth, and fifth week: Skin tumors appeared in eight mice of the cortisone group (Ta), in only one animal of control group Tb—and in two animals of control group Ic. During this period, therefore, the tumor incidence was significantly higher in the cortisone group than in both control groups.
This difference was made even more significant by the death rate of nontumor bearing animals, in as much as eight of the cortisone treated mice died within the first month of the experiment, whereas no death occurred in the control groups during the first two periods of observation. (3) Sixth through 16th week: During these 11 weeks, six of the mice injected with the suspending liquid of Merck's Cortone suspension developed first tumors (group Tb), whereas there was only one new tumor bearer in the cortisone group (Ta) and one in the control group in which injections were omitted (Ic). (4) Seventeenth through 36th week: During this last period, the incidence of first skin tumors was similar in all groups (eight new tumor bearers in group Ia, as well as in group Ic; six new tumors bearers in group Tb). Evidently, the results were considerably more distinctive regarding the time of appearance of first tumors than regarding the final proportion of tumor bearers per group.
Experiments in which the periods of cortisone administration and methyicholanthrene treatment did not coincide:
Table TI shows the weekly number of new tumor bearers and of the mice which died without skin tumors in each week during the course of experiments
II and III.
TABLE
I
T
48
46
Ibf
Jc
0 0
1
0 0
1
0 0
2
1 0
5 4
3
7
8
9 10
11
12
13
14
0
0
0 2 1 0
0 0 0
0 1 0 0 0
0 0 0 0
1 0
0 0
Period
0 0
18
0 0
19
0 0
20
0 0
21
1
1
22
24
25
26
27
28
29
30
31
0 1 0 0 0 1 0 0 0 0 0 0 1 0 0 0 0 0
23
1 1
32
34
35
36 Total
0
0 0 0
0 0 0 0 0 0
0 0
0
0 0 0 3 0 1 0 0 0 0 0 1 0 0 0 —4th Period
2 0 0 0
2 1
—
0 0 1 0
5
11
5
13
0 3 0 1 17 1 0 1 0 14
33
0 0 0 0 0 0 0 0 0 1 0 2 0 2 0 1 0 0 0 0 0 0 0 1 0 1 0 0 1 0 1 0 1 0 0 0 0 0 0 0
0 0
16 17
1 0 0 0 0 0 0 0 0 0 0 0 0 0 1 010 0 0 0 0 0 —* i— 3rd Period
1 0
0 0
15
At beginning of treatment period At end of treatment period
23.7
25.8
Average weight of mice in grams 1a Groups:
26.3 26.7
1W
27.6
26.0
Ic
* Ia: Subcutaneousinjections of cortisone acetate (20 mgm/Kg body weight—Cortone suspension,Merck & Co.) on five days per week, for three consecutive weeks. t Ib: Injectionsof suspendingliquid of cortisone insteadof the cortisone injectionsControl Groups SIc: No injections § T: Number of mice with (grossly visible) skin tumors. ¶ D: Numberof mice dead without (grossly visible) tumor development.
Penod
6
1 0 0 1 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0
5
0 0 0 0
2
4
T 00 00 20 00 00 D —1st-- -- 2nd
D1[
Dlf
T
49
Ia
Weeks:1
of Mice
Groups
No.
Experiment I: External treatment with methyicholanthrene 0.6% in benzene for three weeks. Concomitant injections of cortisone acetate. Occurrence of first skin tumors per week (and weekly death rate of mice without skin tumor)
FACTORS IN TUMOR INCIDENCE IN MICE. II
415
The average weights of the different groups of the mice, recorded at the end of the table,
showed a decline after the injection periods, especially in the cortisone-treated groups (ha and lila); but this weight was regained two weeks after termination of the injections.
The total period of observation was again subdivided into four periods in order to emphasize the pertinent differences in the incidence of first tumors observed in the individual groups.
Experiment II (Cortisone treatment prior to methyicholanthrene applications). There was no significant difference between the tumor incidence observed in the group in which the course of cortisone injections was followed by exposure to the carcinogen (ha) and the incidence observed in either one of the two control groups (JIb and TIc—injected with the plain vehicle of Merck's cortisone suspension and with saline solution, respectively—prior to the carcinogen applications). In all three groups, the incidence showed a "scattered" pattern similar to
that observed in the control group of experiment I, in which injections were omitted (group Ic). Experiment III (Period of cortisone treatment overlapping first week of methyicholanthrene applications). The occurrence of skin tumors was significantly accelerated in the group of animals in which the three weeks' period of cortisone administration overlapped the first week of painting with methyicholanthrene (Table II, group lila). During the four periods of observation, first tumors occurred as follows—in the three groups of this experiment (see Table II): (1) First three weeks: No tumor development. (2) Fourth and fifth week: Skin tumors developed in 12 mice of the cortisone group (lila), against two tumor bearers in the "vehicle" group (11Th) and one tumor bearer in the saline group (Tile). (3) Sixth through 16th week: First skin tumors appeared in six mice of the cortisone group, in three of the vehicle group, whereas no tumor appeared in the saline group. (4) Seventeenth through 36th week: Five additional animals showed tumors in the vehicie group, while there were one new tumor bearer among the cortisonetreated—and two among the saline mice.
It is evident, therefore, that the total number of first tumors observed in group lila, in which the cortisone injections were given two weeks prior to— and one week coinciding with the first week of painting with methylcholanthrene, was significantly higher than in the control groups. The number of tumor bearers of the vehicle group (Tub) somewhat exceeded that of the saline group (IhIc), but this difference was not significant statistically. Description of Tumors. Macroscopically, not more than one third of all the mice with skin tumors in experiment I showed tumors with the characteristics of malignancy, i.e. with nodular configuration, solid consistency, a broad base, and indurated surroundings. The other tumors were either papillomatous and pedunculated, or too small for grossidentification. In experiments II and III, two thirds of the total number of tumor bearers showed skin tumors with grossly distinctive features of malignancy.
37
hIc
111c4
37
1h1b3 37
lIla2 39
TO
40
11b0
00
0
T
D7
T° D7
4 0
0 0 0 0 0
TO D7
Period
0 0
Period
9 10
0 0
0 0 0 0
0 0
0
1
0 0 0 0
0 0
1 0
0 1 1 0 2 0 0 0 0 0
0
1
0 0
1 0
11
0 0
0 0
0 0
1
0
0 0
12
0
0 0
0
0
0 0
14
0
0 0 0
0 0
0
0 0
1 1
0 0
13
0 0
0 0
0 0
0 0
0 0
15
0 0
0 0
0 0
0 0
0 0
16
1 0
0
0
0 0
18
0
0
1
0
19
21
22
23
24
25
26 27
28
29
1 0 0
0 1 0
30
31
32
33
34
35
0 0
0
1
0 0
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0 0
0 0
0 0
0 0
0 0
0
0
0 0
1
0
1
0
1
0
1 0 1 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
1
0
0 0
0
0
0
0
0
0 1
0
0 0
0
0
0
1
0 0
0 0
0 1
0
0
0
0 0
0 0
36
5
19
6
5
7
4
4
8
Total
0 010 0 0
0 0 0 0
0 0
1
0
0 0 2 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0
20
0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 0 1 1 0 1 0 0 0 0
0
0
0
0
0
0
17
0
0
0
0
3rd Period
0
0
0
0
0
1
1
4th Period
0
1
0
0
1
0
00000000000 000010000000001000003 0 0 0 0 0 0 0 0 0 0 0 0 0 s
0 0 1 —1t—-' —2nd--÷
0
8
1 0 0 1 0 0 0 0 0 0
1
1
2
0
1
0 0
0
0
7
1 0 1 0 0 0 0 0 1
1
6
8
0
1
1 0
000 01
0
0 1 0 0
1
0
0
0
0
0 0
1
0 0 0 0
0 0
0 0 0 1 0 0
T 00
0
0
0 0
5
3
4
2
D7
D
TO D7
39
No. of Weeks:1 Miceb
ha1
Groups
TABLE II ExperimentII: Three weeks' period of cortisone treatment preceding three weeks' period of methyicholanthrene applications. ExperimentIII: Three weeks' period of cortisone treatment overlapping first of three weeks of methyicholanthrene applications. Occurrence of first skin tumors per week (andweekly death rate of mice without skin tumor)
I-a
Prior to period of injections After period of injSections
Groups:
25.3 22.7
ha lic 25.4 23.7
24.6
26.4 25.1
23.5
11Th
26.0
lila
Average weight of mice in grams
26.4
Ill,
25.6 24.6
Iflc
1 ha: Subcutaneousinjections of cortisone acetate (20 mgm/Kg body weight—Cortone Suspension,Merck & Co.) on five days per week for three consecutive weeks, followed by paintings with methylcholanthrene (0.6%) in benzene for another three weeks. lila: Subcutaneousinjections of cortisone acetate started two weeks prior to painting with methyicholanthrene and continued until end of first week of painting period of three weeks. JIb and Ilib: Control groups—injectionsof suspending liquid of cortisone, instead of cortisone injections. lic and hlic: Control groups—injectionsof physiologicsaline solution, instead of cortisone injections. 6 In most groups, one or more of the initially employed 40 mice died during the injection periods which preceded the applications of methylcholanthrene. • T: Number of mice with (grossly visible) skin tumors. D: Number of mice dead without (grossly visible) tumor development.
418
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
Microscopically, tumor specimens of 31 mice were examined thus far in experiment I; the characteristics of prickle cell carcinoma were found in at least one tumor per animal in 11; and those of sarcoma in one of the 31 mice, whereas benign tumors were found in the remaining 19 animals. These benign tumors were sebaceous adenomas and/or papillomas. Since in experiments II and III gross observation is still in progress and histologic specimens of skin tumors thus far have been procured only from animals which either were killed when there was tumor formation with grossly unmistakable evidence of malignancy—or had died during the later phase of the experiment, specimens with the microscopic features of malignancy were obtained in nearly all of these mice: Of 28 animals, 23 had at least one
tumor with the characteristics of prickle cell epithelioma, three had tumors suspect of prickle cell carcinoma, one was afflicted with sarcoma, and one died with sebaceous adenoma. DISCUSSION
Due to the chosen schedule of methyicholanthrene applications, the number of mice showing a tumor response was relatively small in most of our experimental groups (Tables I and II). As a consequence, the differences observed between the small numbers of tumor-bearers of certain groups present trends, rather than findings of statistical significance. The acceleration in the occurrence of tumors noted after the concomitant treatment with methyicholanthrene and cortisone (experiment I) is in line with the results of our previous experiments (1—3). As was indicated in our previous report (3), comparison of these findings with the seemingly conflicting observations made by other investigators (Boutwell and
Rusch (4)), Engelbrecht-Holm and Asboe-Hansen (5), Zachariae and AsbocHansen (6), Baserga and Shubik (7), Ghadially and Green (8)) brings out the importance of the time of treatment with cortisone in relation to the periods of exposure to the carcinogen and of tumor development; these investigators obtained a reduction in tumor formation by using the steroid not only during the period of tumor induction, but also for a long time thereafter, or even exclusively after the induction period.
The fact that the cortisone administration which was stopped prior to the course of applications of the carcinogen did not result in a significant change in the incident of tumors (experiment II, Table II) rules out the possibility that withdrawal of the steroid was responsible for the accelerated tumor incidence observed in our other cortisone experiments. The result of experiment II appears comparable with the unaltered tumor response observed in a somewhat similar experiment by Ghadially and Green, in which external cortisone applications were followed immediately by exposure to dimethylbenzanthracene and
subsequent paintings with croton oil (8).
The absence of acceleration in the tumor response in experiment II is in striking contrast with the significant acceleration noted in experiment III (Table II, group IIIa)—which reveals the crucial importance of the brief period of coincidence of cortisone administration and carcinogen applications for the occurrence of an accelerated response. Whereas it is unknown whether or not our cortisone injections during the two weeks prior to painting with methyicholanthrene
somehow contributed to the acceleration (experiment III), it is apparent that
FACTORS IN TUMOR INCIDENCE IN MICE. II
419
the injections during the first of the three weeks of painting were of outstanding significance, since certainly no stronger acceleration was observed in the experiment
in which the cortisone injections were combined throughout the three weeks' period of methyicholanthrene painting (experiment I, Table II). In fact, it appears conceivable that an effect comparable with the inhibitory influence of cortisone on growth formation noted by other investigators after prolonged or "late" administration of the steroid (4, 7, 8) may have set in some time during the second or third week after start of the carcinogen applications and thus interfered with further acceleration of the tumor response. The failure of cortisone to bring forth an acceleration in the tumor response, when employed prior to the carcinogen applications (experiment II), affords evidence that whatever factors may be responsible for the acceleration observed in our other experiments, the effect quickly subsides after cessation of the administration of the steroid. Pertinent in
this connection—and outstanding among the factors possibly responsible, appear the peculiar microscopic findings we previously encountered at the pilo-sebaceous organ of animals subjected to simultaneous administration of cortisone and paintings with methylcholanthrene (2, 9). These microscopic findings, in particular a relative inertia in the regrowth of hair during cortisone administration (as compared with follicular hyperactivity in the control animals treated with methylcholanthrene only), did not outlast the period of steroid treatment. Another indication that cortisone is capable of inactivating the hair follicles of rodents in a rapidly reversible manner is Castor and Baker's observation that (male) rats showed complete cessation of hair growth and a decrease in the size of the sebaceous glands during prolonged external applications of cortisone; but a sudden regrowth and return to normal after the 180th day of painting, even though the treatment was continued (10). Our pertinent specific findings regarding the question of an inter-relationship between the effect of cortisone on the hair-follicular cycle and the accelerated tumor response in our experiments will be described in a subsequent report.
The somewhat accelerated tumor response noted in our control groups Tb, 3rd period, and Tub—in which the diluted vehicle of cortisone was injected— in relation to the response obtained in the other control groups (Ic and Tile Tables I and II), was unexpected and is not readily explained. In view of the fact that no similar difference was apparent iii the corresponding groups of experiment II, in which the injections preceded our course of carcinogen applications, the trend observed in the two other experiments may be not a chance occurrence—even though the differences are not statistically significant—but may perhaps be related to the combined treatment with the vehicle and methylcholanthrene. Recent findings described by K. Setälä, H. Setäla and P. Hoisti (11) might well shed light on the mechanism underlying the trend in question. These authors observed a distinct co-carcinogenic effect of a series of surface active compounds, in particular of non-ionic
detergents including Span 20 (sorbitan monolaurate) and Tween 60 (polyoxyethylene sorbitan monostearate). Tween 60 was considerably more effective than Span 20. When applied to the skin of mice for several weeks, these agents produced not only a spectacular tumor promoting effect in animals subjected to a single painting with dimethyl-benzan-
thracene, but also characteristic histologic alterations of the epidermis ("thickening") and cutis in animals not subjected to any treatment with a carcinogen. Although under our experimental conditions presumably only traces of the Tween incorporated in Merck's
420
T}IE JOURNAL OF INVESTIGATIVE DERMATOLOGY
cortisone suspending liquid reached the site of tumor formation, our observation appears to be in line with the findings of Setala and collaborators, and may thus find a rationale. SUMMARY
(1) Significant acceleration in the occurrence of epidermal tumors was evident in mice painted with methyicholanthrene (0.6 %) in benzene three times a week for three consecutive weeks and concomitantly injected with cortisone (20 mgm/ Kg body weight) five times per week during the three weeks of methyicholanthrene application. (2) When cortisone was administered during the first three weeks of the experi-
ment and methyicholanthrene applied during the following three weeks, no significant difference was observed between the tumor response in the animals treated with cortisone and that in the control animals. This finding negates the possibility that the acceleration obtained with cortisone in our earlier experiments was caused by withdrawal of the steroid. (3) The tumor response was significantly accelerated when the last of the three weeks of cortisone administration coincided with the first week of painting with methyicholanthrene. (4) A slight trend toward acceleration in the tumor response was noted in the control groups in which the cortisone dispersing liquid was injected instead of cortisone—and in which the injections were not discontinued prior to treatment with methylcholanthrene. REFERENCES 1. SULZBERGEE, M. B., HEREMANN, F., PICCAGLI, H. AND FRANK, L.: Incidence of epi-
dermal methylcholanthrene tumors in mice after administration of cortisone. Proc. Soc. Exper. Biol. & Med., 82: 673, 1953. 2. PIcCAGLI, H. W., HEERMANN, F., FEAN, L., ROTHSTEIN, M. J., MORRILL, S. D. AND SULZBERGrn, M. B.: On the development of epidermal methylcholanthrene tumors in
mice receiving cortisone. J. Invest. Dermat., 22: 317, 1954. 3. HERUMANN, F., SHERWIN, R. W., MORRILL, S. D., R0TESTEIN, M. J. AND SULZBERGER,
M. B.: Factors influencing the incidence of epidermal methylcholanthrene tumors in mice treated with cortisone. I. External applications of methylcholanthrene combined with cortisone administration and biopsy trauma. J. Invest. Dermat., 25: 403, 1955. 4. BOUTWELL, R. K. AND RUSCH, H. P.: The effect of cortisone on the development of tumors. Proc. Am. Assoc. Cancer Research, 1: 5, 1953. 5. ENGELBBETH-HOLM, J. AND ASBOE-HANSEN, G.: Effect of cortisone on skin carcinogenesis in mice. Acta path. et microbiol. Scandinav., 32: 560, 1953. 6. ZACHABIAR, L. AND ASBOE-HANSEN, G.: Regression of experimental skin tumors in
mice following local injections of 17-hydroxycorticosterone-21-acetate. Cancer Research, 14: 488, 1954. 7. BASERGA, H. AND SHUBIK, P.: The action of cortisone on transplanted and induced tumors in mice. Cancer Research, 14: 12, 1954. 8. GHADIALLY, F. N. AND GREEN, H. N.: The effect of cortisone on chemical carcinogenesis in the mouse skin. Brit. J. Cancer, VIII: 291, 1954. 9. HERRMANN, F., MOBRILL, S. D., SHERWIN, H. W., ROTRSTEIN, M. J. AND SULZBERGEB,
M. B.: Factors influencing the incidence of epidermal methylcholanthrene tumors in mice treated with cortisone. III. Studies of the hair follicular cycle ("skin cycle") in relation to the incidence of tumors after cortisone administration. Read at the Six-
FACTORS IN TUMOR INCIDENCE IN MICE. ii
421
teenth Annual Meeting of The Society for Investigative Dermatology, Inc., Atlantic
City, N. J., June 5, 1955; J. Invest. Dermat., 25: 423, 1955. 10. CASTOR, C. W. AND BAKER, B. L.: The local action of adrenocortical steroids on epi. dermis and connective tissue of the skin. Endocrinology, 47: 234, 1950. 11. SETALA, K., SETALA, H. AND HoLsrr, P.: A new and physicochemically well-defined
group of tumor-promoting (cocarcinogenic) agents for mouse skin. Science, 120: 1075, 1954.
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
94
linolenic acid extract. Arch. This pdf is a scanned copy UV of irradiated a printed document.
24. Wynn, C. H. and Iqbal, M.: Isolation of rat
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