BIOCHIMIE, 1977, n ° 59, 649-650.

Biosynthesis of [1,2-3H] 5 -androstane-3 ,7 ,17 -triol. R. F. MORFIN + (>, S. DI STI~FANO * * , L. G. BARDOU * a n d H. H. FLOCH *

Laboratoire de Biochimie, F~cult$ des Sciences, 29283 Brest Cedex, France. • Laboratoire de Bioehimie, Faealtd de M~decine, 29279 Brest Cedex, France.

(29-~-1977).

INTRODUCTION.

We have recently shown that in normal and hyper- plastic h u m a n prostate 5~-androstane-3fl,17~i-diol, a metaboli te of testosterone, is specifically t ransformed into 5a-androstane-3~,7~,17,[$-triol by a prostat ie enzyme : the 3~-hydroxysteroid : 7a-hydroxylase [1].

The role played by this neve metabol i te in the me- chanism of androgen action has not yet been deter- mined. Nevertheless it is known that 5ct-androstane-315, 17~-diol main ta ins secretion in organ culture of rat prostate explants [2] and that direct action of this d ihydroxysteroid could not be ascertained.

We suggested tha t its major metabolite, the 5a- androstane-313,7n,17[~-triol might be a l~ey in the tr iggering of nuclear events leading to prostat ic secre- t ion and could therefore bind to specific proteins [1].

Since 'we have shown that canine perianal glands contain a very active 7u-hydroxylat ing enzyme [3], we have used this t issue for the biosynthesis of labelled 5u-androstane-3~,Ta,179-triol f rom high spe- cific activity (S.A.) [1,2~H] 5a-androstane-3[3, 17:[~-diol.

MATERIALS AND METHODS.

[1,2-aH] 5~-Androstane-315,1Z~l-diol.

[1,2-8H] Testosterone (45 Ci/mmole) yeas purchased f rom Ne'w England Nuclear Corporation (Boston, USA) and 'was reduced 'with l i th ium/ l iqu id ammonia in the presence of ethanol. Result ing [1,2-aH] 5c~-androstane- 3~,1713-diol Was purified by th in layer chromatography on siliea gel G (Merck) ",vith two developments in ben- zene /e thanol (9:1, v/v) , and by a lumina column chro- matography elnted ~vith a gradient of ethanol (0-5 per cent) in benzene. A port ion of the purified dihydro- xysteroid 'was diluted wi th authent ic carr ier and crystal l ized to constant S.A. : its radioehemieal pur i ty was 99.4 per cent.

5a-A ndrostane-3~,Ta,17 ~-triol.

This t r ihydroxystero id was prepared by a modifica- t ion of the method of Valcavi el at. [4] : 5-Andro- stene-~l~,17!~-diol (Merck) was acetylated in acetic anhydr ide /py r id in and the resul t ing diacetoxy deri- vative 'was reerystal l ized and treated with Na~CrO, in

** Results presented in this paper are contained in a thesis to he submit ted by S. Di St6fano to the Uni- versity of Brest in part ial fulf i l lment of the require- ments for the degree of Doctorat d 'Universit6.

<> To ~whnm all correspondence should he addressed.

acetic acid/acet ic anhydride. After s t i rr ing for 22 h the react ion was stopped wi th chloroform and the formed 3~, 17~-diaeetoxy-5-androsten-7-one ~vas extrac- ted ~vith chloroform and purified by chromatography on a silica gel 60 column (Merc&) eluted wi th increasing amounts of chloroform in benzene (15-30 per cent). The recrystal l ized pure compound svas dissolved in acetic acid and reduced wi th H2/PtO.~. Both epimeric 3~,17~-diacetoxy,7n-hydroxy- and 315, 17~-diacetoxy, 7,~hydroxy-5ct-androstanes were crystal- lized f rom the reaction mixture and ~vere separated by a lumina column chromatography eluted wi th a gradient of ethanol (1-1.5 per cent) in benzene. Isolated )~,17~-diaeetoxy, 7a-hydroxy-Sa-androstane was crys- tal l ized and then hydrolyzed in methanol ie KOH. Re- suit ing 5tt-androstane-)~,Ta,17.~-triol was crystal l ized and its ident i ty and puri ty were checked by gas-liquid chromatography and mass spectrometry.

Canine perianal 9lands.

Canine perianal glands are androgen-dependent mo- dified sebaceous glands of ui~knowm funct ion located under the slain around the external anal orifice of the dog. Collection of the scrubbed skin containing these glands ~was carried out immedia te ly af ter the death of old male dogs sacrificed for ~wner's convenience. Glands were collected on ice and immediate ly brought to the laboratory for processing and incubations.

Incubat ion procedure.

All subsequent operations were carried out /~ 4°C. The skin containing perianal glands ~was finely cut wi th scissors and minced wi th the aid of an arbor t issue press (Harvard Apparatus). Minces 'were ei ther f rozen in l iquid ni trogen and kept at --70°C or immedia te ly used for incubations.

A benzene /e thanol (9:1, v/v) solution of the [1,2-3H] 5~-andrnstane-3i~,lT:~i-diol substrate (158.3 ~.Ci, 1.4 ~g) was pipetted into a 50 ml round bot tom glass-stoppe- red tube and dried under nitrogen. Five ml of 0.067 M phosphate buffer (pH 7.4) containing 2 mg NADPH 'were added. At the star t of incubat ion 0.4 g of minces were introduced and dispersed in the medium. Incubat ion 'was carried out in air for 30 mn at 37°C in a shaking water bath, s topped by addit ion of 10 ml of chilled acetone and stored at --20°C.

Radioact iv i ty measurements .

Counting of ZH samples in glass vials was carried out 'with a model 3375 Packard Tri Carb liquid scinti l lat ion spectrometer. Quenching ~vas corrected on the basis of external s tandard ratios. Sufficient counting t ime was allo'wed for the samples so tha t the s tandard error did not exceed 0.5 per cent.

47

650 R. F. Mot[in and coll.

RESULTS.

Purification of the biosynthetized 5<t-androstane-3~,Tet,17~-triol.

Digests 'were extracted ~vith 10 ml of ethyl acetate (4 times). The pooled extracts were brought to a known volume and a por t ion ~vas counted for recove- ries (94-95.4 per cent). Volume of the extract 'was reduced under ni t rogen before application of the con- centrated ethyl acetate solut ion to th in layer chro- matography on silica gel H F ~ + ~ developped twice in cyclohexane/e thyl acetate (2:3, v/v). Labelled steroids were visualized by autoradiography for 72 h wi th Kodak BB-54 X-ray film. The radioactive spots were marked on the th in layer and t ransfered into glass vials. Steroids were then eluted f rom the gel 'with ethyl ace ta te /methanol (4:1, v/v). Port ions of the eluates 'were counted for computat ion of the yields.

P re l iminary identification of the radiometabol i tes ~vas based on the i r chromatographic mobili t ies as compared ~with those of authentic reference steroids. Eluate containing the tentat ively identified [1,2-aH] 5c¢-androstane-3)~,7,a,17~-triol 'was evaporated under ni- trogen, applied to a silica gel HF~,+~ thin layer and chromatography 'was carried out successively in two directions, first 'with three developments in benzene/ e thanol (9:1, v/v) , second 'with two developments in cyclohexane/e thyl acetate (2:3, v/v) . A utoradiography as above permit ted to localize ma jo r quant i t ies of a labelled metabol i te coincident wcith the 5,c¢-androstane- 3~,7a,17~-triol s tandard. Eluates of the radioactive areas contained 9.6 and 38.4 per cent of the incubated radioactivi ty 'when f resh and frozen perianal glands were used for incubation, respectively.

Identification and radiochemical parity of [I,2-sH] 5.e~-androstane-31~,7,u,17~-triol.

Eluates containing the major t ihydroxy metabol i te of [1,2-3H]5a-androstane-3i[3,17L[3-diol (7fi.9 ~Ci) 'were pooled, dried under nitrogen and brought to 250 ml in benzene /e thanol (9:1, v/v) . Isotopic di lut ion was carried out by mixing 0.2.5 ml of the solution wi th 11.06 mg of 5a-androstane-31~,7a,171~-triol carr ier in ethyl acetate. Counting of a port ion showed a S.A. of 15,2'33 dpm/mg. The first crystal l izat ion was carried out in methanol / 'water and the S.A. of the resul t ing crystals and mother l iquors fractions (14,912 dpm/ mg and 15,558 dpm/mg, respectively) were computed as previously described [5, 6], except tha t gas chromato- graphic analysis of the 5a-androstane-3:~,7a,17~-triol carrier 'was carried out on t r imethyls i ly l e ther deri- vatives.

S.A. of the second crystals (14,401 dpm/mg) grown in ace tone/hexane did not significantly differ f rom that of the third crystals (15,012 dpm/mg) and third mother l iquors (14,401 dpm/mg) obtained f rom a th i rd crystal l izat ion in acetone/ 'water. Thus, radiochemical puri ty of the biosynthet ized labelled triol was 94.5 per cent.

Levels of endogenous 5(t-androstane-3~,7u, lZ~-triol in canine perianal glands have not yet been measured, but the content of its precursor, 17,~-hydroxy-5a- androstan-~-one, is 5 ng/g of mature prostat ic t issue [7]. If such a value is used for 5,a-androstane- 31~,7u,171]3-triol in perianal glands, the 0.4 g of minces would contain 2 ng of the endogenous t r ihydroxys te- roid and the S.A. of the recovered radioactive triol (15.2 ,~Ci/134 ng or 60.8 i!~Ci/538 ng 'with f resh or f rozen tissue, respectively) would decrease by ei ther 1.5 per cent or 0.3 per cent.

CONCLUSIONS.

A rapid biosynthesis of [1,2-3H] 5c¢-androstane-3~, 7,c~,174~j-triol is described. This steroid of high S.A. may be used in the quest of proteins 'which would bind the 5u-androstane-3.~,7(x,17~-triol in androgen-dependent t issues. If necessary, the 14C-labelled tr iol may also be obtained f rom appropriate substrate and vcith canine per ianal glands by the described procedure.

Acknawtedgements.

"Fhis 'work was supported by a research contract (75-1-068-3) f rom INSERM (France). The authors wish to than~ Dr. J. Lavaud 'who provided access to the canine perianal glands used in this study.

REFERENCES.

1. Morfin, R. F., Di St6fano, S., Charles, J.-F. & Floch, H. H. (1977) Biochimie, 59, 637-644.

2. Robel, P., Lastnitzki, I. ,~ Baulieu, E.-E. (1971) Bio- chimie, 53, 81-97.

3. Morfin, R. F., Lear, I., Ofner, P. ~ Orr, J. C. (1970) Fed. Proc., ~ , 247.

4. Valcavi, U., Martelli, P., Sironi, U. C. a Tcdeschi, S. (1975) Il Farmaco - - Ed. Sc., 30, 464-~78.

5. Morfin, R. F., Berthou, F., Floch, H. H., Vena, R. L. Ofner, P. (1973) J. Steroid Biochem., 4, 381-

391. 6. Morfin, R. F., Bardou, L. a Floch, H. H. (1976) Spec-

tra 2000, 27, 31-38. 7. Gloyna, R. E., Siiteri, P. K. ~ Wilson, J. D. (1970)

J. Clin. Invest., 49, 1746-1753.

BIOCItlMIE, 1977, 59, n ° 7.