Archives of
Arch. Dermatol. Res. 264, 49
Deermatological
54 (1979)
search 9 Springer-Verlag 1979
Effect of Antiandrogens on the Binding of Androgen to Cytosol and Nuclei of Hamster Sebaceous Glands Susumu Takayasu Department of Dermatology, Osaka University School of Medicine, Fukushima-ku, Osaka 553, Japan
Summary. Cyproterone acetate, R2956, Ro 7-2340 and Sch 13521 in the daily dose of 8 mg injected intramuscularly to castrated adult hamsters for 3 weeks were all found to significantly antagonize the stimulatory effect of exogenous testosterone propionate (80 [~g ira, every other day) on the sebaceous gland as well as the prostate and the seminal vesicle. With a view to clarifying the mechanisms of action of these antiandrogenic drugs on the sebaceous gland, the present author carried out both in vivo and in vitro experiments and examined how they would interfere with the binding of 3H-dihydrotestosterone (DHT) to cytosol and nuclei. When 200 - 1000 excess quantities of the antiandrogens were administered to the experimental animals 10min before injection of alltestosterone, all of them except R 2956 reduced the bound radioactivity in t h e nuclei of the sebaceous gland by more than 40 %. Incubation of the cytosol fraction with 1 x 10 7 M of cyproterone acetate caused a 70 % decrease in radioactivity of the specific binding. In contrast, such inhibitory activities of the three other antiandrogens were less conspicuous even at 1 • 10 .6 M. In the homogenate of the sebaceous gland incubated with 1 • 10-s M of DHT, the other drugs than Sch 13521 curbed the characteristic binding of D H T to macromolecules of the nuclei by nearly 70 ~ at 1 x 10- 6 M.
Key words: Antiandrogen - Receptor - Sebaceous gland Zusammenfassung. Cyproteronacetat, R2956, Ro 7-2340 und Sch 13521, welches t/iglich in einer Dosis yon 8 mg intramuskul/ir kastrierten erwachsenen Hamstern fiber 3 Wochen injiziert wurde, verminderten signifikant den stimulierenden Effekt yore intramuskul~ir verabfolgten Testosteronprfiparat. Die untersuchten Parameter waren die Talgdrfisen, die Prostata und die Samenbl~ischen. U m den Wirkungsnaehweis aufzuklfiren, wurden in vivo- und in vitro-Untersuchungen darfiber angestellt, inwieweit die Bindung yon DihydroCyproterone Acetate: 6-chloro-17e-hydroxy1e,2~-methylelene-4,6-pregnadiene-3,20-dione-17acetate; R 2956: 17fl-hydroxy-2,2,7c~-tfimethylestra-4,9,11-triene-3-one;Ro 7-2340: 6c~-bromoq7/3hydroxy-17c~-methyl-4-oxa-5c~-androstane-3-one;Sch 13521: cq~,c~-trifluoro-%methyI-4"-nitro-mpropionotoluidide
0340-3696/79/0264/0049/$1.20
50
S. Takayasu
testosteron an Cytosol und Kernen beeintr~ichtigt wird. Bei Gabe yon Antiandrogenen 10rain vor der Injektion von Testosteron wurde bei allen Pr/iparaten, mit Ausnahme von R 2956 die kerngebundene Radioaktivitfit in den Talgdrtisen mehr ats 40 ~ vermindert. Die Inkubation der Cytosolfraktion mit 10- 7 M Cyproteronacetat bewirkte eine 70 %ige Abnahme der spezifischen Bindung. Die inhibierten Aktivitfiten von drei anderen Antiandrogenen waren sogar bei 10 6 M weniger auff/illig. Im Homogenat der Talgdr/ise, welche mit 10-8 M DHT inkubiert wurde, konnten alle Medikamente mit Ausnahme von Sch 13521 die Bindungsffihigkeit von DHT an Makromolektile der Kerne auf nahezu 70% bei 10-6M begrenzen. Schliisselwiirter: Antiandrogen - Receptor - Talgdrfise
Antiandrogenic agents which inhibit the growth of the sebaceous gland both in man and animals [2, 3, 6, 7, 15] have recently been drawing much attention because of a possibility that they may be of value for the treatment of acne. However, we have little knowledge about their modes of action on the sebaceous gland with a few exceptions [14]. In the past, some investigators including myself demonstrated the presence of specific androgen receptors in the hamsters costovertebral gland consisting of several large sebaceous glands [1, 12, 13]. In the present study, I investigated, with this model system, how four antiandrogens antagonistic with exogenous testosterone would work on the formation of androgen-receptor complexes in the cytosol and nuclear fractions of the sebaceous gland.
Materials and Methods Antiandrogenic Activity on the Growth of Sebaceous Glands', Prostate and Seminal Vesicles Stimulated with Exogenous Testosterone. Young adult male hamsters were used for assessment of the ability of the antiandrogenic drugs, cyproterone acetate, R2956, Ro 7-2340 and Sch 13521, to inhibit the effect of exogenous testosterone. Castration was performed 2 weeks prior to initiation of the treatment. The drugs were dissolved or suspended in either 15 ~ benzene or ethanol-sesame oil. Eight milligrams of each drug was injected i.m. daily for 3 weeks except on Sunday. Testosterone propionate (80 gg) in sesame oiI was administered i.m. every other day. This dose of testosterone propionate was considered suitable for the experiment because it achieved a nearly maximal growth of these organs in our preliminary experiment in which castrated hamsters were given different doses (20, 40, 80 and 160 gg, every other day). Control animals were given 15 ~ benzene-sesame oil without antiandrogens. Twenty-four hours after the last treatment, the animals were sacrificed and the prostate and the seminal vesicles were removed and weighed. The costovertrebral gland composed of several large sebaceous glands were taken out and placed in 10 % formalin for histologic evaluation. Effect of Antiandrogens on the in vivo Binding of 3H-DHT in Hamster Sebaceous Glands. Adult male Syrian hamsters were castrated 16 - 24 h before i.m. injection of 100 gCi (0.6 gg) of 3H-1,2-testosterone (SA 42 Ci/mM, Radiochemical Center). They were medicated with one of the antiandrogens, 600 pg each, 10 rain before administration of 3H-testosterone and sacrified I h after 3H-testosterone injection. The further procedures were described in detail in my previous paper [12l- Briefly, the costovertebral gland was excised and the cytoplasmic fraction (600 x g, supernatant) as well as the 0.4M KC1 extracts from the nuclear fraction (600 x g, sediment) was separated into the portions of bound and free radioactivites by filtration through Sephadex G-25.
Ant•
Action on Sebaceous Glands
Effect of Ant•
51
on the in vitro Binding of 3H-DDT to Cytosol and Nuclei of Sebaceous Glands.
Details of the experimental procedures were depicted elsewhere [13]. For the labeling of the cytosol receptor, the cytosol fraction (I05,000 • g, supernatant) was incubated with I x I0-~M of ~H-L2dihydrotestosterone (SA 49 Ci/mM, New England Nuclear Corp.) for 3 h at 0 ~C in the presence of 1 • ] 0 - 6 M or I • 10-7M of an ant• treated with 0.05% dextran - 0.5% charcoal, and analyzed by sucrose gradient centrifugation. For the intranuclear binding, the homogenate of the costovertebral gland was incubated with 1 x 10 s M of 3H-DHT in the presence of 1 x 10-6 M of an ant• for 1.5 h at 0~ C and it further underwent incubation for 30 min at 25~ C. The nuclear extract was filtered through a Sephadex G-100 column. Protein was measured by the method of Lowry et al, [5]. Cytoproterone acetate was a generous gift from Schering AG, R 2956 from Roussel Uctaff, Inc., Ro 7-2340 from Hoffmann-La Roche, Inc., and Sch 13521 from Schering Co.
Results Effect of Ant• on the Androgenic Activity of Testosterone Propionate in Castrated Hamsters (Table 1). All the a p p l i e d a n t • i n h i b i t e d the a n d r o g e n - i n d u c e d h y p e r t r o p h y o f t h e p r o s t a t e , the s e m i n a l vesicle, a n d t h e sebaceous gland, when given simultaneously with testosterone propionate. C y p r o t e r o n e a c e t a t e a n d Sch 13521 w e r e m o r e e f f e c t i v e t h a n the t w o in c u r b i n g w e i g h t g a i n o f t h e p r o s t a t e , w h e r e a s t h e f o u r d r u g s d i d n o t differ s i g n i f i c a n t l y f r o m o n e a n o t h e r in t h e effect o n t h e size o f the s e b a c e o u s g l a n d ,
In vivo Effect on the Formation of DHT-Receptor Complexes in Nuclei of Sebaceous Glands. P r e v i o u s l y I d e m o n s t r a t e d t h a t t h e specific b i n d i n g o f D H T to m a c r o m o lecules o c c u r r e d in t h e n u c l e i o f h a m s t e r s e b a c e o u s g l a n d s in v i v o w h e n 3Ht e s t o s t e r o n e was a d m i n i s t e r e d to c a s t r a t e d h a m s t e r s [12]. T h i s i n t r a n u c l e a r b i n d i n g is c o n s i d e r e d c h a r a c t e r i s t i c o f a n d r o g e n t a r g e t tissues [4, 8]. T h i s time, t h e r e f o r e , I
Table 1. Effects of antiandrogens on the androgenic activity of testosterone propionate in castrated hamsters Treatment
No.
Control 5 Cyproterone acetate 6 R2956 6 Ro 7-2340 4 Sch 13521 5
Mean body weight (g)
Mean organ weight (rag)
Initial
Seminal vesicles
Prostate
126.6 +, 9,48
Final
] 11
117
302.6 _+ 23_9
129 124 118 121
136 135 124 137
162,7 +_ 8.83" 165,7 _+ ~LI* 199,3 +, 18.0"** 123.2 _+ 13.3"
Sebaceous gland area ( • 10~gz)
35.7 +, 2.50* 55.8 +_ 7.79* 75.8 +, 5.89** 27.6 +, 5.14"
7],9 +, 2.67 31.0 +, 0.79* 32.4 +, 0.95* 44.4 +, 1.85" 29.5 +, 1.38",
*P
Arch. Dermatol. Res. 264, 49
Deermatological
54 (1979)
search 9 Springer-Verlag 1979
Effect of Antiandrogens on the Binding of Androgen to Cytosol and Nuclei of Hamster Sebaceous Glands Susumu Takayasu Department of Dermatology, Osaka University School of Medicine, Fukushima-ku, Osaka 553, Japan
Summary. Cyproterone acetate, R2956, Ro 7-2340 and Sch 13521 in the daily dose of 8 mg injected intramuscularly to castrated adult hamsters for 3 weeks were all found to significantly antagonize the stimulatory effect of exogenous testosterone propionate (80 [~g ira, every other day) on the sebaceous gland as well as the prostate and the seminal vesicle. With a view to clarifying the mechanisms of action of these antiandrogenic drugs on the sebaceous gland, the present author carried out both in vivo and in vitro experiments and examined how they would interfere with the binding of 3H-dihydrotestosterone (DHT) to cytosol and nuclei. When 200 - 1000 excess quantities of the antiandrogens were administered to the experimental animals 10min before injection of alltestosterone, all of them except R 2956 reduced the bound radioactivity in t h e nuclei of the sebaceous gland by more than 40 %. Incubation of the cytosol fraction with 1 x 10 7 M of cyproterone acetate caused a 70 % decrease in radioactivity of the specific binding. In contrast, such inhibitory activities of the three other antiandrogens were less conspicuous even at 1 • 10 .6 M. In the homogenate of the sebaceous gland incubated with 1 • 10-s M of DHT, the other drugs than Sch 13521 curbed the characteristic binding of D H T to macromolecules of the nuclei by nearly 70 ~ at 1 x 10- 6 M.
Key words: Antiandrogen - Receptor - Sebaceous gland Zusammenfassung. Cyproteronacetat, R2956, Ro 7-2340 und Sch 13521, welches t/iglich in einer Dosis yon 8 mg intramuskul/ir kastrierten erwachsenen Hamstern fiber 3 Wochen injiziert wurde, verminderten signifikant den stimulierenden Effekt yore intramuskul~ir verabfolgten Testosteronprfiparat. Die untersuchten Parameter waren die Talgdrfisen, die Prostata und die Samenbl~ischen. U m den Wirkungsnaehweis aufzuklfiren, wurden in vivo- und in vitro-Untersuchungen darfiber angestellt, inwieweit die Bindung yon DihydroCyproterone Acetate: 6-chloro-17e-hydroxy1e,2~-methylelene-4,6-pregnadiene-3,20-dione-17acetate; R 2956: 17fl-hydroxy-2,2,7c~-tfimethylestra-4,9,11-triene-3-one;Ro 7-2340: 6c~-bromoq7/3hydroxy-17c~-methyl-4-oxa-5c~-androstane-3-one;Sch 13521: cq~,c~-trifluoro-%methyI-4"-nitro-mpropionotoluidide
0340-3696/79/0264/0049/$1.20
50
S. Takayasu
testosteron an Cytosol und Kernen beeintr~ichtigt wird. Bei Gabe yon Antiandrogenen 10rain vor der Injektion von Testosteron wurde bei allen Pr/iparaten, mit Ausnahme von R 2956 die kerngebundene Radioaktivitfit in den Talgdrtisen mehr ats 40 ~ vermindert. Die Inkubation der Cytosolfraktion mit 10- 7 M Cyproteronacetat bewirkte eine 70 %ige Abnahme der spezifischen Bindung. Die inhibierten Aktivitfiten von drei anderen Antiandrogenen waren sogar bei 10 6 M weniger auff/illig. Im Homogenat der Talgdr/ise, welche mit 10-8 M DHT inkubiert wurde, konnten alle Medikamente mit Ausnahme von Sch 13521 die Bindungsffihigkeit von DHT an Makromolektile der Kerne auf nahezu 70% bei 10-6M begrenzen. Schliisselwiirter: Antiandrogen - Receptor - Talgdrfise
Antiandrogenic agents which inhibit the growth of the sebaceous gland both in man and animals [2, 3, 6, 7, 15] have recently been drawing much attention because of a possibility that they may be of value for the treatment of acne. However, we have little knowledge about their modes of action on the sebaceous gland with a few exceptions [14]. In the past, some investigators including myself demonstrated the presence of specific androgen receptors in the hamsters costovertebral gland consisting of several large sebaceous glands [1, 12, 13]. In the present study, I investigated, with this model system, how four antiandrogens antagonistic with exogenous testosterone would work on the formation of androgen-receptor complexes in the cytosol and nuclear fractions of the sebaceous gland.
Materials and Methods Antiandrogenic Activity on the Growth of Sebaceous Glands', Prostate and Seminal Vesicles Stimulated with Exogenous Testosterone. Young adult male hamsters were used for assessment of the ability of the antiandrogenic drugs, cyproterone acetate, R2956, Ro 7-2340 and Sch 13521, to inhibit the effect of exogenous testosterone. Castration was performed 2 weeks prior to initiation of the treatment. The drugs were dissolved or suspended in either 15 ~ benzene or ethanol-sesame oil. Eight milligrams of each drug was injected i.m. daily for 3 weeks except on Sunday. Testosterone propionate (80 gg) in sesame oiI was administered i.m. every other day. This dose of testosterone propionate was considered suitable for the experiment because it achieved a nearly maximal growth of these organs in our preliminary experiment in which castrated hamsters were given different doses (20, 40, 80 and 160 gg, every other day). Control animals were given 15 ~ benzene-sesame oil without antiandrogens. Twenty-four hours after the last treatment, the animals were sacrificed and the prostate and the seminal vesicles were removed and weighed. The costovertrebral gland composed of several large sebaceous glands were taken out and placed in 10 % formalin for histologic evaluation. Effect of Antiandrogens on the in vivo Binding of 3H-DHT in Hamster Sebaceous Glands. Adult male Syrian hamsters were castrated 16 - 24 h before i.m. injection of 100 gCi (0.6 gg) of 3H-1,2-testosterone (SA 42 Ci/mM, Radiochemical Center). They were medicated with one of the antiandrogens, 600 pg each, 10 rain before administration of 3H-testosterone and sacrified I h after 3H-testosterone injection. The further procedures were described in detail in my previous paper [12l- Briefly, the costovertebral gland was excised and the cytoplasmic fraction (600 x g, supernatant) as well as the 0.4M KC1 extracts from the nuclear fraction (600 x g, sediment) was separated into the portions of bound and free radioactivites by filtration through Sephadex G-25.
Ant•
Action on Sebaceous Glands
Effect of Ant•
51
on the in vitro Binding of 3H-DDT to Cytosol and Nuclei of Sebaceous Glands.
Details of the experimental procedures were depicted elsewhere [13]. For the labeling of the cytosol receptor, the cytosol fraction (I05,000 • g, supernatant) was incubated with I x I0-~M of ~H-L2dihydrotestosterone (SA 49 Ci/mM, New England Nuclear Corp.) for 3 h at 0 ~C in the presence of 1 • ] 0 - 6 M or I • 10-7M of an ant• treated with 0.05% dextran - 0.5% charcoal, and analyzed by sucrose gradient centrifugation. For the intranuclear binding, the homogenate of the costovertebral gland was incubated with 1 x 10 s M of 3H-DHT in the presence of 1 x 10-6 M of an ant• for 1.5 h at 0~ C and it further underwent incubation for 30 min at 25~ C. The nuclear extract was filtered through a Sephadex G-100 column. Protein was measured by the method of Lowry et al, [5]. Cytoproterone acetate was a generous gift from Schering AG, R 2956 from Roussel Uctaff, Inc., Ro 7-2340 from Hoffmann-La Roche, Inc., and Sch 13521 from Schering Co.
Results Effect of Ant• on the Androgenic Activity of Testosterone Propionate in Castrated Hamsters (Table 1). All the a p p l i e d a n t • i n h i b i t e d the a n d r o g e n - i n d u c e d h y p e r t r o p h y o f t h e p r o s t a t e , the s e m i n a l vesicle, a n d t h e sebaceous gland, when given simultaneously with testosterone propionate. C y p r o t e r o n e a c e t a t e a n d Sch 13521 w e r e m o r e e f f e c t i v e t h a n the t w o in c u r b i n g w e i g h t g a i n o f t h e p r o s t a t e , w h e r e a s t h e f o u r d r u g s d i d n o t differ s i g n i f i c a n t l y f r o m o n e a n o t h e r in t h e effect o n t h e size o f the s e b a c e o u s g l a n d ,
In vivo Effect on the Formation of DHT-Receptor Complexes in Nuclei of Sebaceous Glands. P r e v i o u s l y I d e m o n s t r a t e d t h a t t h e specific b i n d i n g o f D H T to m a c r o m o lecules o c c u r r e d in t h e n u c l e i o f h a m s t e r s e b a c e o u s g l a n d s in v i v o w h e n 3Ht e s t o s t e r o n e was a d m i n i s t e r e d to c a s t r a t e d h a m s t e r s [12]. T h i s i n t r a n u c l e a r b i n d i n g is c o n s i d e r e d c h a r a c t e r i s t i c o f a n d r o g e n t a r g e t tissues [4, 8]. T h i s time, t h e r e f o r e , I
Table 1. Effects of antiandrogens on the androgenic activity of testosterone propionate in castrated hamsters Treatment
No.
Control 5 Cyproterone acetate 6 R2956 6 Ro 7-2340 4 Sch 13521 5
Mean body weight (g)
Mean organ weight (rag)
Initial
Seminal vesicles
Prostate
126.6 +, 9,48
Final
] 11
117
302.6 _+ 23_9
129 124 118 121
136 135 124 137
162,7 +_ 8.83" 165,7 _+ ~LI* 199,3 +, 18.0"** 123.2 _+ 13.3"
Sebaceous gland area ( • 10~gz)
35.7 +, 2.50* 55.8 +_ 7.79* 75.8 +, 5.89** 27.6 +, 5.14"
7],9 +, 2.67 31.0 +, 0.79* 32.4 +, 0.95* 44.4 +, 1.85" 29.5 +, 1.38",
*P
