Comments Inhibition of Testosterone Conversion to Dihydrotestosterone in Men Treated Percutaneously by Progesterone PIERRE MAUVAIS-JARVIS, FREDERIQUE KUTTENN, AND NICOLE BAUDOT Biochemical Department, Faculty of Medicine Pitie-Salpetriere, 75634 Paris, France ABSTRACT. Unlabeled progesterone was added topical treatment and incubated with the same at different doses to human skin homogenates procedure as during in vitro experiments. An inhi- originating from the pubic area of normal men. bition of testosterone 5a-reduction to dihydrotes- The transformation of radioactive testosterone to tosterone was also observed after this percuta- dihydrotestosterone was inhibited by this in vitro neous administration of progesterone. Thus pro- addition of progesterone. In other experiments gesterone, when percutaneously applied upon the 100 mg of progesterone dissolved in alcohol was skin of human beings, might be useful as an applied upon the skin of 2 normal men. A skin antiandrogen. (/ Clin Endocrinol Metab 38: 142, specimen was obtained before and after this 1974) TJECENT studies upon metabolism in human ically, the inhibition of testosterone 5a-reduction •*•*• skin have demonstrated the transformation by progesterone might be considered as an anti- of several steroid hormones in this organ and androgenic effect of endocrinological and derma- there is increasing evidence that these transfor- tological significance, since progesterone admin- mations are of endocrinological importance. In istered percutaneously is largely metabolized in particular, the 5a-reduction of testosterone to situ (8) before reaching the peripheral circula- 17(3-hydroxy-5a-androstan-3-one (dihydrotestos- tion. terone, DHT) which seems to be an essential In this study, the results of experiments relat- prerequisite for the action of testosterone in ing the inhibition by progesterone of testoster- male accessory organs (1,2) has been also ob- one 5a-reduction by human skin are presented. served in vitro in human skin (3,4). In this lab- These studies have been carried out both in oratory (5,6), the reduction'of testosterone to vitro by addition of unlabeled progesterone to DHT and 5a-androstane-3q,17(3-diol (andro- skin homogenates incubated with radioactive stanediol) by human skin wjas also suggested, testosterone and "in vivo plus in vitro" by incu- since in normal man, testosterone was a better bation of skin from normal men previously sub- precursor of androstanediol when the labeled tes- mitted to a percutaneous treatment with proges- tosterone was administered .through the skin terone. than when the radioactive precursor was injected Both experiments resulted in an inhibition of in the peripheral circulation. i the 5a-reduction of testosterone to DHT. On the other hand, the Sa-reduction of proges- terone to 5a-pregnane-3,20-dione (dihydropro- Materials and Methods gesterone) was observed both in vitro (7) and Chemicals in vivo (8) by comparing, as for testosterone, Testosterone-1,2-:{H (SA 42.75 C/mmole) was the fate of radioactive progesterone simulta- obtained from New England Nuclear Corp. and neously administered by percutaneous and intra- purified in the laboratory. Its purity was found to venous routes. Moreover, Voigt, Fernandez and be at least 95% after chromatography and isotope Hsia (9) have found that a number of steroids dilution. Reference steroids were purchased from inhibited the 5a-reduction of testosterone by Roussel Uclaf and Ikafarm laboratories; glucose-6- microsomal preparation of human,skin; among phosphate, glucose-6-phosphate dehydrogenases, these, the most potent inhibitor was progester- NADF and NADPH were obtained from Boehringer one which competed effectively with testosterone chemical corp. for the active site of the enzyme. Thus theoret- Specimens Six normal men were studied. They were 30-40 Received June 18, 1973. yr old. In 4 cases pubic skin samples were obtained 142 The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 17 February 2015. at 10:44 For personal use only. No other uses without permission. All rights reserved. COMMENTS .143 at laparotomy for urologic nonendocrine diseases. reduction of testosterone to DHT, the identification None of the patients studied had renal failure. In of the other metabolites of testosterone was not the 2 other cases, skin was obtained after a topical performed. The chromatographic systems used in application on the same pubic area of 100 mg of this study are described in Table 1. Dried extracts unlabeled progesterone dissolved in absolute ethanol were submitted to a first paper chromatography. (prepared by Besins-Iscovesco laboratories). The Reference steroids were visualized by the Zimmer- total application of progesterone was fractionated mann reaction. Labeled testosterone, DHT and into 4 percutaneous applications of progesterone for androstenedione were located by scanning with a 24 hr between 2 biopsies of pubic skin performed radiochromatogram scanner Packard model 7200 and under saline anesthesia. All skin specimens were im- eluted. An aliquot of each spot was counted in a mediately stored at —20 C, approximately for 2 liquid scintillation spectrometer Packard model weeks, because NADPH does not seem to penetrate 3376. The remaining steroid material was purified the fresh skin samples (10). by 2 thin-layer chromatographies and 1 paper chromatography, as described in Table 1; and identi- Homogenization of skin samples fied by addition of the unlabeled carrier steroid The general procedure employed was very simi- and recrystallization to constant specific activity lar to that described by Voigt, Fernandez and Hsia (Table 2). (9). Approximately 100 mg of skin was used The final specific activity of material from the after removal of subcutaneous fat. Skin samples area corresponding in the first paper chroma- were minced with fine scissors and homogenized in tography to testosterone and androstenedione was Krebs Ringer phosphate buffer with an ultra 95% of the calculated value. Turrax until there was no visible piece of intact In the ligroin-propanediol chromatographic sys- skin and then transferred into a Tenbroeck homog- tem, DHT and epiandrosterone have a very close enizer. All operations were carried out at —4 C mobility. So, the material of this peak was examined for less than IS min. by reverse isotopic dilution with DHT and epiandros- terone. The data indicated that 85% of the DHT Incubation peak was effectively DHT (Table 2) and 15% could be epiandrosterone. The same rate had been obtained The medium consisted of 10 ml of Krebs Ringer by Gomez and Hsia (3). Phosphate buffer (pH 7.4) with addition of Peni- r> cillin G, 50 U/ml; glucose anhydre, 10~ mole/ml Results and either NADPH 1 nmole/ml or NADP 1 n-mole/ ml with or without glucose-6-phosphate, 5 M-mole/ml Effect of cofactors (Table 3) and glucose-6-phosphate dehydrogenase, 1 U/ml. In one experiment only 0.79% and 0.13% of Immediately prior to incubation, radioactive tes- radioactive testosterone was recovered as DHT tosterone (15 X 10(i dpm = 158 pmoles) in 0.1 ml and androstenedione in the absence of cofactors. of ethanol was added to each flask and evaporated In the presence of NADP the transformation of to dryness. The incubation was carried out for 1 hr at 37 C in a Dubnoff shaking incubator. In one case testosterone into DHT and androstenedione was the incubation was performed for 1, 2 and 3 hr. not significantly greater. By contrast, in the • presence of a NADPH generating system, there Extraction was more androstenedione-(22.9%) than DHT (8:4%) formed from radioactive testosterone. All incubations were terminated by addition of acetic acid in order to have a pH close to 5.0. The optimal reduction of testosterone to DHT Mixtures were then kept in a freezer at —10 C was obtained when NADPH was added to the overnight. Steroids were extracted with ethyl acetate, medium (10.5 to 15.3% of the incubated radio- washed with sodium carbonate (10%) and water, active testosterone). In that case, only 1.8 to then evaporated to dryness under vacuum. Non- 6.1% of testosterone was oxidized to androstene- steroid lipids were separated from steroids with dione. 709? aqueous methanol/heptane system. The re- covery of tritium in the methanol phase was greater TABLE 1. Solvent systems used in chromatography than 90%. Paper partition chromatography: Chromatography and identification of steroids 1. Ligroin:Propanediol 1:1 2. Cyclohexane:Benzene 1:1 In the present experiment, only testosterone, DHT and androstenedione were identified and mea- Thin-layer chromatography: sured. The essential object of this study being the 1. Ethyl acetate:Benzene 1:1 inhibitory action of progesterone upon the 5a- 2. Chloroform:Methanol 99:1 The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 17 February 2015. at 10:44 For personal use only. No other uses without permission. All rights reserved. 144 COMMENTS JCE & M• 1974 Vol 38 •No 1 TABLE 2. Characterization of testosterone, andros- 00 r^ tenedione and D H T I •—I CM \O 1) Testosterone Specific x No. activity Solvents crystallization dpm/nmole Isooctane-methanol 1540 o-o Dichloromethane-hexane 2 525 Ethyl acetate-benzene 3 530 80 mg of unlabeled testosterone were added to an aliquot containing 150,000 dpm from the area of the O O O O peak corresponding to testosterone. The calculated specific activity (SA) was 541 dpm/nmole. The data indicate that 95% of the peak was effectively unme- tabolized testosterone. 2) Androstenedione a Specific E No. activity Solvents crystallization dprn/nmole f-4 — OC t/j O Isooctane-benzene 1651 O '~- Dichloromethane-hexane 2 633 p 0.8 Ethyl acetate-benzene 3 625 25 mg of unlabeled androstenedione were added to O io an aliquot containing 55,000 dpm of the peak of q o androstenedione. The calculated SA was 639 dpm/ d d M-mole. The data indicate that 95% of the peak was effectively androstenedione. 3) DHT Specific No. activity Solvents crystallization dpm/mnole Isooctane-benzene 1356 2 >^ Dichloromethane-hexane 2 302 o a Ethyl acetate-benzene 3 300 o 40 mg of unlabeled DHT were added to an aliquot containing 43,300 dpm from the area of the peak cor- responding to DHT.