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The effects of X-irradiation and adriamycin on proliferating and nonproliferating hair coat of the mouse
Int. J. Rudiurion Oncology Bid. Phys., Vol. 5, pp. 1261-1264 o Pergamon Press Ltd., 1979. Printed in the U.S.A.
??Adriamycin
THE EFFECTS OF X-IRRADIATION AND ADRIAMYCIN ON PROLIFERATING AND NONPROLIFERATING HAIR COAT OF THE MOUSEt M. L. GRIEM,
M.D., G. S. DIMITRIEVICH, R. M. LEE, B.S.
M.S.,
A.M.
and
Department of Radiology, University of Chicago, Chicago, IL 60637, U.S.A. Dose-response curves for proliferating, nonproliferating, and nonproliferating hair coat stimulated 24 hours after treatment show similar D,, of 135 rad and a D,, of 500, 900, and 1400 rad respectively. Using a priming dose of 650 rad X-rays, a drug-dose-response curve was estahlished in the proliferating hair coat. Drugdose-response curve was estahlished for the nonproliferating, stimulated coat with priming doses of 1200 and 1400 rad. NO drug effect could he seen in the nonproliferating hair coat left unstimulated. Cyclic interaction hetween drug and irradiation was found in the proliferating hair coat. Adriamycin,
Radiotherapy,
Chemotherapy,
Proliferating
INTRODUCTION We have evaluated chemotherapeutic drugs in a in proliferative normal tissue with wide variations activity. The rapidly proliferating induced by plucking stimulation. mice, after 18 days, proliferative
anagen
tissue, Nonproliferating
tissue.
coat conditions (see Fig. 1). Fourteen days after plucking, graded doses of drug and graded doses of irradiation were tested with varying drug dose, radiation dose and time interval between drug and irradiation. The drug was administered intravenously by tail vein route. Irradiation was administered with 45 KV X-rays with a focal-skin distance of ll.5 cm and with a 2 mm added aluminum filtration. A 2 cm circle was irradiated. The animals were unanesthetized and the side being irradiated was randomized. The mice were prepared by gently clipping the hair coat and they were scored for hair density by photography, using dark field epi-illumination and a 4x objective. Eighteen photographs were made on the nonirradiated side and 18 on the X-irradiated side. There are about 10,000 hairs in the 2 cm diameter test area. Linagraph Shellburst film was used and processed through an X-Omat film processor. Each untreated hair density photograph has about 100 hair per frame or 42 hair per mmz. In the unstimulated telogen experiments, the mice were plucked, and on the 21st day after plucking, drugs and irradiation were administered in a fashion similar to the anagen experiments detailed. On the 41st day after plucking, the hair coat, which had been unperturbed, was gently clipped and hair density measurements were made. A third series of ex-
hair coat is
In our strain of activity ceases and a nonproliferating telogen state lasts for a period of approximately one month, when spontaneously the anagen hair coat again occurs.‘,’ The resting or telogen hair coat can be restimulated by a second plucking during the resting phase which, again, induces the proliferating coat.
METHODS AND MATERIALS Carworth Farms #l female mice, 12 weeks of were age, were used. The backs and haunches plucked. Those mice which could not be plucked cleanly were discarded at this point, since this is an indication that the mouse hair coat is already in the anagen phase. The animals were doubly ear-tagged and assigned to cages-each containing one animal from each treatment group. Five animals per point were used. Data analysis used a second-order analysis of variante. We looked for cage and treatment effect and the standard error of the mean was calculated for each treatment group. Adriamycin and radiation effects were studied in \~it~o in three hair i-This research was supported by grant No. CA 20692 from the National Cancer Institute and was conducted in the University of Chicago Cancer Research Center sup-
ported by grant No. CA 14599. Reprint requests to: M. L. Griem. M.D. 1261
Radiation Oncology 0 Biology 0 Physics
1262
ANAGEN-PROLIFERATING 0
45
EXPERIMENTS:
14 DAYS
KVp
X-RAYS
21 OAYS
t PLUCK
4ugust 1979, Volume 5, Number 8
I TREATMENT
PHOTOGRAPH
TELOGEN-NON-PROLIFERATING
t+
UNS-WAULATED
+___+
TELOGEN STIMULATEO 24 HRS AFTER X-RAYS ANAGEN
W
TELOGEN
EXPERIMENTS:
21 DAYS
0
41 DAYS I I PHOTOGRAPH
t PLUCK
TREATMENT
s-
TELOGEN-STIMULATED
i?.-
EXPERIMENTS:
21 22 DAYS
0
36
OAYS
i 7G-
PLUCK
TREATdNT
PLUCK
(STIMULUS)
PHOTOGRAPH
s4-
Fig. 1. Experimental
design.
3-
Z-
the unstimulated telogen experof plucking, drug, and radiation treatment on the 21st day after plucking. Twentyfour hours later the resting, treated hair coat was stimulated by plucking. On the 41st day after the first plucking (and after gentle clipping) hair density photographs in the treated and untreated areas were made. periments, similar iments, consisted
to
10-L 0
500
1000
Fig. 2. Dose-response
IN
2000
2500
RAD
curves for irradiation.
ANAGEN
RESULTS Figure 2 demonstrates the dose-response curves for graded doses of irradiation administered in the three hair coat states described. For the anagen hair coat, the Do is 135 rad and the D, is 500 rad, For the unstimulated telogen hair coat, the D, is approximately 135 rad and the D, is 1400 rad. For the stimulated telogen, the D, is approximately 135 rad and the D, is 900 rad intermediate in radiosensitivity between the proliferating and nonproliferating states. By injecting drugs one hom prior to a priming dose of 650 rad, a drug-dose-response curve was made for the anagen hair coat. Graded doses of drug produced the hair density measurements in Fig. 3. With the anagen hair coat, the time interval between drug administration and irradiation was varied. Two experiments were conducted with the drug administered prior to irradiation and one was made with the drug administered after irradiation. The drug dose was 20 mg/m2. Figure 4, a composite of those three experiments, shows the cyclic interaction between drug and irradiation. A maximum hair loss was observed when the drug was administered severa1 hours prior to irradiation. Using a priming dose of 1600 rad, graded doses of Adriamycin were administered to groups of mice. The drug-dose-response curve in Fig. 5 was deter-
1500
DOSE
HAIR
100 9 6 ‘1
I 650
RAD
X -RAY
ADRIAMYCIN
4-
(660
RAD
PRIMING
DOSE-RESPONSE X-RAY
GIVEN
DOSE CURVE WITH
DRUG1
3-
2-
ADRIAMYCIN DOSE ( I.V., SINGLE INJECTION,mg/m*)
Fig. 3. Dose-response
curve for drug in the anagen
hair
Effects of X-irradiation
ANAGEN
GRIEM
RAD
ct
1263
cl/.
UNSTIMULATED
HAIR
ADRIAMYCIN 650
and Adriamycin 0
I.V
TELOGEN
HAIR
ZOmg/m*
,oi_____
X-RAY
_-.?---------_D.
T-( 6-
5-
.
4-
3,600
x 2-
RAD
X-RPIY
C--.
EXPERIMENT
1
-
EXPERIMENT
11
PRIMING
DOSE
10-‘9:6543*-
10
I
10-2’
12
10
6
6
4
2
0
2
4
HOUW
Fig. 4. Tempora1
response of the anagen hair coat when
the time between
drug and irradiation
STIMULATED
I
TELOGEN
is varied.
HAIR
.
1200
X-RAY PRIMING
DOSE
o
1400 RAD X-RAY PRIMING Telogen Stimuloted 24 t.r. after X-RAY
RAD
DOSE
10-2 1
10-3
( 0
l
I
l
30
60
90
ADRIAMYCIN DOSE SINGLE INJECTION,
Fig. 6. Dose-response
(I.V., mg/m2)
curve for drug in the stimulated telogen hair coat.
20
30
ADRIAMYCIN DOSE SINGLE INJECTION,
Fig. 5. Dose-response
40
50
60
(1. V., mg/m*)
curve for drug in the unstimulated telogen hair coat.
mined. Two experiments were conducted. NO drug effect was observed even at the highest doses of drug tested. With the stimulated telogen hair coat, a priming dose of 1200 and 1400 rad of X-ray was administered, and graded doses of drug given one hour prior to irradiation. Drug-dose-response curves were seen in these as demonstrated in Fig. 6. DISCUSSION Cyclic interaction between drug and irradiation is seen in the proliferating hair coat with a maximum hair loss observed when the drug is administered several hours prior to irradiation (Fig. 4). This is similar to the observations made by Phillips et al. on gut epithelium and presented in this symposium. However, we were unable to get a drug-doseresponse curve in the unstimulated, unperturbed telogen hair coat. A possible explanation for the above might be that the resting hair follicle matrix cells do not receive the drug at the time of irradiation, since the follicle resides closer to the skin surface and may be avascular. In the final series of experiments (Fig. 6), however, the follicle in the unstimulated state was treated with drug and X-irradiation, and stimulated 24 hours afterwards. The results showed frank drug-dose-response effects. It may wel1 be that in the
1264
Radiation
Oncology
0
Biology
0 Physics
unperturbed state the drug effect is gradually repaired, thus accounting for the observations we have presented in the unstimulated telogen experiments. The observations reported by Twentymen and Kallman in this symposium on three animal tumors show very little drug-radiation interaction. If our unstimulated telogen observations represent a non-
August
1979, Volume
5, Number
8
proliferating or G,, state, tumors with a low growth fraction may likewise show very little drugirradiation interaction. We plan to extend observations on the unstimulated hair coat by changing the time of the growth stimulus to study further this very interesting phenomenon.
REFERENCES the hair follicle. In Ad\~rnw.s in Radicltion Re.srurch. 1. Dry, F.W.: The coat of the mouse (Mus musculus). J. Biologie md Mrdicinr, Vol. 2, ed. by Duplan, J.T., Genrt. 16: 287-340, 1926. Chapiro, A., New York, Gordon & Breach Scientific 2. Fry, R.J.M., Weber, C.L., Kisieleski, W.E., Griem, Publishers, 1973, pp. 853-860. M.L., Malkinson, F.D.: Study of cel1 prohferation of
??Adriamycin
THE EFFECTS OF X-IRRADIATION AND ADRIAMYCIN ON PROLIFERATING AND NONPROLIFERATING HAIR COAT OF THE MOUSEt M. L. GRIEM,
M.D., G. S. DIMITRIEVICH, R. M. LEE, B.S.
M.S.,
A.M.
and
Department of Radiology, University of Chicago, Chicago, IL 60637, U.S.A. Dose-response curves for proliferating, nonproliferating, and nonproliferating hair coat stimulated 24 hours after treatment show similar D,, of 135 rad and a D,, of 500, 900, and 1400 rad respectively. Using a priming dose of 650 rad X-rays, a drug-dose-response curve was estahlished in the proliferating hair coat. Drugdose-response curve was estahlished for the nonproliferating, stimulated coat with priming doses of 1200 and 1400 rad. NO drug effect could he seen in the nonproliferating hair coat left unstimulated. Cyclic interaction hetween drug and irradiation was found in the proliferating hair coat. Adriamycin,
Radiotherapy,
Chemotherapy,
Proliferating
INTRODUCTION We have evaluated chemotherapeutic drugs in a in proliferative normal tissue with wide variations activity. The rapidly proliferating induced by plucking stimulation. mice, after 18 days, proliferative
anagen
tissue, Nonproliferating
tissue.
coat conditions (see Fig. 1). Fourteen days after plucking, graded doses of drug and graded doses of irradiation were tested with varying drug dose, radiation dose and time interval between drug and irradiation. The drug was administered intravenously by tail vein route. Irradiation was administered with 45 KV X-rays with a focal-skin distance of ll.5 cm and with a 2 mm added aluminum filtration. A 2 cm circle was irradiated. The animals were unanesthetized and the side being irradiated was randomized. The mice were prepared by gently clipping the hair coat and they were scored for hair density by photography, using dark field epi-illumination and a 4x objective. Eighteen photographs were made on the nonirradiated side and 18 on the X-irradiated side. There are about 10,000 hairs in the 2 cm diameter test area. Linagraph Shellburst film was used and processed through an X-Omat film processor. Each untreated hair density photograph has about 100 hair per frame or 42 hair per mmz. In the unstimulated telogen experiments, the mice were plucked, and on the 21st day after plucking, drugs and irradiation were administered in a fashion similar to the anagen experiments detailed. On the 41st day after plucking, the hair coat, which had been unperturbed, was gently clipped and hair density measurements were made. A third series of ex-
hair coat is
In our strain of activity ceases and a nonproliferating telogen state lasts for a period of approximately one month, when spontaneously the anagen hair coat again occurs.‘,’ The resting or telogen hair coat can be restimulated by a second plucking during the resting phase which, again, induces the proliferating coat.
METHODS AND MATERIALS Carworth Farms #l female mice, 12 weeks of were age, were used. The backs and haunches plucked. Those mice which could not be plucked cleanly were discarded at this point, since this is an indication that the mouse hair coat is already in the anagen phase. The animals were doubly ear-tagged and assigned to cages-each containing one animal from each treatment group. Five animals per point were used. Data analysis used a second-order analysis of variante. We looked for cage and treatment effect and the standard error of the mean was calculated for each treatment group. Adriamycin and radiation effects were studied in \~it~o in three hair i-This research was supported by grant No. CA 20692 from the National Cancer Institute and was conducted in the University of Chicago Cancer Research Center sup-
ported by grant No. CA 14599. Reprint requests to: M. L. Griem. M.D. 1261
Radiation Oncology 0 Biology 0 Physics
1262
ANAGEN-PROLIFERATING 0
45
EXPERIMENTS:
14 DAYS
KVp
X-RAYS
21 OAYS
t PLUCK
4ugust 1979, Volume 5, Number 8
I TREATMENT
PHOTOGRAPH
TELOGEN-NON-PROLIFERATING
t+
UNS-WAULATED
+___+
TELOGEN STIMULATEO 24 HRS AFTER X-RAYS ANAGEN
W
TELOGEN
EXPERIMENTS:
21 DAYS
0
41 DAYS I I PHOTOGRAPH
t PLUCK
TREATMENT
s-
TELOGEN-STIMULATED
i?.-
EXPERIMENTS:
21 22 DAYS
0
36
OAYS
i 7G-
PLUCK
TREATdNT
PLUCK
(STIMULUS)
PHOTOGRAPH
s4-
Fig. 1. Experimental
design.
3-
Z-
the unstimulated telogen experof plucking, drug, and radiation treatment on the 21st day after plucking. Twentyfour hours later the resting, treated hair coat was stimulated by plucking. On the 41st day after the first plucking (and after gentle clipping) hair density photographs in the treated and untreated areas were made. periments, similar iments, consisted
to
10-L 0
500
1000
Fig. 2. Dose-response
IN
2000
2500
RAD
curves for irradiation.
ANAGEN
RESULTS Figure 2 demonstrates the dose-response curves for graded doses of irradiation administered in the three hair coat states described. For the anagen hair coat, the Do is 135 rad and the D, is 500 rad, For the unstimulated telogen hair coat, the D, is approximately 135 rad and the D, is 1400 rad. For the stimulated telogen, the D, is approximately 135 rad and the D, is 900 rad intermediate in radiosensitivity between the proliferating and nonproliferating states. By injecting drugs one hom prior to a priming dose of 650 rad, a drug-dose-response curve was made for the anagen hair coat. Graded doses of drug produced the hair density measurements in Fig. 3. With the anagen hair coat, the time interval between drug administration and irradiation was varied. Two experiments were conducted with the drug administered prior to irradiation and one was made with the drug administered after irradiation. The drug dose was 20 mg/m2. Figure 4, a composite of those three experiments, shows the cyclic interaction between drug and irradiation. A maximum hair loss was observed when the drug was administered severa1 hours prior to irradiation. Using a priming dose of 1600 rad, graded doses of Adriamycin were administered to groups of mice. The drug-dose-response curve in Fig. 5 was deter-
1500
DOSE
HAIR
100 9 6 ‘1
I 650
RAD
X -RAY
ADRIAMYCIN
4-
(660
RAD
PRIMING
DOSE-RESPONSE X-RAY
GIVEN
DOSE CURVE WITH
DRUG1
3-
2-
ADRIAMYCIN DOSE ( I.V., SINGLE INJECTION,mg/m*)
Fig. 3. Dose-response
curve for drug in the anagen
hair
Effects of X-irradiation
ANAGEN
GRIEM
RAD
ct
1263
cl/.
UNSTIMULATED
HAIR
ADRIAMYCIN 650
and Adriamycin 0
I.V
TELOGEN
HAIR
ZOmg/m*
,oi_____
X-RAY
_-.?---------_D.
T-( 6-
5-
.
4-
3,600
x 2-
RAD
X-RPIY
C--.
EXPERIMENT
1
-
EXPERIMENT
11
PRIMING
DOSE
10-‘9:6543*-
10
I
10-2’
12
10
6
6
4
2
0
2
4
HOUW
Fig. 4. Tempora1
response of the anagen hair coat when
the time between
drug and irradiation
STIMULATED
I
TELOGEN
is varied.
HAIR
.
1200
X-RAY PRIMING
DOSE
o
1400 RAD X-RAY PRIMING Telogen Stimuloted 24 t.r. after X-RAY
RAD
DOSE
10-2 1
10-3
( 0
l
I
l
30
60
90
ADRIAMYCIN DOSE SINGLE INJECTION,
Fig. 6. Dose-response
(I.V., mg/m2)
curve for drug in the stimulated telogen hair coat.
20
30
ADRIAMYCIN DOSE SINGLE INJECTION,
Fig. 5. Dose-response
40
50
60
(1. V., mg/m*)
curve for drug in the unstimulated telogen hair coat.
mined. Two experiments were conducted. NO drug effect was observed even at the highest doses of drug tested. With the stimulated telogen hair coat, a priming dose of 1200 and 1400 rad of X-ray was administered, and graded doses of drug given one hour prior to irradiation. Drug-dose-response curves were seen in these as demonstrated in Fig. 6. DISCUSSION Cyclic interaction between drug and irradiation is seen in the proliferating hair coat with a maximum hair loss observed when the drug is administered several hours prior to irradiation (Fig. 4). This is similar to the observations made by Phillips et al. on gut epithelium and presented in this symposium. However, we were unable to get a drug-doseresponse curve in the unstimulated, unperturbed telogen hair coat. A possible explanation for the above might be that the resting hair follicle matrix cells do not receive the drug at the time of irradiation, since the follicle resides closer to the skin surface and may be avascular. In the final series of experiments (Fig. 6), however, the follicle in the unstimulated state was treated with drug and X-irradiation, and stimulated 24 hours afterwards. The results showed frank drug-dose-response effects. It may wel1 be that in the
1264
Radiation
Oncology
0
Biology
0 Physics
unperturbed state the drug effect is gradually repaired, thus accounting for the observations we have presented in the unstimulated telogen experiments. The observations reported by Twentymen and Kallman in this symposium on three animal tumors show very little drug-radiation interaction. If our unstimulated telogen observations represent a non-
August
1979, Volume
5, Number
8
proliferating or G,, state, tumors with a low growth fraction may likewise show very little drugirradiation interaction. We plan to extend observations on the unstimulated hair coat by changing the time of the growth stimulus to study further this very interesting phenomenon.
REFERENCES the hair follicle. In Ad\~rnw.s in Radicltion Re.srurch. 1. Dry, F.W.: The coat of the mouse (Mus musculus). J. Biologie md Mrdicinr, Vol. 2, ed. by Duplan, J.T., Genrt. 16: 287-340, 1926. Chapiro, A., New York, Gordon & Breach Scientific 2. Fry, R.J.M., Weber, C.L., Kisieleski, W.E., Griem, Publishers, 1973, pp. 853-860. M.L., Malkinson, F.D.: Study of cel1 prohferation of