Metabolism of androgens by isolated human beard hair follicles

Metabolism of androgens by isolated human beard hair follicles

367 Laboratoired'ErdocrinAogie, H6pitalNotre-D~~ DSpar&mentdeM&kcine, Uni~siSde&xkr&il, MontSal, Canada. Received: 5/31/71 Ikrnan beard hair follicl...

819KB Sizes 0 Downloads 11 Views

367

Laboratoired'ErdocrinAogie, H6pitalNotre-D~~ DSpar&mentdeM&kcine, Uni~siSde&xkr&il, MontSal, Canada. Received: 5/31/71

Ikrnan beard hair follicleswere incubatedin vitro with testostero1-~-7-3~, ar&ostenedione-1,2-3~ ti dehydroepi4J4c. The principalme~litesof testosteronewereardrostenedione, 5a-androstanedione,5a-dihydrotestisteroneard ardrosterone.Theconversionof androstensdioneinti testisteroneand5u+Wqdrotestosteronewasvery limited. Dehydroepiardrost was metabolizedTV 5-androstene-38,17Bdiol, 7a-hydroxy-dehydroepiardrosterone ard androstenedione, kith rates of formationfar excesdingthosereportedfor hunan skin. The siqnificance of thesemetabolictransformations is discussedwith specialreferencetothemechanisnofactionofa&rcgens.

As a resultof nrmerousinvestigations, it has beaxne firmly establishedthat hunan skin is an active site of arxdrogametabolism. TeStOsterone,androstenedione and dehydroepiardrosterone are metabolizedextensivelyby survivingpreparations of this tissue (1,2,3,4,5). The transformationoftestosteroneinti [email protected] (DlTJ?) has beendanonstratedto occur inhuman skin (2,6). This kichisanorepotentaklrogen

than

steroid,

testosterone, is probablytheactive

formof this hormoneina number of axkogen-sensitivetissuessuchas the ventralprostate,seminal

vesicles, preputialglands,epipidymisard

the canb of chicks (7,8,9). For a betterundersizrding of the role of hormonemetabolitesin themechanismof actionof ardrogens,it

is important to study themeta-

kolisnof these hormonesin a numberof targetorgans. It is well tiwn that the growthard distributionof body hair is controlledby androgens. I&ever, theirmetabolismardmodeofaction in the hair folliclerenainsto be elucidated. For our studieswe selectedthe folliclesof the ~WW organ

for testosterone.

beard which are obviouslya target

STEROIDS

368

We

184

have thereforeexaminedthe _invitro metabolismof testosterone

-7-3H,ar&ostenedione-1,2-3H ard dehydroepia&rosterone-4-14C by the isolatedbeardhair folliclesobtainedEm

t&o &althymales andone

hirsutefemalepatient.

Beard hair follicleswere pluck& freshlyfran TV males (ages: 34 a& n years) ax-drrcma~ (age: 32 years)withidiopathic hirsutism. Afterweightmeasurenent,thefolliclesweretransferredintiflasks contai.ningtbelabell&steroidsubstrate. In each flask, 20 follicleswere incubatedin 1.5 ml of Irtn;rbations. Krebs-Ringer-Ptisphate solution,pH 7.3, containing200 ng% glucoseand 50 pg eachof NAD, NADP, NADHard NADPH. The incubations were carried autbyshakingthesamplesinairatmosphereat37Cfor5hcrurs. In thoseexperiments,wheretheeffectof cyprotsroneacetatewasinvestigated, itms added to the flasksin 0.02mlof etharolattheonsetof the incubation(20 pg of cyproteroneacetate/flask). SteroFds, Radioactivesteroidspurcha~franNew~l~NLbclearcOrp., Boston,Mass., werepurifisdbeforeuseb.ypaperchranatographyinthe Bush A system (IIf. Steroidsusedas substrateshad the followingspe58.8 r&X&M; testistecific a tivities: dehydroepiar&osterone-4-14C: 9H: 25 Ci/rrM;4-arrdrostene-3,17-dione-l,2-3H: 48 [email protected]% rone-7Peferae steroidswere obtainedcmercially fran Ikapti (Pamat Can, Israel)or SteraloidsInc., Pawling,N.Y. 7cr_hydroxydehydroepiandrosterone-diacetatewas agiftfmthe SteroidPeferenceCollection, MRC,I.otion,-land. lbe followingquantitiesof labelledsteroidswere used for the incubations: dehydroep&&rosterone: 0.3 IrCi,5220.83wflask; testosterone: 1 uCi, 40.0 PM/flask;ardrostenedione:1 UCi, 20.83 @l/flask. Par use as internalstardards,5cr-androstandione-4-14C was synthesized fran 5a-dihydrotestosterone-4-W by oxidationwith Q-03 ard 5-androstene-3b,17B-diol-3H frcm dehydroepiarrlrosterone-7-3H by reduction with scdi.Lan borohydride(10). Isolationard identification of metabolites. After incubation,the volume of the incubationmixturewas broughtto 10 ml with distilledwater and upon additionof 100 Figeach of nonradioactive authentictestisteroneand arxdrostenedione, the ste~XG& were extract& with 2 x 20 ml of methylene chlorideandlx 2Oml benzene. The organicpbaseswerecanbined ard evaporatsdto dryness. Steroidsin thedry residuewere first resolved byaninitialpaper chranatographic step. Theextracts of testosterone arrlarxlrX?stenedione incubateswere run in Push A, tbile tise of the [email protected]&rosterone ir~~W9ons in the Bush Bl system (11). After scan~~c~~~, theradioactivezonescorrespon%_ngto therunningrateofauthenticrefere.ncesteroidswereelutedand identifiedby furtherchrcmatographic separationsinpaperandvarious thinlayerchron-atographic (TLC)systemsand serialcrysiallizationticonstantspecific activityor isotoperatio. V&never applicable,authenticradioactive internalstandardswere used for the identification of tiividual steroids. Thesewereaddedafter the initialpaperchranatcgraphic step. Results

Oct. 1971

STEROIDS

369

weremrrecttrlfcx lossesooxrredduringextraction~ the initialpaperchrcxnatographic stepbasedontherecoveries of au-tic mnradioactive testosteronearrdardrostenedione. 0xntitativemeasurementof radioactivitywasperfomed ina Packard LiquidScintillationSpectr~. The fol.lmingTLC systanswereused: for silica-gel-Gplates:TICla: cyclohexane-ethyl acetate (60:40,v/v)f4l;TIC-GH: ~~of~~~ml (98.25:1.'75, v/v)(2f;for alumjnaplates: TIC-E: n-hexam-ethylacetate-glacial acetic acid-a&. ethanol (20:210:1:10, v/v/v/v>;TLC-F: nhexane-ethylacetate-glacial aceticacid-&s. ethanol (10:210:1:20, v/v/ v/v,(3). Fre-coatedTLC sheetswere us&, obtainedfran Merck A.G., lkmstadt, w. Gexmany. FtEsuLTs

ILncbuations with testoste.rone-7-'H. The follawingmetaboliteswere isolated(Table1): 1.

I-Androstene-3,17-dione. Thissteroidwaselut&ifranthe

initialchrcxnakgmmrun in theBushA system. After theadditionof 42,500DPM of authenticandrostenedione-4-14C ard 8,900 DPM of Eja-dihydrotestisterone-4-14 C, ardrostenedione was separatedin the systemWGE. Theidentityof the steroidwas furtherestablishedbyserialcrystallizationto constantisotoperatio. This caqxnmd was ccxnpletely se2..5a-Ardrostahe-3,17-dione. paratedby the firstpaperchranatographyintheBushA

system. After

the additionof 11,000DPM of authentic5a-ardrostane-3,17~ione-4-14C and ~s~tc~~~~

inTIC-la itwas crystallizedtoconstant

specificactivity. 3. 5a-Dihydrotestistemme.J&sr dionein'lU=a,

separationfmna&rostene-

this steroidwas furtherseparatedfmnakimsterone in

the Bush A systemand identifiedby serialcrystallization(Table5). 4. Pndrosteronewasseparatedfrandihydrotestisteroneinthe 14C labelledandrosterone was available,the BushAsysten. Since m losseswere correctedas for dihydrotestosterone. Lfter elutionfran the paper, 4Omgof

authenticsubstancewereaddedand the steroidwas crys-

tallizedto constantspecificactivity. SItall guantitiesof radioactivity refrained with 5a-a.xIrostane-3f3, 17~-dioland 5a-ardrostam-3a,17f3-diol during serialcrystallization. These steroidsweremtregarded as identified, but their presencewas indicatedas furthermetabolitesof 5a-dihydrotestosterone in the hair

f&4

STEROIDS

370

CcmpaundIsolated Ardrostenedione

M-l-CA

M-l

M-2

H.F.

76.31l6.70) 59.8Ol5.27) 198.0(17.39) 81(5.33)

%-findrostanedione

8.3110.73)

6.54(0.57)

25.28(2.22)

5.5(0.36)

Androstemne

0.59(0.05)

0.34(0.03)

0.54(0.05)

1.70fO.U.)

5a-Dih#rotestosterone 4.21cO.37)

2.67tO.23)

8.88tO.78)

1.78(0.12)

Quantitiesofradianetabolites [email protected]%&s

fome.i/loomg tissue/hr.

IQ_nrbers in bracketsrepresentpercentageof substrateconverted/incubation flask. M: male H.F,: hirsutef&e CA: cyproteroneacetateadded, 20 ug/flask.

mBm2 m-4-14 MEIlAE0LITFSOFDETJYDKEE'IANDROS

canplzx3

Isolated

M-l

7a-HydroxpDHA

1,654U.U) 5-Andmxten~3B,17B-diol2,500(1.68)

Ardrostenedione

196(0.13)

C~WITHBEZU?DHAIRlX&LI~S

M-l-CA

M-2

H.F.

670(0.47) 1,450(0.97)

575tO.38)

1,340(0.90) 2,350(1.58)

1,060(0.53)

160(0.10)

267cO.18)

Quantitiesof radianetabolites are expressedas poles form&/100 q

192fO.13) tissue/hr.

i&mh.rsin bracketsrepresentpercentageof substrateconvert~/incubation flask. M: male H.F.: hirsute

female

CA: cyproteroneacetateadded,20 ug/flask.

Oct. 1971

371

STEROIDS

MlmAEfxITEScF4-m

RXXENE-3,17-DIoNE-1,2-3H I.mJBmmwITHm HAIRFo.TLI~

ccmpaurd

Isolated

M-2

H.F.

[email protected]

12.45(2.09)

0.60(0.076)

5a-Adrostandione

13.07(2.20)

4.80(0.60)

5a-Dihykotestmtne

0.50(0.08)

traces

An3_roslzercme

0.48(0.08)

traces

Leged as in Y&de 1.

mBLE4 ISCXATIQNAND IDENCIE'I~IONW

7a-HYDROXY-DBAFRa'[email protected])

OF DEBYD~El'IAN>ROSTEW3NE_4_14C WITH =

Chr~tographic Bush Bl

TLC-F

systen

Derivative

Specific Activity

Freesteroid ,I

125 ug carrier added

TLC-la

3,7-diacetate

'IX-E

Freeste?zoid II

Bush B5

HAIR FOLLICLES

62 DpM/ug 46 47

" II

" II

48

"

"

STEROIDS

372

18:4

follicles. larger quantities of tissuearenecessary

inorder toobtain

eraoughradioactivity for their ccmplete identification, Incubations with 4-ar&ostene-3,17-dione-l,2-3H. 'IWOsuch incubations me

carried out, one with the beard follicles

of a male (M-2) anclthe other with those of the hirsute female patient. Testosterone, k-dihydrotestosterone and S-ardrostenedione were isolated fran these incubations. The methods me internalstardardswereused, Incubations

as outlined; carbon-14-labelled

followed by serial crystallization (Table 3).

with dehydroepiandrosterone-414C (Table 2). 1 5-Pnkostene-36,17B_diol was isolated by the initial paA.

per chrcmatography in the Bush Bl system. After adding authentic 5-androstene-3fi,17f3-diol-3H the steroid was identified as outlined in Table 5. 2. 7a-Hydroxy-dehyzdroepiarrdrosterone. After separation in Bush Bl, 125 pg of authentic nonradioactive carrier was added to the samples and the [email protected]

was identified along the lines shown in Table 4.

3. 4->&rostene-3,17-dione. Pfter

elution fran the initial

chrcmatcgram in Bl and addition of 72,000 DFM of authentic ardrostenedione-3H themixturewas

run in the BushA

system. The region corresponding

to ardrostenedionewas eluted ard purified in TLC-GH, followed by crystallization to constant isotope ratio (Table 5). Small quantities of radioactivity were found to remain with 5a-androstanedione and androsterone during serial crystallization. Lowever, these caqzounds were not regarded as identified. I;0radioactivity was associated with testosterone or 5c(-dihydrotestosterone after several steps of crystallization. In addition to the above described steroids, two further derivatives of dehydroepiandrosterone,with minor radioactivity were localized by scanning on the initial Bl strips. These steroids were not identified, but according to their polarity, they were oxygenated derivatives of DHA. Tables 1, 2 ard 3 show the quantities of metabolites formed fran the three substrates. Tables 4 and 5 illustrate examples of the identification sequence of various steroids.

373

STEROIDS

Oct. 1972

WIT

Steroid Identified

Al&OS~&iO~

TestoSteI-0~

Substrate

D-IA

DB

M-2

M-l

M-2

8I900 DPM Dwr-b%

72,100DFM ardr stsH enedione-1,2-

Ixubation Inwstardard

Ardrosteraediol

Chrunatxqraphic Sysm

BWhA

Bush

TLC-GH

BushA

BUshA

TLC-GH

Serialcxystalli-

R:

B1

52,00013pMandxostenediol-3H Bush Bl TLC-E !t!LC-la(diacetate)

3H#/14c

R:3H,?4&

Zati0Z.l

CR

solvent:

ML

CR

ML

CR

ML

1. methawltwater

1.53

-

80.4

-

5.20

4.30

2. acetone:xwthawl:

1.52

3.20

82.3

43.0

5.40

4.90

1.51

2.03

84.4

69.0

5.51

5.20

water

3. cyclohexane:ethgl acetate

4. hleGUK?:benZelle

1.49

1.53

82.5

8165

5.53

5.49

5. acetxxwwater

1.50

1.45

81.0

82.3

5.53

5.50

6. metharwl:water

1.45

1.45

81.0

82.0

7. cyclohexarwethyl

1.51

1.51

acetate

M-male;

E-t-l-&i0;

CR-crystals;

ML-xwthfxliqwr;

[email protected]

18:4

STEROIDS

374

DISCtJSSION The rate of transformation of tes&Xkerone into Su-dihydrotestostegenerally less

roneby ~eh~~~~fo~icles~s

tknthatfound

with human skin (Table6). The values publishedby Wilson ti Walker (12) hcrwwerseem to be exceedinglyhigh, which might be due tc contamination by androsterone.On the other hand, the metabolicactivityof pubic skin slices (13)was less than that of the hair follicles. In thebeard hair folliclesthe major productswere androstenedione ard anlrostaneione followedby 5cr-dihydrotestost. probablylargelydement

The rate of DHT formationis roost

on the type of tissueinvolved. Northakt

etal. (15)have shown avery

intensiveDHT formationby hLnnanpubichair

follicles,while accordinglo anotherreport the scalp hair follicles formedvery littleDHT (16). Similarly,human skin samples,t&en frcan perinealareas exhibit&dexcessivelyhigh rates of this conversioncanpared to the activityof skin samplesoriginatingfran variousother sites (12). The oxidationof testosteroneto ar&osten&ione is an inactivation step when the steroidloses its [email protected], which is responsiblefor ankogenic activity. This transformationisweaklyreversible in the hair folliclesas shown by the i.ncu&tionswith androstenedione and might serve as a regulativeprocess to set the levelsof testosteroneavailablefor Sa-reduction.Tissueswhich are not arrdrogendeperrkntdo rotformDHT - testosteroneis metabolizedexclusivelytr, androstenedione and 5a-akkrostanedione in canine urinarybladders (17) ard in the submaxillarygland of the dog (18). Dehydroepiardrosteronewas intensivelymetakolizedbythebeard follicles. One of the three major metakolites,5-ard.rostene-3B,178-diol is amorepotenta&rogentbanDHA

(19)and itcanbeconvertedinto

testosteronedirectlybythe actionof 3Pol-dehydrogenase-isaseas shown w Rosner ax-dMacane with hunan testiculartissue (20). The rates of formationof 5-ar&ostene-3B,17B-diol and 7ol-hydroxy-DHA were found toben~chhigher thanthoseby humanskin (Table6). Thedifference for anlrostenediol is SO-100fold and for 7a-hydroq-DHAabout tenfold in favourof the follicles. Therefore,thesetwocanpoun% appear IXIbe even zore characteristic of the beard hair folliclesthan DHT since the

STEROIDS

Oct. 1971

375

Fkard follicles Product

substrate

5a-DHT

!&stas+zenme

TkStYXterone

Androsten&ione

mle

skinarea

fenale I various prepuce scrotun

4.2 8.9

1.8

12.5

0.60

46 -_ 1-2

382 2

534 ___

(12) ( 6) (13)

454

--

--

( 4)

--

--

(14) ( 5) ( 3)

--

--

( 5) ( 3)

-c

--

(10) ( 3) (14)

l 5-Androstene3&17wiol

DHA

2,500 2,350

1,060

10-105 20 90

7wCH-DHA

DHA

1,654 1,450

575

140 80

196 267

192

ArdrosMoneDm

Metabolicactivities areexpressedas

Wles

wference

20 50 80-1,500

of steroid fomed/lOOmg

tissue/lx

STEROIDS

376

synthesisof the latter is canparativelyless praninentin this tissue (Table6). The

transformation of dehydroepiardrosterone into ardrostenedione

occurredat rates within the range describedfor hunan skin (Table6). metabolismof this steroidtowardsandrosteroneobviouslytakes

Further

place,but,with decreasedintensity. This pathwaymight have significance in pathologicalstatesas, for example,hirsutism. The intensiveformation of ardrosteroneby the beard folliclesof the hirsutefemaleunderlines this possibility(Table1). Thepresenceof theactiveanIAar&ogencyproterone acetateinthe incubationsresultedin sune inhibitionof the conversionof testosterone to all its metaboliteswith no selectiveeffecton 5c&ihydrotestosterone formation. This is ccnsistentwiththeobservationsthatcyproterone ard cyproteroneacetateantagonizethe actionof testisteroneby ccmpeting for intracellular birxding proteinsof male accessoryreproductive glards (22,23). Theeffectof this antiandrogenwas~reprorrouncedon the 7o-hydroxylation of DHA and formationof 5-ardrostene-38,17B_diol. The metabolismof testosterone by the beard folliclesof the hirsute fenaledifferedquantitatively frcm that by the male follicles. The formation of DEE was relativelyless intensivewith an androstenedione:DHT ratio of 45.5, in contrastto 22.3 and 18.1 in the male cases. Synthesis of androsteronefrcxntestosterone was more intensive,while transformation of ardrostensdione into testosterone was 20 times less efficientthan in the male follicles. Similarshiftsofmet&.olic intensitiescanbeobserved in the utilizationof DHA, where with less formationof 7a-hydroxy-DHA ard [email protected],17B-diol the conversioninto arxlrostenedione was equivalentin intensityto one of the male values (Table2). In conclusion,the presentwork shows that the hair folliclesof the hm

beard intensivelymetabolizeax&ogens. Scme of thesemetabo-

lites,such as 5cl-dihydrotestosterone ard 5-ar&ostene-3B,17B-diol, are biologicallyn-ore~tentardrogensthantheparentaxqou.rx%. These fir& ings favour thehpthesis

thatthemetabolites formed in the targetorgan

might serve as the active forms of circulatingardrogens(7,24). ibwever, in order to establisha causal relationship betweenhair grcwthard the metabolitesformed in the hair follicles,the effect of these steroids

STEROIDS

Oct. 1971

377

I

*..I:

.*. J % '0

%

*

STEROIDS

378

has to be examined

18~4

on protein synthesis ard keratinization in this tissue.

Studiesof this Mturearecurrentlyurrlerkayinourlaboratory.

This investigationwas sqqxxted byagrantfran

theM&icalResearch

Ccnmdl of the Province of Quebec (no. 2541). The excellent tedmica assistance of Miss Judith Sylvester is gratefully ackncwl&g&.

1.

Rmgone, E.L.: Steroids, 7_,489, 1966.

2.

Ganez, E.C. and Hsia, S.L.: Biochenistry,2, 24, 1968.

3.

Far&in, I., Fazekas, A.G., K&ai, K., !lbth,I. [email protected] Julesz, M.: Eur. J. Steroids, 2_,223, 1967.

4.

Faredin, I., Tbth, I., Fazekas, A.G., Kdcai, K. ard Julesz, M.: Int. J. of Dermtology, 2, 147, 1970.

5.

Far&in, I., Fazekas, A.G., !lbth,I., Kdcai, K. ad J. Invest. Dermatology, 52, 357, 1969.

6.

Voigt, W., Fernardez, E.P. ard Hsia, S.L.: J. E%iol.Chem., I245 5594, 1970.

7.

Bruchovsky, N. ad

8.

Gloyna, R.E. and Wilson, J.D.:

9.

Anderson, K.M. and Liao, S.: Nature, 219, 277, 1968.

Wilson, J.D.: J. Biol. Chm.,

Julesz, M.:

243, 2012, 1968.

J. Clin. Erdocrinol., 2,

970, 1969.

10.

Faredin, I., Kokai, K., Tbth, I., Fazekas, A.G. ard Julesz, M.: Acta Med. Acad. Sci., Hung., 27_,95, 1970.

11.

Bush, I.E.: Biochm. J., 50. 370, 1952. Wilson, J.D. and Walker, J.D.: J. Clin. Invest., 48, 371, 1969. Jenkins, J.S. and Ash, S.: J. Erdocr., 2, 515, 1971.

12. 13. 14.

Chakrabxty, J., !&cmson, J., MacSween, M.P., Muir, A.V., Calman, K.C., Grant, J.K. ard Milne, J.A.: Br. J. Derm., 83, 477, 1970.

15.

Northcott, R-C., Iskuu3, 2, 422, 1969.

16.

Sansone-Bazzano,G., Reisner, R.M., Bazzam, G.: 169, 1971. (Abstr.)

17.

bbrfin, R.F., i?liapaulios,M.E,.,Chambeslain, Esxk3cxinology, 87_,394, 1970.

18.

Weiner, A.L., Ofner, P. ad 1970.

19.

Dxfman, R.I. an3 Dorfman, 1962.

D.P. and Liddle, G.W.:

J. Clin. Endocr., Clin. Res., -' 19

J. and Ofner, P.:

Sweeney, E.A.: Enckxrinology,

87, 406, -

A.S.: Acta Endccrinol., Suppl., 74,

3,

Oct. 1971

STEROIDS

379

20. Rosner, J.M. and Macome,J.C.: Steroids, 15, 181,

1970.

21. Pochi, P.E. and Strauss, J.S.: J. Invest. Derm., 52, 32, 189. 22.

Stern, J.M. and Eisenfeld, A.J.:

Science, l66, 233, 1(&I.

23.

Fang, S. and Liao, S.: Mol. Pharmacol., 5, 420, 1969.

24. Baulieu, E.E.: Rev. Europ. Etudes Clin. et Biol., 15, 723, 1970.

25. Systematic nomenclature: Androstenedione: bandrostene-3,17-dione 5a-Androstandione: 5c+androstane-3,17-dione ?a-Dihydrotestosterone(DHT): 17P-hydroxy-5a,-androstan-j-one Androsterone: jcz-hydroxy-f&z-androstan-17-one Androstenediol: 5-androstene-$,17j3-diol Dehydroepiandrosterone(DHA): 3p-hydroxy-5-androsten-17-one 7a-Hydroxydehydroepiandrosterone:3/3,17a-dihydroxy-5androsten-17-one Testosterone: 17fi-hydroxy-4-androsten-j-one Cyproterone acetate: 1,2a-methylene-6-chloro-17-hydroxy-k,6pregnadiene-3,20-dione-17'-acetate