- Email: [email protected]
5α-Androstane-3β, 17β-diol and 5α-androstane-3β,7α, 17β-triol in the human hyperplastic prostate
Sa-ANDROSTANE-3B, 17b-DIOL AND Sa-ANDROSTANE-3fl,7cr,17/?-TRIOL IN THE HUMAN HYPERPLASTIC PROSTATE R. F. MORFIN’, s. DI S-rEFANot,J.-F. CHARLES# and H. H. FLockIt lLaboratoire de Biochimie. ERA 784 du C.N.R.S., Facultk des Sciences, 29283 Brest Cedex tLaboratoire de Biochimie, ERA 784 du C.N.R.S.. FacultC de MCdecine. 29279 Brest Cedex, and $ervice de Chirurgie GCntrale, C.H.U., 29200 Brest, France SUMMARY Slices
of human prostates with benign hypcrplasia were separated after 60min incubation from the NADPH-supplemented medium containing [4-‘*CJ-5a-androstane-3/3,17fi-diol and/or [1,2-‘HI-Suandrostane-3j.7a.17fi-triol. Metabolism and retention of Sa-androstane-3/Xl7/%diol and [email protected],l7P-trio1 in the slices were derived from the quantities of labelled substrates and metabolites in medium and slices. Accordingly. 5z-androstane-38.17b-dial was transformed into Sz-androstane3B.7z.17fl-triol. Sa-androstane-3a.l7fi-diol and 17b-hydroxy-5c+androstan-3-one. Minor transformation of 5z-androstane-3/I.7r.l7fi-triol into31,7a-dihydroxy-Sol-androstan-17-one was noticed. Retention and metabolism of 5a-androstane-3B.l7fi-diol in tissue were about four times larger than for Sa-androstane-3,%7z.l7fi-triol. In both cases. the largest quantities of substrate and metabolites were found in incubation medium. These results and the absence of binding of the See-androstane-3/?,7a.l7/?-trio1 by prostatic cytosol proteins. suggest an active excretion of the trihydroxysteroid from the tissue.
lNTRODUCllON
Using mince and homogenate preparations of normal and hyperplastic human prostates, we have previously demonstrated that NADPH-dependent hydroxylations occur at the 6/% and predominantly ?'K- positions of 5a-androstane-38.1 ‘I/?-diol (3/?-diolQ) and isoandrosterone [l. 23. Hydroxylations at the 65- and 7{- positions of 3/Ldiol were also reported with canine prostate C3.43. Recent work by Ofner et al.[4] with serum-free cultures of canine prostate explants showed that hydroxylated metabolites of 38-diol and isoandrosterone were released into the medium. Disposal of androgens from the prostate by their 3/3-reduction coupled with hydroxylations was suggested [4]. The incubation of slices of hyperplastic human prostate in a medium containing [1,2-3H]-7c(-triol and/or [4-“Cl-3fi-dial followed by analysis of the labelled metabolites and substrates in the slices separated from the medium provided means for extending to human the results found in canine. We now present evidence that hydroxylation of 3j?-diol into 7z-trio1 may serve for the disposition of 3jLdiol precursors from the human prostatic tissue. EXPERIMENTAL
lithium/liquid ammonia in the presence of ethanol. The resulting [4-’ 4q-5cc-asdrostane-3fl, 17$diol was purified by thin layer chromafography and by alumina column chromatography eluted with a gradient of ethanol (&S%) in benzene. A portion of the purified 3/3-diol was diluted with authentic carrier and crystallized to constant specific activity. Radiochemicat purity was 99.6%. [1,2-3H]-5a-Androstane-3~,7a,17/?-triol (45 Ci/ mmol) was biosynthesized and purified as previously described [S] with a radiochemical purity of 94.5%. Carrier steroids and solvents
All solvents were of reagent grade or distilled before use. All carrier steroids were purchased from Merck and Sigma but the 7a-trio1 which was prepared from 5-androstene-3/?,17B-dial as previously described [S]. Prostatic tissues
Hyperplastic prostate glands were removed by the transvesical approach from patients undergoing surgery. As soon as obtained, the glands were chilled in a beaker surrounded with ice before transport into a cold room. At 4”C, tissues were finely sliced with scissors and 2-g portions prepared for incubations.
Labelled steroids
Incubation procedure
[4-‘Q-Testosterone (50 mCi/mmol) was purchased from C.E.A. (France) and was reduced with
Benzene-ethanol (9: 1, v/v) solutions of [4-14c7-3fldiol (7 ug) and/or [1,2-3H]-7a-triol (10.36 ng) were transferred into incubation flasks and dried under nitrogen. In three cases of mixed substrates, 6.99 pg of non-radioactive ‘lo+trio1 were added. Incubation medium consisted in 50 ml of 0.067 M phosphate buffer (PH 7.4) containing 5 mg NADPH. Incubations
S Abbreviations used: Sz-DHT. 17/l-hydroxy-5cc-androstan-3-one: isoandrosterone. 3/?-hydroxy-5a-androstan-17one; androsterone. 3a-hydroxy-Sg-androstan-17-one; 3fi-diol, 5a-androstane-3p.l7#?-dioi ; 3a-diol, SK-androstane3a..l7B-diol: 7a-triol. 5a-androstane-3j.7ar.17/S-triol.
529
R. F. MORFINet al.
530
sliced tissues were carried out at 37°C in the presence of air in a shaking water bath. After 60 min. tissue slices were quickly separated from the medium and rinsed three times with 2ml of chilled buffer. The three rinses and 20ml of acetone were added to the medium. The mixture and slices in acetone were stored at -20°C prior to extraction. with 2 g of the
All operations were carried out at 2°C. After incubation with [I.2-3H]-7z-triol. the washed slices were suspended in 5 ml of buffer and homogenized with a Polytron homogenizer (five times at 10,000 rev./min for 5 s with intervals of IO s for cooling). The supernatant recovered after centrifugation of the homog~ate at 600 g for 10 min was ultracentrifuged for 60 min at 105,CQOgand yielded the cytosol fraction. A portion of the cytosol was applied on a Sephadex G-25 column eluted with 0.067 M Phosphate buffer for separation of the free steroids from those bound with cytosoiic proteins. Separation
of sreroid metaboiites
Incubation media. slices and fractions eluted from Sephadex G-25 were extracted with ethyl acetate. Prior to thin-layer chromatography, 1Ogg of non radioactive steroid carriers (5x-DHT. 3x-dial. 3&diol, 7r-trio]) were added to the extracts. Separation of steroids was carried out by thin-layer chromatography on silica gel HFr5*+,e6 (Merck) in benzeneethanol (9: 1. v/v) twice. Autoradiography was used for locating the “C-labelled or ‘H/W-labelled steroids on the chromatograms. Carrier-diluted ‘H-labefled steroids were detected under U.V. (336nm). The separated spots were scrapped from the plate directly into scintillation vials prior to the addition of three drops of methanol and 10 ml of Instagel (Packard). Rlf(lio~Icticit~
mettstrrements
A Packard Tricarb, model 3375 liquid ~intiIlation spectrometer was used. Counting of 3H. “‘C and ‘H/‘4C was carried out on preset windows. Ail counts were expressed in d.p.m.. quenching being corrected on the basis of external standard ratios. 1. Average per cent k standard deviation of the f4-‘%Wabel associated with the 3B-diol substrate and its
Table
mctabolites recovered from prostatic slices and incubation medium
Table 2. Average per cent + standard deviation of the [I,2-‘H] label associated with the 7x-trio1 substrate and its metabolites recovered from prostatic slices and incubation medium Identified steroids [Z]
Medium
S~-Androstane-3~.l7&diol So-Androstane-36.7x.17~.trioi 5x-Androstane-3;.17j-dial 5x-Dihydrotestosterone
53.6 8.6 10.5 0.9
-i_ 18.8 + 0.9 z 9.9 -1:0.4
Total recovered steroids
77.9 + 14.2
Slices 17.3 f 1.6 + 1.9 I 0.3 f
ft.4 0.8 1.6 0.2
23.1 & 14.1
Two grams of slices from four hypetptastic prostate glands were incubated for 60 min in 50 ml 0.067 M phosphate buffer (pH 7.4) containing 7 pg of [4-‘aC]-3#Ldiol and NADPH (5 mgl.
Slices
5z-Androstane-3/I,7a.I7&trioI 3~,7z-~hydroxy-Saandrostan- I7-one Polar unknowns
87.0 k 8.9 2.9 * I.9
3.8 k 1.2 0.4 & 0.2
1.4 * 0.2
0.2 i: 0.2
Total recovered steroids
93.7 rf: 4.3
6.3 k 4.2
Two grams of slices from four hyperplastic prostate glands were incubated for 60min in 50 ml 0.067 M phosphate buffer (pH 7.4) containing 10.36 ng of [1.2-‘HI-71trio1 and NADPH (5 mg).
RESULTS
incubation
of 7 /,tg [4-14C]-3#3-dioi
In control incubations where tissue slices were omitted, the 3j?-diol substrate remained unchanged. Therefore, metabolites found in the medium after incubation with slices were produced by the tissue from the 3/I-dial which penetrated the ceils. The prostatic metabolites of 3#?-dioi previously identified as 7x-triol. 3a-dioI and Sa-DHT [Z] were found in slices and medium (Table 1). From the data presented in table 1, the total radioactivity associated with metabolites found in the medium was 24.3% of the incubated label. Since 22.1% of the total label was found in slices, we may consider that 46.4% of the incubated 3&dioI were retained and/or transformed by the slices, and that most of its transformation products were liberated into the medium. Iltcubation of 10.36 ng [1,2-3H]-7r-triol In control incubations where tissue slices were omitted, the 7x-trio1 substrate remained unchanged. After incubation with slices. 877; of the incubated lable was found unchanged in the medium (Table 2). The difference (13%) mostly included the unchanged 7x-trio1 found in the slices and the few metabolites extracted from slices and medium. As expected the previously identified 3&7x-dihydroxy-%androstan-1Fone [2] was one of these metabolites. Other minor poiar metabohtes were not identified and no transformation into 3/Ldiol was noticed. Iucubatiort
Identified steroids [2]
Medium
qf [ 1,2-3H]-7z-trioli[4-‘*C]-3~-diol
Two sets of experiments were carried out. One. with prostates A. B and C, incubated the slices with a mixture of 7 .ug [l.2-3H]-7x-trioI and 7 1-18 [4-‘“Cl-3& diol (Table 3). The second set used prostates D and E with a mixture of 10.36 ng [I&‘HI-7z-trio1 and 7~g[4-L4C]-3/I-dio1 (Table 4). In both cases. the ‘H/“C ratios were measured in extracts from medium and slices and in the 7z-trio1 fraction isolated from each extract (Tables 3 and 4). In addition, recovered ‘H and “C d.p.m. were used to compute per
Sa-Androst~e-3~,17&diol
and 5~-androstane-3~,7cl?&triol
531
Table 3. Per cent yields and ‘H/‘*C ratios of total steroids and isolated 5%.androstanc-3/3.7a.l7p-triol
in medium and
slices from three different hyperplastic prostate glands Prostate A Total Isolated steroids trio1 Incubated Medium Slices
JH/“C per cent { pet cent jH/W per cent per cent JH/‘Y
‘H ‘V ‘H ‘*C
4.12 92.0 82.3 4.6 8.0 17.7 1.87
Prostate B Total Isolated steroids trio1 9.75 94.8 86.4 10.47 5.2 13.6 3.76
8:6 16.4 21.78 7.6 2.7 Il.65
Prostate C Total Isolated steroids trio!
879 18.4 46.57 4.6 3.1 14.53
9.91 97.0 92.3 10.41 3.0 7.6 3.86
8:6 8.2 97.26 2.0 0.5 40.55
Two grams of slices were incubated for 60 min in 5Oml 0.067 M phosphate buffet (pH 7.4) supplemented with NADPH (5 mg) and containing a mixture of ~4-‘4C]-5a-androstane-3~.17/?-diol (7 @g)and [l.2-3H]-Sa-androstane-3/3,7a,17j3-triol
yields of the labels in medium and dices (Tables 3 and 4). In both experiments, the incubated 3H/i4C ratios were increased in incubation media and decreased in slices. On the other hand total steroids from slices always contained at least twice more “C than ‘H. These data are in support for a higher tissular retention and metabolism of the ‘*C-labelled 3&diot than that of C3HJ-7a-trio1 at two widely different concentrations. Correlatively, decrease of the 3H/14C ratio in isolated 7a-trio1 was larger in slices than in medium and the per cent of neoformated [‘4Cj-7a-triol was 6 to 16 times larger in medium than in slices. These data reflect the low retention and meta~iism of %-trio1 in the tissue and support the excretion of the neoformated [4-‘*C’j-7e(-trio1 from the slices.
DkSCUSSON
cent
fnvestigarion 0f[l.t-~H]-7a-triol prostotic cytosol
binding to proteins of
In four cases, after incubation for 60min with 10.36ng of [l.2-3HJ ?a-trioi, prostatic cytosol was isolated from the slices. The cytosol fractions contained from 0.9 to 4.6% of the incubated 3H label. The labelled free steroids were separated from the cytosol proteins by gel filtration on sephadex G-25. No significant amount of tritium iabel was found associated with the isolated proteins.
The recent work by Ofner et ul.[4] has shown that cultures of canine prostate explants disposed of radiotestosterone by a predominant transformation into 3fl-hydroxy-Sa-reduced-7<-hydroxylated radiometabolites which were released into the medium. Using a different approach with incubations of human prostatic minces, we have proved that the 3j?-diol, a prostatic metabolite of testosterone, was a precursor for 7a-triol[23. We now report that the 3/Ldiqi is more retained and metabolized than the ‘la-trio1 in the human hyperplastic prostate. Evidences supporting this conclusion are: (i) The larger quantities of metabolites formed from 3@diol than from 7a-triol. (ii) The irrtversible prostatic transformation of 3j%diol into 7a-triol. (iii) The absence of binding of 7a-trio1 in prostatic cytosol while 3jSdiol is a precursor for Sa-DHT. (iv) The decrease of 3H/14C ratio in slias when [3H}-7a-trioV[‘6q3B-diol is incubated. On the other hand, we suggest that ‘la-trio1 is a terminal metabolite actively formed from 3/?-diol and excreted from the human prostate. Supports for this suggestion are: (i) The irreversible prostatic transformation of 3/l-dial into 7a-triol. (ii) The small quantities of ‘la-trio1 transformed by the prostate. (iii) The much larger quantities of %-trio1 found in medium than in
Table 4. Pet cent yields and 3H,‘14C ratios of rota1 steroids and isolated Srr-androstane3/X72.17/3-trio1in medium and slices from two different hyperplastic prostate glands Prostate E Isolated Total trio1 steroids
Prostate D Isolated Total trio1 steroids Incubated Medium Slices
3H/‘4C per cent ‘H per cent ‘Y aH/‘4C { per cent 3H ( per cent ‘%I. 3H,“*C
9.83 96.5 93.2 10.17 3.5 6.8 5.04
8;7 12.9 68.19 3.1 1.7
17.44
9.96 94.5 89.9 IO.47 5.5
8& 13.4 65.96 4.7
10.1 5.44
I.6 29.41
TWO grams of slices were incubated for 60 min in 50 ml 0.067 M phosphate buffer (PH 7.4) supplemented with NADPH (5 mg) and containing a mixture of [4-“a-5a-andrdstane3fi.l7P-dial (7 pg) and [1,2-‘H]Jcc-androstane-3fi.7a.l7~-triol
(10.36 ng).
532
R.
F.
MORFIN et al.
slices after incubation of 3@-dioi. (iv) The decrease of 3H!L”C ratio in isolated 7cx-trio1 after incubation of [3H]7~-triol/[1JC]-3P-diol, this decrease being more extensive in slices than in medium. Our findings confirm and complete those reported by Morst er ~~[.[6-j who injected [3H]-3@dioI in six live patients 30 min prior to prostatectomy. The radioactivity associated with the 38-diol and its metaboiites was found six times larger in I ml of plasma than in I g of prostatic tissue. The major identified metabolites were Sa-DHT, 3u-diol and isoandrosterone but their quantities were also larger in plasma than in prostatic tissue. These results agree with an
elevated clearance of the neoformated Scr-DHT and 3*-diol from the prostate [73 and with our finding of larger quantities of these metabolites in medium than in tissue slices. Our use of NADPH in incubation medium did not favor the formation of isoandrosterone and comparison with injection [6] and superfusion [7] studies aie not possible in that respect. In contrast. neither Horst et a[.[61 nor Malathi and Gurpide[7] mentioned polar metabolites which could account for the 7z-trio1 formed from the 3/?-dial substrate. Nevertheless. recent work by Jacolot and Berthou @ersonat communications carried out to complete the findings of Mauvais-Jar&s er u/.@} showed that following injection of [JH]-3/?-diol the urines of human males contained labelled sulfate and glucuroconjugated derivatives of isoandrosterone. androsterone. 3z-diol. 7r-trio1 and 3&%-dihydroxy-Sz-androstan- 17-one. These findings and our evidences suggest an elevated ckarance of 3/?-dial metabolites from the target tissues to androgens. Implica-
tions are that the human prostate gland may dispose of intracellular androgens by a coupled 3/l-hydroxysteroid oxidoreductase-7a-hydroxyiase system. The estrogen inhibition of the terminal hydroxylation proved in canine [4] remains to be investigated in human prostate.
REFERENCES
I. Morfin R. and Floch H.: 7a-Hydroxylation du 5a-androstane-3fl, 17/3-diol dans la prostate humaine normale et hyperplasique. C.R. mud. Sci. Paris 235 f I977) 897-900. 2. Morfin R. F.. Di Stefano S.. Charles J.-F. and Floch H. H.: Precursors for 6/?- and 7a-hydroxylations of 5a-androstane-3fl,178-diol by human normal and hyperplastic prostates. Biochimie 59 (1977) 637-644. 3. Ofner P.. Vena R. L. and Motlin R. F.: Acetylation and hydroxylation of 5u-androstane-38.178diol by prostate and epi~dymi~ Steroids 24 (1974) 261-279. 4. Ofner P, Vena R. L.. Leav I. and Hamilton D. W.: Metabolism of CtP-radiosteroids by explants of canine prostate and epididymis with disposition as hydroxylated products: a possible mechanism of androgen inactivation. J. sreroid &&em. 11 (1979) 1367-1379. 5. MO&I R. F.. Di Stefano S.. Bardqu L G. and Floch H. H.: Biosynthesis of [ 1.2-3H]-5a-androstane3~.7al7~-~io~. ~j~~i~ie 59 (1977) 649-650. 6. Horst H.-J.. Dennis M, Kaufmann J. and Voigt K. D.: In ttitu, uptake of [‘H J-5a-androstane-3s. 17_&diol and of [JH]-5a-androstane-38.178-diol by human prostatic hypertrophy. Acta endow., Copenh. 79 (1975) 39&tO2. I. Malathi K. and Gurpide E.: Metabolism of h-dihydrotestosterone in human benign hyperplastic prostate. J. sreroid Bioclrern. 8 (1977) 14l-l45. 8. Mauve-J~is P., Guiilemant S.. Corvof P.. F&h H. H. and Bardou L.: Metabolism of radioactive 5a-androstane-3fi,l78-diol. Steroids 16 ( 1970) l73- 18I.
of human prostates with benign hypcrplasia were separated after 60min incubation from the NADPH-supplemented medium containing [4-‘*CJ-5a-androstane-3/3,17fi-diol and/or [1,2-‘HI-Suandrostane-3j.7a.17fi-triol. Metabolism and retention of Sa-androstane-3/Xl7/%diol and [email protected],l7P-trio1 in the slices were derived from the quantities of labelled substrates and metabolites in medium and slices. Accordingly. 5z-androstane-38.17b-dial was transformed into Sz-androstane3B.7z.17fl-triol. Sa-androstane-3a.l7fi-diol and 17b-hydroxy-5c+androstan-3-one. Minor transformation of 5z-androstane-3/I.7r.l7fi-triol into31,7a-dihydroxy-Sol-androstan-17-one was noticed. Retention and metabolism of 5a-androstane-3B.l7fi-diol in tissue were about four times larger than for Sa-androstane-3,%7z.l7fi-triol. In both cases. the largest quantities of substrate and metabolites were found in incubation medium. These results and the absence of binding of the See-androstane-3/?,7a.l7/?-trio1 by prostatic cytosol proteins. suggest an active excretion of the trihydroxysteroid from the tissue.
lNTRODUCllON
Using mince and homogenate preparations of normal and hyperplastic human prostates, we have previously demonstrated that NADPH-dependent hydroxylations occur at the 6/% and predominantly ?'K- positions of 5a-androstane-38.1 ‘I/?-diol (3/?-diolQ) and isoandrosterone [l. 23. Hydroxylations at the 65- and 7{- positions of 3/Ldiol were also reported with canine prostate C3.43. Recent work by Ofner et al.[4] with serum-free cultures of canine prostate explants showed that hydroxylated metabolites of 38-diol and isoandrosterone were released into the medium. Disposal of androgens from the prostate by their 3/3-reduction coupled with hydroxylations was suggested [4]. The incubation of slices of hyperplastic human prostate in a medium containing [1,2-3H]-7c(-triol and/or [4-“Cl-3fi-dial followed by analysis of the labelled metabolites and substrates in the slices separated from the medium provided means for extending to human the results found in canine. We now present evidence that hydroxylation of 3j?-diol into 7z-trio1 may serve for the disposition of 3jLdiol precursors from the human prostatic tissue. EXPERIMENTAL
lithium/liquid ammonia in the presence of ethanol. The resulting [4-’ 4q-5cc-asdrostane-3fl, 17$diol was purified by thin layer chromafography and by alumina column chromatography eluted with a gradient of ethanol (&S%) in benzene. A portion of the purified 3/3-diol was diluted with authentic carrier and crystallized to constant specific activity. Radiochemicat purity was 99.6%. [1,2-3H]-5a-Androstane-3~,7a,17/?-triol (45 Ci/ mmol) was biosynthesized and purified as previously described [S] with a radiochemical purity of 94.5%. Carrier steroids and solvents
All solvents were of reagent grade or distilled before use. All carrier steroids were purchased from Merck and Sigma but the 7a-trio1 which was prepared from 5-androstene-3/?,17B-dial as previously described [S]. Prostatic tissues
Hyperplastic prostate glands were removed by the transvesical approach from patients undergoing surgery. As soon as obtained, the glands were chilled in a beaker surrounded with ice before transport into a cold room. At 4”C, tissues were finely sliced with scissors and 2-g portions prepared for incubations.
Labelled steroids
Incubation procedure
[4-‘Q-Testosterone (50 mCi/mmol) was purchased from C.E.A. (France) and was reduced with
Benzene-ethanol (9: 1, v/v) solutions of [4-14c7-3fldiol (7 ug) and/or [1,2-3H]-7a-triol (10.36 ng) were transferred into incubation flasks and dried under nitrogen. In three cases of mixed substrates, 6.99 pg of non-radioactive ‘lo+trio1 were added. Incubation medium consisted in 50 ml of 0.067 M phosphate buffer (PH 7.4) containing 5 mg NADPH. Incubations
S Abbreviations used: Sz-DHT. 17/l-hydroxy-5cc-androstan-3-one: isoandrosterone. 3/?-hydroxy-5a-androstan-17one; androsterone. 3a-hydroxy-Sg-androstan-17-one; 3fi-diol, 5a-androstane-3p.l7#?-dioi ; 3a-diol, SK-androstane3a..l7B-diol: 7a-triol. 5a-androstane-3j.7ar.17/S-triol.
529
R. F. MORFINet al.
530
sliced tissues were carried out at 37°C in the presence of air in a shaking water bath. After 60 min. tissue slices were quickly separated from the medium and rinsed three times with 2ml of chilled buffer. The three rinses and 20ml of acetone were added to the medium. The mixture and slices in acetone were stored at -20°C prior to extraction. with 2 g of the
All operations were carried out at 2°C. After incubation with [I.2-3H]-7z-triol. the washed slices were suspended in 5 ml of buffer and homogenized with a Polytron homogenizer (five times at 10,000 rev./min for 5 s with intervals of IO s for cooling). The supernatant recovered after centrifugation of the homog~ate at 600 g for 10 min was ultracentrifuged for 60 min at 105,CQOgand yielded the cytosol fraction. A portion of the cytosol was applied on a Sephadex G-25 column eluted with 0.067 M Phosphate buffer for separation of the free steroids from those bound with cytosoiic proteins. Separation
of sreroid metaboiites
Incubation media. slices and fractions eluted from Sephadex G-25 were extracted with ethyl acetate. Prior to thin-layer chromatography, 1Ogg of non radioactive steroid carriers (5x-DHT. 3x-dial. 3&diol, 7r-trio]) were added to the extracts. Separation of steroids was carried out by thin-layer chromatography on silica gel HFr5*+,e6 (Merck) in benzeneethanol (9: 1. v/v) twice. Autoradiography was used for locating the “C-labelled or ‘H/W-labelled steroids on the chromatograms. Carrier-diluted ‘H-labefled steroids were detected under U.V. (336nm). The separated spots were scrapped from the plate directly into scintillation vials prior to the addition of three drops of methanol and 10 ml of Instagel (Packard). Rlf(lio~Icticit~
mettstrrements
A Packard Tricarb, model 3375 liquid ~intiIlation spectrometer was used. Counting of 3H. “‘C and ‘H/‘4C was carried out on preset windows. Ail counts were expressed in d.p.m.. quenching being corrected on the basis of external standard ratios. 1. Average per cent k standard deviation of the f4-‘%Wabel associated with the 3B-diol substrate and its
Table
mctabolites recovered from prostatic slices and incubation medium
Table 2. Average per cent + standard deviation of the [I,2-‘H] label associated with the 7x-trio1 substrate and its metabolites recovered from prostatic slices and incubation medium Identified steroids [Z]
Medium
S~-Androstane-3~.l7&diol So-Androstane-36.7x.17~.trioi 5x-Androstane-3;.17j-dial 5x-Dihydrotestosterone
53.6 8.6 10.5 0.9
-i_ 18.8 + 0.9 z 9.9 -1:0.4
Total recovered steroids
77.9 + 14.2
Slices 17.3 f 1.6 + 1.9 I 0.3 f
ft.4 0.8 1.6 0.2
23.1 & 14.1
Two grams of slices from four hypetptastic prostate glands were incubated for 60 min in 50 ml 0.067 M phosphate buffer (pH 7.4) containing 7 pg of [4-‘aC]-3#Ldiol and NADPH (5 mgl.
Slices
5z-Androstane-3/I,7a.I7&trioI 3~,7z-~hydroxy-Saandrostan- I7-one Polar unknowns
87.0 k 8.9 2.9 * I.9
3.8 k 1.2 0.4 & 0.2
1.4 * 0.2
0.2 i: 0.2
Total recovered steroids
93.7 rf: 4.3
6.3 k 4.2
Two grams of slices from four hyperplastic prostate glands were incubated for 60min in 50 ml 0.067 M phosphate buffer (pH 7.4) containing 10.36 ng of [1.2-‘HI-71trio1 and NADPH (5 mg).
RESULTS
incubation
of 7 /,tg [4-14C]-3#3-dioi
In control incubations where tissue slices were omitted, the 3j?-diol substrate remained unchanged. Therefore, metabolites found in the medium after incubation with slices were produced by the tissue from the 3/I-dial which penetrated the ceils. The prostatic metabolites of 3#?-dioi previously identified as 7x-triol. 3a-dioI and Sa-DHT [Z] were found in slices and medium (Table 1). From the data presented in table 1, the total radioactivity associated with metabolites found in the medium was 24.3% of the incubated label. Since 22.1% of the total label was found in slices, we may consider that 46.4% of the incubated 3&dioI were retained and/or transformed by the slices, and that most of its transformation products were liberated into the medium. Iltcubation of 10.36 ng [1,2-3H]-7r-triol In control incubations where tissue slices were omitted, the 7x-trio1 substrate remained unchanged. After incubation with slices. 877; of the incubated lable was found unchanged in the medium (Table 2). The difference (13%) mostly included the unchanged 7x-trio1 found in the slices and the few metabolites extracted from slices and medium. As expected the previously identified 3&7x-dihydroxy-%androstan-1Fone [2] was one of these metabolites. Other minor poiar metabohtes were not identified and no transformation into 3/Ldiol was noticed. Iucubatiort
Identified steroids [2]
Medium
qf [ 1,2-3H]-7z-trioli[4-‘*C]-3~-diol
Two sets of experiments were carried out. One. with prostates A. B and C, incubated the slices with a mixture of 7 .ug [l.2-3H]-7x-trioI and 7 1-18 [4-‘“Cl-3& diol (Table 3). The second set used prostates D and E with a mixture of 10.36 ng [I&‘HI-7z-trio1 and 7~g[4-L4C]-3/I-dio1 (Table 4). In both cases. the ‘H/“C ratios were measured in extracts from medium and slices and in the 7z-trio1 fraction isolated from each extract (Tables 3 and 4). In addition, recovered ‘H and “C d.p.m. were used to compute per
Sa-Androst~e-3~,17&diol
and 5~-androstane-3~,7cl?&triol
531
Table 3. Per cent yields and ‘H/‘*C ratios of total steroids and isolated 5%.androstanc-3/3.7a.l7p-triol
in medium and
slices from three different hyperplastic prostate glands Prostate A Total Isolated steroids trio1 Incubated Medium Slices
JH/“C per cent { pet cent jH/W per cent per cent JH/‘Y
‘H ‘V ‘H ‘*C
4.12 92.0 82.3 4.6 8.0 17.7 1.87
Prostate B Total Isolated steroids trio1 9.75 94.8 86.4 10.47 5.2 13.6 3.76
8:6 16.4 21.78 7.6 2.7 Il.65
Prostate C Total Isolated steroids trio!
879 18.4 46.57 4.6 3.1 14.53
9.91 97.0 92.3 10.41 3.0 7.6 3.86
8:6 8.2 97.26 2.0 0.5 40.55
Two grams of slices were incubated for 60 min in 5Oml 0.067 M phosphate buffet (pH 7.4) supplemented with NADPH (5 mg) and containing a mixture of ~4-‘4C]-5a-androstane-3~.17/?-diol (7 @g)and [l.2-3H]-Sa-androstane-3/3,7a,17j3-triol
yields of the labels in medium and dices (Tables 3 and 4). In both experiments, the incubated 3H/i4C ratios were increased in incubation media and decreased in slices. On the other hand total steroids from slices always contained at least twice more “C than ‘H. These data are in support for a higher tissular retention and metabolism of the ‘*C-labelled 3&diot than that of C3HJ-7a-trio1 at two widely different concentrations. Correlatively, decrease of the 3H/14C ratio in isolated 7a-trio1 was larger in slices than in medium and the per cent of neoformated [‘4Cj-7a-triol was 6 to 16 times larger in medium than in slices. These data reflect the low retention and meta~iism of %-trio1 in the tissue and support the excretion of the neoformated [4-‘*C’j-7e(-trio1 from the slices.
DkSCUSSON
cent
fnvestigarion 0f[l.t-~H]-7a-triol prostotic cytosol
binding to proteins of
In four cases, after incubation for 60min with 10.36ng of [l.2-3HJ ?a-trioi, prostatic cytosol was isolated from the slices. The cytosol fractions contained from 0.9 to 4.6% of the incubated 3H label. The labelled free steroids were separated from the cytosol proteins by gel filtration on sephadex G-25. No significant amount of tritium iabel was found associated with the isolated proteins.
The recent work by Ofner et ul.[4] has shown that cultures of canine prostate explants disposed of radiotestosterone by a predominant transformation into 3fl-hydroxy-Sa-reduced-7<-hydroxylated radiometabolites which were released into the medium. Using a different approach with incubations of human prostatic minces, we have proved that the 3j?-diol, a prostatic metabolite of testosterone, was a precursor for 7a-triol[23. We now report that the 3/Ldiqi is more retained and metabolized than the ‘la-trio1 in the human hyperplastic prostate. Evidences supporting this conclusion are: (i) The larger quantities of metabolites formed from 3@diol than from 7a-triol. (ii) The irrtversible prostatic transformation of 3j%diol into 7a-triol. (iii) The absence of binding of 7a-trio1 in prostatic cytosol while 3jSdiol is a precursor for Sa-DHT. (iv) The decrease of 3H/14C ratio in slias when [3H}-7a-trioV[‘6q3B-diol is incubated. On the other hand, we suggest that ‘la-trio1 is a terminal metabolite actively formed from 3/?-diol and excreted from the human prostate. Supports for this suggestion are: (i) The irreversible prostatic transformation of 3/l-dial into 7a-triol. (ii) The small quantities of ‘la-trio1 transformed by the prostate. (iii) The much larger quantities of %-trio1 found in medium than in
Table 4. Pet cent yields and 3H,‘14C ratios of rota1 steroids and isolated Srr-androstane3/X72.17/3-trio1in medium and slices from two different hyperplastic prostate glands Prostate E Isolated Total trio1 steroids
Prostate D Isolated Total trio1 steroids Incubated Medium Slices
3H/‘4C per cent ‘H per cent ‘Y aH/‘4C { per cent 3H ( per cent ‘%I. 3H,“*C
9.83 96.5 93.2 10.17 3.5 6.8 5.04
8;7 12.9 68.19 3.1 1.7
17.44
9.96 94.5 89.9 IO.47 5.5
8& 13.4 65.96 4.7
10.1 5.44
I.6 29.41
TWO grams of slices were incubated for 60 min in 50 ml 0.067 M phosphate buffer (PH 7.4) supplemented with NADPH (5 mg) and containing a mixture of [4-“a-5a-andrdstane3fi.l7P-dial (7 pg) and [1,2-‘H]Jcc-androstane-3fi.7a.l7~-triol
(10.36 ng).
532
R.
F.
MORFIN et al.
slices after incubation of 3@-dioi. (iv) The decrease of 3H!L”C ratio in isolated 7cx-trio1 after incubation of [3H]7~-triol/[1JC]-3P-diol, this decrease being more extensive in slices than in medium. Our findings confirm and complete those reported by Morst er ~~[.[6-j who injected [3H]-3@dioI in six live patients 30 min prior to prostatectomy. The radioactivity associated with the 38-diol and its metaboiites was found six times larger in I ml of plasma than in I g of prostatic tissue. The major identified metabolites were Sa-DHT, 3u-diol and isoandrosterone but their quantities were also larger in plasma than in prostatic tissue. These results agree with an
elevated clearance of the neoformated Scr-DHT and 3*-diol from the prostate [73 and with our finding of larger quantities of these metabolites in medium than in tissue slices. Our use of NADPH in incubation medium did not favor the formation of isoandrosterone and comparison with injection [6] and superfusion [7] studies aie not possible in that respect. In contrast. neither Horst et a[.[61 nor Malathi and Gurpide[7] mentioned polar metabolites which could account for the 7z-trio1 formed from the 3/?-dial substrate. Nevertheless. recent work by Jacolot and Berthou @ersonat communications carried out to complete the findings of Mauvais-Jar&s er u/.@} showed that following injection of [JH]-3/?-diol the urines of human males contained labelled sulfate and glucuroconjugated derivatives of isoandrosterone. androsterone. 3z-diol. 7r-trio1 and 3&%-dihydroxy-Sz-androstan- 17-one. These findings and our evidences suggest an elevated ckarance of 3/?-dial metabolites from the target tissues to androgens. Implica-
tions are that the human prostate gland may dispose of intracellular androgens by a coupled 3/l-hydroxysteroid oxidoreductase-7a-hydroxyiase system. The estrogen inhibition of the terminal hydroxylation proved in canine [4] remains to be investigated in human prostate.
REFERENCES
I. Morfin R. and Floch H.: 7a-Hydroxylation du 5a-androstane-3fl, 17/3-diol dans la prostate humaine normale et hyperplasique. C.R. mud. Sci. Paris 235 f I977) 897-900. 2. Morfin R. F.. Di Stefano S.. Charles J.-F. and Floch H. H.: Precursors for 6/?- and 7a-hydroxylations of 5a-androstane-3fl,178-diol by human normal and hyperplastic prostates. Biochimie 59 (1977) 637-644. 3. Ofner P.. Vena R. L. and Motlin R. F.: Acetylation and hydroxylation of 5u-androstane-38.178diol by prostate and epi~dymi~ Steroids 24 (1974) 261-279. 4. Ofner P, Vena R. L.. Leav I. and Hamilton D. W.: Metabolism of CtP-radiosteroids by explants of canine prostate and epididymis with disposition as hydroxylated products: a possible mechanism of androgen inactivation. J. sreroid &&em. 11 (1979) 1367-1379. 5. MO&I R. F.. Di Stefano S.. Bardqu L G. and Floch H. H.: Biosynthesis of [ 1.2-3H]-5a-androstane3~.7al7~-~io~. ~j~~i~ie 59 (1977) 649-650. 6. Horst H.-J.. Dennis M, Kaufmann J. and Voigt K. D.: In ttitu, uptake of [‘H J-5a-androstane-3s. 17_&diol and of [JH]-5a-androstane-38.178-diol by human prostatic hypertrophy. Acta endow., Copenh. 79 (1975) 39&tO2. I. Malathi K. and Gurpide E.: Metabolism of h-dihydrotestosterone in human benign hyperplastic prostate. J. sreroid Bioclrern. 8 (1977) 14l-l45. 8. Mauve-J~is P., Guiilemant S.. Corvof P.. F&h H. H. and Bardou L.: Metabolism of radioactive 5a-androstane-3fi,l78-diol. Steroids 16 ( 1970) l73- 18I.