BIOCHIMIE, 1977, n ° 59, 637-644.

Precursors for and 7 -hydroxylations of 5 -androstane-3 ,17$-diol by human normal and hyperplastic prostates,

R. F. MORFIN +~, S. DI STEFANO * ('), J . -F. CHARLES ++ a n d H. H. FLOCH *

+ Laboratoire de Biochimie, Facult~ des Sciences, 29283 Brest Cedex, France. Laboratoire de Biochimie, Facultd de Mddecine, 29279 Brest Cedex, France. ++ Service de Chirurgie G~n~rale << Bichat ~>, C.H.U., 29200 Brest, France.

(29-4-1977).

Summary. Minces and homogenates f rom h u m a n no rma l and hyperp las t ic prosta tes were incuba ted w i t h 14C-labelled C~,,O~-steroids in the presence of NADPH. Thus, testoste- rone, isoandrosterone, 3a-andros taned io l and 31[~-androstanediol Were t r an s fo rmed into radioact ive metabol i tes 'which 'were identified by c rys ta l l iza t ion to cons tan t specific acti- vity. Among polar metabol i tes derived f rom these substrates , f o r m a t i o n of 5ct-androstane- 313,7c~,17i~-triol by bo th t issues 'was proved and t h a t of 5a-androstane-3[~,613,17[~-triol s t rongly suggested. A specificity of 7ct-hydroxylat ion for 3[~-hydroxylated subs t ra tes xvas proposed since only m a j o r metabol i tes of isoandrosterone and 3[3-androstanediol were 7~-hydroxylated.

Fu r t he r m or e it was confirmed t h a t C~O,~-3[~-hydroxysteroid subs t ra tes only lead to minor quant i t i es of 3-oxometabol i tes in uitro and in the presence of NADPH. In contrast , 3 t t -androstanediol is actively t r ans formed , in the same condi t ions, in to 5a-d ihydrotes to- ~terone, th is metabol i te giving successively r ise to 3[3-androstanediol and hydroxy la ted metabol i tes . Involved enzymic act ivi t ies allo~ved to complete the pros ta t ic tes tos te rone me tabo l i sm pa thways and to showy tha t no qua l i ta t ive difference occurs be tween normal and hyperp las t ic h u m a n glands.

It r ema ins to be found to which extent quan t i t a t ive differences of enzymic act ivi t ies may occur beSween bo th t issues and to de te rmine the role wh ich may be played by 5a-androstane-3~,7cx,17~-triol in the mode of act ion of androgenic steroids on prosta t ic secret ion or hyperp las ia .

I N T R O D U C T I O N .

B o t h in vivo [1, 2] a n d in vitro [3, 4, 5~ s t u d i e s h a v e sho~vn t e s t o s t e r o n e to be m a i n l y t r a n s f o r - m e d b y t h e h u m a n b e n i g n h y p e r p l a s t i c p r o s t a t e i n t o 4 - a n d r o s t e n e - 3 , 1 7 - d i o n e ( a n d r o s t e n e d i o n e o) a n d v a r i o u s 5 a - r e d u c e d s t e r o i d s i n c l u d i n g 5a-d i - h y d r o t e s t o s t e r o n e , 3 a - a n d r o s t a n e d i o l a n d 3~- a n d r o s t a n e d i o l . P r e s e n c e of N A D P H or N A D H - d e - p e n d e n t A4-3 - oxos t e r o i d - S t t - r educ t a s e a n d 17[~-,

° List of abbrev ia t ions and t r iv ia l names. BPH = Benign Pros ta t ic Hyperplas ia . tes tos terone = 17,~-hydroxy-4-androsten-3-one. 5o.-dihydrotestosterone = 17[~-hydroxy-5(t-androstan- 3-one. andros tened ione = 4-androstene-3,17-dione. andros te rone = 3ct-hydroxy-Sa-androstan-17-one. isoandrosterone ---- 3:13-hydroxy-5(x-androstan-17-one. 3c~-androtanediol = 5c¢-androstane-3a, 171~-diol. 3~-andros tanedio l = 5a-androstane-3!~, 17~-diol. progesterone = 4-pregnene-3,20-dione. DHEA = 3~-hydroxy-5-androsten-17-one. S.A. = Specific Activity. (-~ Resul ts presented in th is paper are conta ined in

a thesis to be submi t t ed by S. Di Stdfano to the Uni- vers i ty of Brest in par t i a l fu l f i l lment of the requi re- men t s for the degree of Doctorat d 'Universi t~.

<> To 'whom all correspondence should be addressed.

3ct- a n d 3 ~ - h y d r o x y s t e r o i d o x i d o r e d u c t a s e s in B P H t i s s u e ~ ,as t h e r e f o r e a s c e r t a i n e d a n d f u r t h e r p r o v e d w i t h t he use of 5 c ~ - d i h y d r o t e s t o s t e r o n e , 3 c t - a n d r o s t a n e d i o l a n d 3 8 - a n d r o s t a n e d i o l sub - s t r a t e s [5, 6, 7, 8, 91.

Fe 'w p a p e r s de l t w i t h o t h e r t r a n s f o r m a t i o n s of t h e s e a n d r o g e n i c s t e r o i d s . E v i d e n c e s fo r 26- a n d 6 ~ - h y d r o x y l a t i o n s of A~-C19Oo-steroids i n t h e h u m a n p r o s t a t e w e r e r e p o r t e d y e a r s ago [3, 10, H I a n d r e c e n t l y e x t e n d e d to 6~- a n d 7 ~ - h y d r o x y - l a t i o n of 5 ~ - r e d u c e d s u b s t r a t e s [6].

F u r t h e r e v i d e n c e s c o n c e r n i n g t e s t o s t e r o n e m e - t a b o l i s m a n d h y d r o x y l a t i o n of 5 a - r e d u c e d - C m O 2- s t e r o i d s in vitro b y B P H a n d n o r m a l p r o s t a t i c t i s s u e p r e p a r a t i o n s a r e n o w r e p o r t e d i n t h i s p a p e r .

M A T E R I A L S AND M E T H O D S .

Carrier steroids and solvents.

All s o l v e n t s w e r e of r e a g e n t g r a d e o r d i s t i l l e d b e f o r e use. T e s t o s t e r o n e , 5 a - d i h y d r o t e s t o s t e r o n e , isoandrosterone, 3 a - a n d r o t a n e d i o l a n d 3:~-andro-

45

638 R. F. M o r f i n

stanediol were purchased from Merck. 3~,7a-Dihy- droxy-5a-androstan-17-one was obta ined from iso- andros terone incuba t ions wi th Rhizopus nigri- cans [12] ; fur ther potassium borohydr ide reduc- t ion yielded 5¢t-androstane-3~-7a,176-triol. The 5a-androstane-3;~,6~,lZ~-triol was obtained by LiA1H~ reduct ion of 3i~,lZ~-dihydroxy-5a-andro- stan-6-one k ind ly provided by Sir Ewar t R. H. Jones (Oxford Universi ty , U.K.).

Labelled steroids.

[4-1~C] testosterone (50 and 57.5 mCi /mmole) was purchased from C.E.A. (France) and N.E.N. (Boston, USA), respectively. Purif icat ions were carr ied out by th in layer chromatography on silica gel GF254 wi th one development in benzene / 95 per cent ethanol (9:1, v /v) . Crystal l izat ion to constant S.A. of a car r ie r -d i lu ted por t ion of the eluted testosterone permi ted to measure radio-

and coll.

l iquid ammonia , or by incuba t ion wi th female rat l iver microsomes as previously described ['13]. Purif icat ion as above was followed by co lumn chromatography on a lumina eluted wi th a gra- dient mixture of ethanol (0-6 per cent) in benzene. Crystal l izat ion to constant S.A. showed a 98 per cent r ad iopur i ty for the chemical ly- reduced testosterone and a 97.4 per cent r ad iopur i ty for the biological ly-prepared 5a-dihydrotestosterone. [4-14C] 3a-Androstanediol was p repared from 5a- dihydrotes tos terone digests wi th female rat l iver microsomes [:13]. Successive puri f icat ions were by th in layer chronla tography and column chro- matography as above. A 96.9 per cent r ad iopur i ty yield was measured. [4-~C] 3~-androstanediol was obtained by complete reduct ion of [4-14C] testosterone by l i t h i u m / l i q u i d ammonia in pre- sence of ethanol. Puri f icat ions as above yielded rad iopure (99.4 per cent) 3~-androstanediol as

C R U D E

Non ident i f ied p o l a r s . . . . . . . . . . . . . . .

5 a - A n d r o s t a n e - 3 f 3 , 7 a , 17~3-triol . . . . .

5a -Andros tane -3~3 , 6~3,17f3-triol . . . . . .

3f3, 7 a - D i h y d r o x y - 5 ~ x - a n d r o s t a n - 1 7 - o n e

3f3 - A n d r o s t a n e d i o l . . . . . . . . . . . . . . . . . .

3a - A n d r o s t a n e d i o l . . . . . . . . . . . . . . . . . .

T e s t o s t e r o n e . . . . . . . . . . . . . . . . . . . . . . .

I s o a n d r o s t e r o n e . . . . . . . . . . . . . . . . . . .

A n d r o s t e r o n e . . . . . . . . . . . . . . . . . . . . . .

5a - D i h y d r o t e S t o s t e r o n e . . . . . . . . . . . . .

Andro s t ened ione . . . . . . . . . . . . . . . . . . .

5a - A n d r o s t a n e - 3 , 17-di one . . . . . . . . .

EXTRACTS 1 \ \ % \ \

FIRST TLC SECOND

(systom I} R f = 0 - 0 . 1 2

Rf -- 0. 15 ---~ CR

Rf = 0 .15 l(sy~ternZ) Rf = 0. Z2 --~ CR

........ ~Rf = 0 .41 RfRf == 0.0"3738 ] [ R f = 0 .46

Rf = 0.51

Rf = 0.56

Rf = 0.6Z

Rf = 0.62

Rf = 0.77

Rf = 0.88

TLC

--~ CR

--~ CR

--2 CR

---,~ CR ( ~ y s t e m 3) 1

.Rf = 0.59 }--~ CR

FIG. 1. - - Use of successive thin layer chromalographies (TLC) /or the separation of testosterone metabolites formed by human prostate.

The flow-sheet underlines ~,hich steroids were eluted for a second TLC and where crystallization to constant S.A. (CR) took place for identification. See the text for TLC systems.

pur i ty yields of 98.8 and 99.4 per cent respecti- vely. [4-1~C] 5a-Dihydrotestosterone was obtained either after exposure for a short t ime of a solut ion of [4-14C] testosterone in d ioxane to l i t h i u m /

BIOCHIMIE, 1977, 59, n ° 7.

ind ica ted by crystal l izat ion to cons tant S.A. of a carr ier -di lu ted por t ion. [4-14C] Isoandros terone was b iochemica l ly-prepared as previously descri- bed [llg] wi th a pur i ty of 95.1 per cent.

Pros ta t i c h y d r o x y l a t i o n s of 5u-androstane-3~, lT~-diol . 639

Prostatic tissues.

Tissues ~ i t h BPH Were r emoved by the trans- vesical approach f rom pat ients undergo ing sur- gery. Normal human prosta te was obtained f rom an 18 years old donnor used for k idney trans- plant. As soon as obtained, tissues were chi l led in a beaker su r rounded w i t h ice before por t ions were taken for h is topa thologica l examinat ions and t ranspor t to a cold room. At 4°C, tissues ~vere finely cut w i t h scissors and forced through the 1 mm d iamete r holes of a sieve wi th an a rbor tissue press (Harvard Apparatus) . Obtained min- ces ~were ~veighed on a mic roba lance and 200 mg por t ions used for incubat ions. In one case of BPH, minces were fur ther homogenized in 0.067 M phosphate buffer (pH 7.4) wi th a Poly t ron homo- genizer (15 sec at 10,000 rpm). 0.2 g-equivalent por t ions ~vere used for incubat ions. Less than one hour elapsed between obta inment of the tissues and incubat ion.

Incubation procedure.

Benzene /e thano l (9:1, v / v ) solutions conta in ing 0.7 ~g of the labelled steroid snbstrate were p ipe t ted into glass-stoppered 50 ml tubes and dr ied under ni trogen. A freshly p repa red 0.5 mM solution of NADPH in 0.067 M phosphate buffer (pH 7,4) and 200 mg of the tissue p repara t ion w e r e mixed in the incuba t ion tube. F ina l vo lume was 5 ml. Incubat ions were car r ied out in a shaking 'water bath for 30 rain. Time was increased to 60 mn for homogenates All incuba- t ions w e r e s topped by addi t ion of chi l led acetone (10 ml) and cooling at - -20°C. Fu r the r ex t rac t ion wi th ethyl acetate (10 m], 4 times) gave a c rude ext rac t w h i c h was concen t ra ted under ni trogen. Recover ies of the 14C-labelled steroids (94.5-98 per cent) w e r e computed after measurement of the rad ioac t iv i ty in por t ions of the extracts.

Thin layer chromatography.

Exper imen ta l processes leading to rad iometa- bolites separat ion and ident i f icat ion by crystal l i - zation to constant S.A. are out l ined in figure 1. A first separa t ion of rad ios te ro ids conta ined in the c rude extract was ach ieved on si l ica gel HFe54 + 3~6 after two developments in benzene / 95 per cent e thanol (9:1, v /v ) (system 1). Metabo- lites ~vere located on the ch romatogram by auto- r ad iog raphy prev ious to elution of the deact iva ted zones w i t h ethyl acetate or ethyl ace ta t e /me thano l (5:1, v /v ) . Separat ion of the 3u-androstanediol f rom the 3~-epimer in the eluted mix ture ~vas ach ieved on a lumina G after two developments in benzene /95 per cent e thanol (97:3, v /v ) (system 2). Eluates conta in ing both andros te rone and 5~-di-

BIOCHIMIE, 1977, 59, n ° 7.

d ihydro tes tos te rone were appl ied on a lumina G. T~vo developments in d i c h l o r o m e t h a n e / e t h e r (9:1, v /v ) (system 3) separated the two metaboli tes.

Radioact ivi ty measurements.

A Packard Tr icarb , model 3375 l iquid scint i l la- tion spec t romete r was used. All count ing rates exceeded 5 t imes the background and sufficient counts were al lowed to accumulate so that the

log A.S

4.3- 0 o 4.2- A ' ~ ' ~ * ~ - o O B ' ~ : - - * ° - o O

41- . 1 o j o

4.0- D ~ , ~ ® _ O O

39- 3.8" K ---'b - o °

3.7- ~o C " ~ ' P

36" E "~.,,°-- qP- Oo 3.5 ~ 34'

3 3 32.

o /o 3.1 L . ~ , ~ o - - o o 30 F j ° 29 " ~ , j o

2 8 ~"D - e °

2.7 2 6 / °

M / 25"~

24 - i . o 23 - "~O~o 0 22-

G . ~ . - - ~ ) - o o

j .__.o~O-oO H " - - 'o -o°oO ~o

o ,,o

/ P - \ • ,'~ '~ j o 0

e . j o e ~ o O 0

~ e - o o

Fto. 2. - - Crystallizations to constant S.A. of uuchan- .tied steroid subslrates and Cl~,02-metabolites obtained from incubations with minces of human normal and hyperplastic prostates.

In each case the log S.A. of the crystals (O) and mother liquors (O) is expressed from left to right for consecutive crystallizations of the initial solution con- taining authentic carrier.

In all cases, the first, second, third and fourth crys- tallization 'was carried out in ethyl acetate/n-hexane, acetooe/n-hexane, acetone/water and methanol/water, respectively.

A = unchanged 3~-androstanediol substrate, normal prostate. B ---- unchanged testosterone substrate, nor- mal prostate. C = 5¢t-dihydrotestosterone from testo- sterone substrate, normal prostate. D ----- unchanged 5a-dihydrotestosterone substrate, normal prostate. E -- 3(t-androstanediol from 5c~-dihydrotestosterone substrate, normal prostate. F ~ 3~J-androstanediol from 5ct-dihydrotestosterone substrate, normal prostate. G = unchanged 3(~-androstanediol substrate, BPH. H = unchanged testosterone substrate, BPH. J = 5~-dihydrotestosterone from testosterone substrate, BPH. K ---- unchanged 5a-dihydrolestosterone sub- strate, BPH. L = 3a-androstanediol from 5ct-dihydro- testosterone substrate, BPH. M = 3fJ-androstanediol from 5¢t-dihvdrotestosterone substrate, BPH. N = unchanged iso-androsterone substrate. BPH. O ----- 5a-dihydrotestosterone from 3a-androstanediol sub- strate, BPH. P = 5ct-dihydrotestosterone from 3[~- androstanediol substrate, BPH.

count ing e r ro r did not exceed _ 2.5 per cent. Quench ing was cor rec ted on the basis of external s tandard ratios.

46

640 R. F. Mor[in and coll.

Ide:dification of radiomelabolites.

E x c e p t 'when spec i f i ca l ly u n d e r l i n e d , iden t i f i - ca t ion of r a d i o m e t a b o l i t e s was c a r r i e d out as s h o w n in f igure 1 b y c r y s t a l l i z a t i o n to c o n s t a n t S.A. a f t e r i s o t o p i c d i l u t i o n of t he e lu ted r ad io - s t e ro ids w i t h a p p r o p r i a t e p u r e ca r r i e r s . C o m p u - t a t ion of spec i f i c ac t i v i t i e s in c ry s t a l s and m o t h e r l i quo r s f r a c t i o n s w a s based on gas - l iqu id c h r o m a - t o g r a p h y m e a s u r e m e n t s a c c o r d i n g to the p re - v ious ly d e s c r i b e d m e t h o d [113, 14] e x c e p t tha t t r i m e t h y l s i ly l e t he r d e r i v a t i v e s of t r i h y d r o x y - s t e ro ids w e r e f o r m e d p r e v i o u s to ana lys i s .

R E S U L T S .

Incubation of minces from BPH and normal prostatic tissues.

Subs t r a t e s w e r e e i t h e r [4-14C] t e s t o s t e r o n e o r ~4-14C] 5 a - d i h y d r o t e s t o s t e r o n e o r E4-14C] 3~- a n d r o s t a n e d i o l o r [4-14C] i s o a n d r o s t e r o n e . Yie lds

Both n o r m a l and BPH t issues p r e s e n t e d ex t en - s ive 5 a - r e d u c t i o n of t e s tos t e rone , h i g h e s t y i e ld s bve ing , for these t w o cases, in f a v o u r of the BPH digests . T h e m a j o r 5 a - r e d u c e d m e t a b o l i t e of tes to- s t e r o n e ~was 5 a - d i h y d r o t e s t o s t e r o n e w h i c h was c ry s t a l l i z ed to cons t an t S.A. (fig. 2) a f t e r s epa ra - t ion f r o m the a n d r o s t e r o n e c o n t a m i n a n t . P u r i t y y i e ld s of 100 p e r cen t w e r e o b t a i n e d in b o t h cases. I d e n t i t y of t he u n c h a n g e d t e s t o s t e rone sub- s t ra te w a s c o n f i r m e d by c r y s t a l l i z a t i o n to c o n s t a n t S.A. (fig. 2). S l ight d e c r e a s e in S.A. of the c rys t a l s f r a c t i o n s m a y be due to the p r e s e n c e of isoandro- s t e rone or 3o . -andros tanedio l c o n t a m i n a n t s . I d e n - t i ty of the o t h e r t e s t o s t e rone m e t a b o l i t e s w a s o n l y based on t h e i r c h r o m a t o g r a p h i c m o b i l i t i e s c o m p a r e d w i t h those of a u t h e n t i c s t a n d a r d s and p r e v i o u s e x p e r i e n c e s .

I n c u b a t i o n of [4-14C] 5 ~ - d i h y d r o t e s t o s t e r o n e led in b o t h cases to t he f o r m a t i o n of m a j o r quan- t i t ies of 3a- and 3~ -and ros t aned io l s ' w h i c h w e r e s e p a r a t e d by th in l a y e r c h r o m a t o g r a p h y and c r y s t a l l i z e d to c o n s t a n t S.A. (fig. 2). At c o n s t a n c y ,

TABLE I.

r4-~Cl testosterone, 5a-dihydrotestosterone, 3~-androstanediol and isoandrosterone metabolism by NADPH-supplemented minces of human normal and hyperplastic prostates.

Steroid metabolites

Non identified polars * 5a-Androstane-3~, 7a,17,~-

triol * 5a-Androstane-3 ~,6,~,173-

triol *39, 7~--Dihydroxy-5~-andros-

tan-17-one * 3~-Androstanediol * 3:t-Androst anediol * T or Iso A. Androsterone * 5~-Dihydrotestosterone Androstenedioue 5a-Androstane-3,17-dione

Normal prostate

[4-~4C] substrates

T IsoA

1.2

1.7

0 **1.5 **6.9 59.0

**1.6 *'25.1

2.1 0.9

DflT 3~-0L

0.8 3.0

I 2.4 +18.2

+ ~ l . 3

0 0 **4.9 **70.8

*'19.1 **4.0 0 1.3

**6.2 **0.2 **65.2 **1.3

0 0 1.3 0

2.3

0.1

5.6

1.0

90.1

0.1

0 0.8

tlyperplastie prostate

[4-t4C] substrates

T I DHT 3~-0L IsoA

1.7

1.3

0 **1.7 **7.8 37.8

**1.8 *'45.1

1.4 1.4

1.3

i 2.2

0 **4.1

*'13.1 0

**5.7 **70.1

fi 2.3

2.2

+15.3

+ 4 2 . 0

0 *'77.1

**0.4 1.1

**0.1 **0.9

0 0.8

2.8

0.1

6.4

1.7

85.4

2.8

0 0.8

Values are expressed as percentage contribution of the listed steroids to the total recovered radioactivity. Incubation conditions 'were : 0.7 I~,g substrate/0.2 g minces incubated for 30 min at 37°C in 5 ml 0.067 M phosphate buffer (pH 7.4) containing 0.5 mM NADPH.

T = testosterone, DHT : 5a-dihydrotestosterone, 3(~-OL ---- 31[~-androstanediol, IsoA = isoandrosterone. * Identified by crystal l ization to constant S.A. + Based on crystal l izat ion to constant S.A. ** Based on second thin layer chromatography separation.

o f s e p a r a t e d r a d i o m e t a b o l i t e s table I.

BiOCHIMIE, 1977, 59, n ° 7.

a re s h o w n in the s l igh t d e c r e a s e s in S.A. led to n e w c o m p u t a - t ions of the y i e ld s ' w h i c h ~Tere for the n o r m a l

P r o s t a t i c h g d r o x g l a t i o n s of 5~t-androstane-315,17~-diol. 641

prostate and BPH of 3.6 and 2.1 per cent for the 3B-androstanediol and 18.3 and 12.8 per cent for the 3ct-androstanediol, respect ively . There fore highest yields in 3~t- and 3B-reduction w e r e obtained v¢ith the normal prostate.

Use of [4-~C1 3I -andros tanedio l substrate led to extensive format ion by BPH and normal tissues of a single polar rad ioac t ive metabol i te separated by thin layer ch roma tog raphy (table I). Half of each cluate was added to authent ic 5ct-andro- stane -3~,7.a,17~-triol ca r r i e r and crystal l ized to constant S.A. (fig. 3). When cons tancy was

Io9.S.A.

4.0 3.9 3.8 3.7 36 3.5 3.4 3.3 32 3,1 3.0 2.9 2.8 2.7 2.6 25 2.4

B. ~ '° E / o H /°

F ~ o

Fro. 3. - - Crystallizations to constant S.A. of C,O,- .,'leroids oblained from incubations of labelled precur- sors !with minces and homogenates of human normal and hgperplastic prostates.

In each case the log S.A. of the crystals (O) and mother liquors (O) is expressed from left to right for consecutive crystallizations of the initial solution con- taining authentic carrier.

In crytallization of the 5ct-andro~tane-3~,6~,17B- triol and 3~,7c~-dihydroxy-5.tx-androslan-17-one, the first, second~ third and fourth crystallization was carrfed out in ethyl acetate/n-he'xane, acetone/n- hexane, acetone/~vater and methanol/water, respecti- vely. Crystallizations of 5a-androstane-3,~,7u,17~-triol were carried out successively 'with acctone/n-hexane, acetone/water and methanol/water.

A = 5ct-androstane-31f~,7a,l%-triol from 3r~-andro- stanediol substrate, BPH homogenate. B ---- 5a-andro- stane,-3B,7a,17~-triol from 3i~-androstanediol substrate, BPH homogenate. C = 5a-androstane-3B.7a,17~-triol from 5(t-dihydrotestosterone substrate, BPH homoge- nate. D = 5a-androstane-3B,7a,17;~-triol from 3B-andro- stanediol substrate, normal prostate minces. E = 5~t- androstane-3~,6~,176-triol from 3~-androstanediol sub- strate, normal prostate minces. F = 5et-androstane- 3~,7ct,17B-triol from 3~-androstanediol substrate, BPH minces. G = 5ct-androstane-3B,6~8,17{~-triol from 3B- androstanediol substrate, BPH minces. H = 3~,7a- dihydroxy-5~rt-androstan-17-one from iso-androsterone substrate, BPH minces.

15.3 per cent in BPH. The other half of each eluate was invest igated for the presence of 5a- androstane-3,~,6~,17,~-triol. In that order authen- tic ca r r i e r was added and 4 crysta l l izat ions ca r r i ed out (fig. 3). Extens ive drops in S.A. to the 4th crystals f rac t ion did not al low accurate measure- ments of S.A. in a fifth crystal l izat ion. Never- theless, even that constancy was not formal ly reached, only small differences exist be tween the four th crystals and mother l iquors S.A. and expe- r ience showed that three crysta l l izat ions w i t h a wrong ca r r i e r resulted in complete loss of radio- ac t iv i ty in the last crystals f ract ion. Accord ing ly the ident i ty of E4-14C] 5u-androstane-3~-6~,176- tr iol present in small quant i t ies is s t rongly sug- gested. Computed yields are then < 1.3 per cent and < 2.0 per cent for the normal and hyper - plast ic prostates, respect ively . L imi ted quant i t ies of the t r iol ca r r i e r did not pe rmi t other crystal l i - zations. In both cases, the r ecovered [4-14C] 3~-androstanediol substrate non separa ted f rom possible 3a-androstanediol con taminan t ~'as crys- tal l ized to constant S.A. (fig. 2). Ident i ty of o ther E4-14C] 3~-androstanediol rad iometabol i tes was solely based on the i r ch roma tog raph ic mobil i t ies.

Both normal and BPH tissue minces t ransfor- med the E4-14C] isoandrosterone substrate into major quant i t ies of a single metabol i te w h i c h was isolated by thin layer ch roma tog raphy (table I). This rad iometabol i te der ived f rom BPH digests was identif ied in 74.1 per cent pur i ty y ie ld as 3~, 7a-dihydroxy-5a-androstan-17-one by crystal l i - zation to constant S.A. (fig. 3). The isoandroste- rone substrate ~vas also r ecove red and its radio- pur i ty checked by crys ta l l iza t ion to constant S.A. (fig. 2). Rad iocon taminan t s ~vere found to amount to 6.5 per cent and main ly cor responded ~ ' i th impur i t ies conta ined in the or iginal substrate. Minces f rom two other BPH tisues were used for ident i f icat ion of 5a-dihydrotes tos terone der ived f rom [4-14C] 3a- and 3~-androstanediol substrates. Because of low yields of t r ans format ion of the substrates and to avoid losses due to th in layer ch roma tog raphy and elution, 5a-dihydrotestoste- rone was not separated f rom possible androste- rone contaminant . Crystal l izat ion to constant S.A. of the 5a-dihydrotes tos terone ehmtes obta ined f rom [4-14C1 3ct- and 3~-androstanediol substrates (fig. 2) showed r ad iopur i ty yie lds of 49.7 and 57.3 per cent, respect ive ly , and suggested the pre- sence of the labelled isopolar 17-oxo contaminant .

reached, decrease in S.A. pe rmi t t ed to ca]culate yields of pure [4-~4C] 5a-androstane-3~,7a,17'~- t r i o l : 18.2 per cent in normal prostate and

Incubat ions of BPH homogenates.

Substrates for 60 min incubat ions of BPH tissue homogenates were e i ther [4-14C] 5a-dihydro- tes tosterone or [4-14C] 3¢t-androstanediol or

BIOCHIM1E, 1977, 59, n ° 7.

642 R. F. Morfin and coll.

TABLE II.

[4-s*C] 5a-dihgdrotestosterone, 3~-androslanediol and 3~-androstanedio~ metabolism by NADPH-supplemented homogenates of human BPH.

Steroid metabolites

Non identified polars * 5a-Androstane-3 ~, 7c¢, 17,~-trio] 3~-Androstanediol 3a-Androstanediol lsoandrosterone Androsterone * 5~-Dihydrotestosterone 5a-Androst ane-3,17-dione

[~.-l~C] substrates

DIlT 3c~-OL

5.8 4 .2 + 5 . 3 + 2 . 5

** 18.1 ** 2 .0 ** 57.9 ~* 84.3

1.3 0 .6

8 .4 6.5

2 .9 1.3

3~-OL

4 .0 +12 .3

** 79.9 ** 2 .6

0 .7

0.7

0 .2

Values are expressed as percentage con t r ibu t ion of the l is ted steroids to the total recovered radioact iv i ty . Incuba t ion condi t ions 'were : 0.7 ~g subs t r a t e / 0.2 g bomogcneized t issue incuba ted for 60 rain at 37°C in 5 ml wi th 0.067 M phospha te buffer (pH 7.41) con ta in ing 0.5 mM NADPH.

DHT = 5c~-dihydrotestosterone, 3.ct-OL = 3a-andros tanedio l , 3,[~-OL = 3~-- andros tanedio l .

* Identified by c ry ta l l i za t ion to cons tan t S.A. + Based on c rys ta l l iza t ion to cons tant S.A. "" Based on second t h i n layer ch roma tog raphy separat ion.

[4-14C] 3 i~ -and r os t aned i o l . R a d i o m e t a b o l i t e s y i e l d s a re s h o w n in t a b l e II .

P r e l i m i n a r y i d e n t i f i c a t i o n of l a b e l l e d t r a n s f o r - m a t i o n p r o d u c t s w a s b a s e d u p o n c h r o m a t o g r a - p h i c m o b i l i t i e s c o m p a r e d ~ i t h t h a t of a u t h e n t i c s t e r o i d r e f e r e n c e s . R a d i o a c t i v e z o n e s c o r r e s p o n - d i n g w i t h 5a -andros tane -3 [~ ,7cd7 i~- t r io l 'were i n d i - v i d u a l l y e lu ted , d i l u t e d w i t h n o n r a d i o a c t i v e c a r r i e r a n d c r y s t a l l i z e d to c o n s t a n t S.A. (fig. 3).

A f t e r t h i n l a y e r c h r o m a t o g r a p h y a 7 p e r c e n t r a d i o a c t i v i t y y i e l d 'was f o u n d a t t h e l eve l of t r i h y d r o x y m e t a b o l i t e s d e r i v e d f r o m 5c~-dihydro- t e s t o s t e r o n e i n c u b a t i o n s . C r y s t a l l i z a t i e n d a t a s h o w e d r a d i o p u r e 5a-andros tane-35 ,7(x ,17 '5- t r io l to c o r r e s p o n d w i t h a 5.3 p e r c e n t y i e ld . T h e d i f fe - r e n c e m a y a c c o u n t f o r o t h e r p o l a r r a d i o m e t a - b o l i t e s i n c l u d i n g t h e 5(~-androstane-3~,6 ,~, lT~-t r io l ' w h i c h w a s no t c h a r a c t e r i z e d in t h i s case.

S i m i l a r l y t h e 3.3 p e r c e n t a n d 15.3 p e r c e n t r a d i o a c t i v i t y y i e l d s m e a s u r e d a t t h e l eve l of 5a- audrostane-3i~,7,c~,17,fi-triol a f t e r t h i n l a y e r c h r o m a - t o g r a p h y of e x t r a c t s f r o m [4-1~C] 3a- a n d 3~- a n d r o s t a u e d i o l d i ge s t s ~ e r e r e s p e c t i v e l y b r o u g h t to 2.5 a n d 12.3 p e r c e n t a f t e r c r y s t a l l i z a t i o n to c o n s t a n t S.A.

D I SCU SSI O N .

P r e v i o u s r e p o r t s h a v e a l r e a d y e s t a b l i s h e d t h e f i r s t s t e p s of t h e c a t a b o l i c p a t h w a y s i n v o l v e d i n t e s t o s t e r o n e t r a n s f o r m a t i o n s b y t h e h u m a n p r o - s t a t e g l a n d [1-9]. T h u s , a r e d u c t i v e pathvv-ay, i n c l u d i n g t h e 1 7 ~ - h y d r o x y m e t a b o l i t e s , i n v o l v e d a

BIOCH1MIE, 1977, 59, n ° 7.

OH O

°! o

FI(I. 4 . - Testosterone catabolic pathtwa.tls in human normal and hyp.erplastic prostates,

@ 17~-Hydroxysteroid, 178-oxidoreduetase. (2) A4-3-Oxosteroid, 5.tx-reductase. @ 3a-Hydroxysteroid, 3,¢t-oxidorednetase. !~4) 3~5-Hydroxysteroid, 3~[3-oxidoreductase. (5) Steroid-6~-hydroxylase. (@ 3.~-Hydroxysteroid, 7~-hydroxylase.

* Identified by crystal l izat ion to constant S.A.

A ~ - 3 - o x o s t e r o i d - 5 ~ - r e d u c t a s e a n d 3~- a n d 3,~- h y d r o x y s t e r o i d o x i d o r e d u c t a s e s a n d l ed to ep i - I n e r i c 5 a - a n d r o s t a n e d i o l s . T h e s e t r a n s f o r m a t i o n s w e r e o p p o s e d to a n o x i d a t i v e p a t h w a y l e a d i n g to

Pros ta t i c h g d r o x y l a t i o n s o[ 5a-androslane-3i3,17~-diol. 643

the c o r r e s p o n d i n g 17-oxosteroids by a 17~-hydro- xys t e ro id ox idoreduc tase .

Evidences r epo r t ed here for 6r0- and 7a-hydro- xy la t ions of 5a-androstane-3~,17!~-diol in h u m a n norma l and h y p e r p l a s t i c p ros ta te comple te and conf i rm a p rev ious r epo r t [~]] and fur ther ex tend the known tes tos terone me tabo l i sm in h u m a n prosta te . Accord ing ly , the comple ted p a t h w a y s are p re sen ted in figure 4.

Prev ious w o r k s have shown the steroid-6~- h y d r o x y l a s e f rom human pros ta te not to be ve ry specif ic for the s te ro id substrate . Thus testoste- rone [3], and ros t ened ione ~3], p roges te rone [15] and 5u-pregnane-3,20-dione [!1~] we re h y d r o x y - la ted at the 6-posit ion. I t is the re fore not su rp r i - s ing to obta in a 5a-androstane-3~,6~,17,~-triol f rom the 3i~-androstanediol substrate .

Unident i f ied po la r metabol i tes of 3~-androsta- ned io l and 5ct-dihydrotes tosterone may con ta in 6~-hydroxyl der ivat ives . In m a r k e d contras t , the 7~-hydroxyla t ion in pros ta te or o ther ta rge t t issues [~, '17] has never been desc r ibed on ste- ro ids o ther than those w i t h a 31~-structure, even tha t 7a -hydroxy la t ion of tes tos terone, andros te - ned ione and DHEA has been shown in ra t test is w h e r e i t is suggested to have a r egu la to ry effect on tes tos terone b iosyn thes i s [i18]. Our resul ts show that h ighes t y ie lds of 7¢t-hydroxylat ion by h u m a n p ros t a t i c t issues were ob ta ined f rom the 3,~-androstanediol and isoandrosterone substra tes . Lo'w y ie lds ob ta ined f rom testosterone, 5a-dihy- d ro les tos te rone and 3a -andros taned io l incuba t ions suggest that p r e l i m i n a r y act ion of the 3~-hydro- xys t e ro id dehyd rogenase is a p re requ i s i t e and tha t a 3,~-structure is r e q u i r e d for ac t ion of the p ros t a t i c s t e ro id -7a-hydroxy lase .

The observed 6~- and 7a -hydroxy la t ions of 5a- androstane-3~, 17~-diol in p ros ta t e may occur in o ther species w h i c h we re used as models , inclu- d ing canine w h e r e NADPH-dependen t 6~- and 7~-hydroxyla t ions 'were demons t ra t ed [6] and mur ine where 3B-androstanediol , isoandrosterone and 5o.-dihydrotestosterone incuba t ions led to the extens ive fo rmat ion of un iden t i f i ed p o l a r metabo- l i tes [19, 20].

S tudy of p r ecu r so r s for 7a -hydroxy la t ion ra ises a po in t on the mutua l t r ans fo rma t ions of 3B- andros taned io l , 5ct -dihydrotes tos terone and 3a- andros taned io l . In vivo s tudies of the fate of these subs t ra tes in h u m a n pros ta te have shoxvn 5ct-dihy- d ro tes tos te rone to be t r ans fo rmed into ma jo r quant i t ies of e p i m e r i c d iols w i th h ighe r conver - s ion y ie lds for the 3a-ep imer [1, 2], and both diols to be p r ecu r so r s of 5a -d ihydro tes tos t e rone [7].

These resul ts a re to be co r re l a t ed wi th p rev ious in vivo w o r k on ra t p ros ta te whe re s imi la r resul ts we re ob ta ined [21]. Imp l i ed t r ans fo rma t ion of 3~B-androstanediol into 5a -d ihydro tes tos t e rone must be opposed to in vitro data ob ta ined by Robel et al. and Roy et al. [19, '20] wi th ra t p ros ta te organ cul tures w h e r e only t race amounts of 5ct-di- hydro tes tos t e rone we re found.

Our f indings that 3a -andros taned io l is t r ans for - med into 5ct-dihydrotes tos terone and 3~-andro- s taned io l before 7a -hydroxy la t ion agrees wi th the c i ted in vivo and in vitro results . In contras t , t r ans fo rma t ions of our 30-andros tanedio l into p o l a r metabol i tes and t race amounts of ident i f ied 5a -d ihydro tes tos te rone only suppor t the in vitro works .

A recent s tudy of the 3or-reduction of 5et-dihy- dro tes tos te rone in human pros ta te d i scussed of d i f ferent op t ima l pH for the 3a- and 3~-hydro- xys t e ro id ox ido- reduc tases [8] w h i c h might expla in , in add i t i on to di f ferent subce l lu la r loca- t ions and cofac tor ava i lab i l i ty , the lower y ie lds of t r ans fo rmat ions by the 3~-enzyme. These diffe- rences may also be involved in the low fo rma t ion of 3-oxo metabol i tes f rom 3~-hydroxys te ro id sub- s trates. In such case, resul ts ob ta ined by in vivo s tudies w h e r e 3-oxo metabol i tes may be fo rmed exogeneous ly to the p ros ta te [2] must be diffe- r en t i a ted f rom in vitro s tudies w h e r e t issue pre- pa r a t i ons incuba ted at a k n o w n pH [19, 20] do not seem to oxid ize the 3~-hydroxy substrates .

Only smal l quant i t ies of 3~-andros tanedio l are found af ter tes tos terone incuba t ions w i th h u m a n prosta te . Major 7u-hydroxy la t ion of the 3~-andro- s taned io l may exp la in such low y ie lds and impl ies a ne'w metabo l ic p a t h w a y for this d ihy- d roxys t e ro id . Robel et al. have shown in ra t p ros ta te organ cul ture [19] that the 3~-androsta- ned io l ma in t a ine d the ep i the l i a l cell he ight and s t imula ted sec re to ry act ivi ty . In con t ras t w i t h 5ct-dihydrotestosterone, i t d id not p romote hype r - p las ia . I t was conc luded tha t full ma in t enance ~xithout h y p e r p l a s i a of p ros t a t i c cells may be based on the fo rma t ion of 3~-andros tanedio l and that the 5a-d ihydro tes tos te rone /3 :~-andros taned io l ba lance could p l ay a role in the occurence of p ros t a t i c h y p e r p l a s i a r a t h e r than the absolute concen t ra t ion of 5ct-dihydrotestosterone.

Actual suppor t for the invo lvement of 5a-andro- stane-3.~,7a,17i~-triol in androgen ac t ion is the fa i lure to detect th is t r i h y d r o x y s t e r o i d above 1 ~g l imi t in 24 h u r ines f rom n o r m a l human males by gas- l iquid c h r o m a t o g r a p h y (F. Berthou, pe r sona l communica t ion ) .

BIOCHIMIE, 1977, 59, n ° 7.

644 R. F. Morfin and coll.

T h e r e f o r e , e f f ec t s of t h e 3 ~ - a n d r o s t a n e d i o l a n d of i t s 7 a - h y d r o x y l a t e d m e t a b o l i t e o n n u c l e a r re -

c e p t o r s of r a t a n d h u m a n p r o s t a t e r e m a i n y e t to be s t u d i e d a n d a s c e r t a i n e d . I t a l so r e m a i n s to be s e e n i f s e c r e t o r y a c t i v i t y i n p r o s t a t i c e p i t h e l i a l ce l l s i s t r i g g e r e d b y t h e s e s t e r o i d s a n d to ~ h i c b

e x t e n t t h e p r o s t a t i c 3 ~ - h y d r o x y s t e r o i d o x i d o r e -

d u c t a s e a n d t h e 3 t ~ - h y d r o x y s t e r o i d 7 a - h y d r o x y l a s e a c t i v i t i e s a r e i n v o l v e d i n h o r m o n e a c t i o n a n d h y p e r p l a s i a .

Aeknotwledgements.

This work was suppor ted by a research contract (75-1-068-3) f rom INSERM (France). The a u t h o r s wish to t h a n k Dr. C. Moreau (Brest) for supply ing the pure Rhizopus nigrieans s t ra in and Sir Ewar t R. H. Jones (Oxford) for the gif t of C~O~-steroids.

R~svM~.

Des h roya t s et des homogdna t s de pros ta tes h u m a i - nes normale on hype rp la s iques sen t incub6s en pre- sence de NADPH avec des st~roides en C~.~O: m a r q u i s au ~4C. Ainsi la tes tos terone, la 5.c~-dihydrotestostd- rone, l ' i soandros t6rone , le 3(~-androstanediol et le 3,[3- andros taned io l p rodu i sen t des mdtabol i tes radioact i fs den t l ' identi t~ est prouvde par c r i s ta l l i sa t ion "h activit~ sp6cifique constante . Parrot les m6tabol i tes polaires de ces subs t ra t s , l ' ident i t4 du 5ct-androstane-3#,7a, 171~5-triol form5 par les deux types de t i s sus est prou- v~e et celle du 5(~-androstane-3i[~,61[5,17~[5-triol for te- m e n t suggdr~e. Une spdeifieit6 de la 7¢t-hydroxylat ion pros ta t ique pout" les subs t r a t s 3:[~-hydroxyl6s est pro- posde car seuls le 31~J-androstanediol et l'isoandroste- rone ont pour m6tabol i tes p r ine ipaux leurs d6riv~s 7et-hydroxyl6s respeet ifs . I1 est d ' au t re par t v6rifi6 que les subs t r a t s en C~.,O: hydroxylds en 3~ ne donnent , in vitro et en pr6sence de NADPH, que de tr6s faibles quant i tds de 3-oxostdroides. Pa r centre, et dans les m~mes condit ions, le 3c~-androstanediol est ac t ivement t ransfor ln6 en 5c¢-dihydrotestostdrone, pr~curseur du 3,~-androstanediol et de ses ddriv6s hydroxyl~s . La d~- m o n s t r a t i o n de ces activit~s e n z y m a t i q u e s pe rmet de pr6ciser les votes mdtabol iques de la tes tos tdrone dans la pros ta te et de m o n t r e r qu 'el les res ten t qua l i ta t ive- m e n t semblahles dans la g lande no rma le et hyper - plasique.

De possibles diffdrences quan t i t a t ives d 'act ivi tds e n z y m a t i q u e s ent re les deux t i s sus , le r61e du 5(x- androstane-3l~,7(x,17il]-triol et son in te rven t ion dans

les m~can i smes d 'ae t ion des st6roides androg~nes con- d u i s a n t h la sderdtion p ros ta t ique ou h l ' hyperp las ie res ten t h ~tre ddtermin~s.

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BIOCHIMIE, 1977, 59, n ° 7.