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The effect of five synthetic progestational compounds on 5α-reductase activity in genital skin fibroblast monolayers
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THE EFFECT OF FIVE SYNT~TIC PROGESTATIONAL OONPOUNDS ON 5a-R.gDUCTASK ACTIVITY IN GENITAL SKIN FIBROBLAST HOHOI,kYSRS. H . J . l)ean and J . S . D . ~ t n t e r D e p a r t - ~ n t of P e d i a t r i c s ,
U n i v e r s i t y of Manitoba, Winnipeg, N a n l t o b a , Canada.
Received 9-20-83 ABSTKAL~ The s u g g e s t i o n has been mule t h a t p r e n a t a l e x p o s u r e to s y n t h e t i c p r o g e s t o g e n e c o n t r i b u t e s t o an i n c r e a s e d i n c i d e n c e of h y p o s p a d l u . One p o t e n t i a l n e c h a n i s n f o r such an e f f e c t ~Lght be i n h i b i t i o n of 5 a r e d u c t a s e , a key enzyme in norm .I s a l e s e x u a l d i f f e r e n t i a t i o n . We have e x s a t n e d the e f f e c t of p r o g e s t e r o n e and of f l v e s y n t h e t i c p r o s e s t o s e n s upon 5 a - r e d u c t a n e a c t i v i t y i n f l b r o b l n e t I o n o l a y e r s from 12 genltal s k l n cell lines o b t a i n e d f r o a n e r s a l newborn i n f a n t s , boys w l t h p h i m o s l s and h y p o s p a d i s s and a u o r m ~ a d u l t m a l e , B a s a l enz~ae a c t i v i t i e s ranged from 0 . 8 - 1 2 . 1 p l o l e s 5 a - r e d u c e d p r o d u c t / ~ S DNA/hour. P r o g e s t e r o n e and n o r e t h l n d r o n e i n h i b i t e d 5 a - r e d u c t a n e a c t i v i t y i n a d o s e dependent manner to • uu~isnm of 95Z and 50Z o f b e s a l l e v e l s r e s p e c t i v e l y a t 10- 5 H. S i a t l a r c o n c e n t r a t i o n s (10 - 5 N) of n o r e t h y n o d r e l , e t h i s t a r o n e , d l - n o r g e s t r a l and d - n o r g e s t r e l had l i t t l e or no e f f e c t . S t u d i e s of c e l l v i a b i l i t y showed t h a t t h e e f f e c t s of p r o g e s t e r o n e and n o r e t h t n d r o n e were s p e c i f i c f o r 5 ~ - r e d u c t a s e and n o t
non-specific toxic effects. INTRODUCTION The a t c r o s o a a l enzyme r e s p o n s i b l e f o r the c o n v e r s i o n of testosterone
to d i h y d r o t e s t o s t e r o n e (DHT), ~ - r e d u c t a s e ,
d e t e r a t n a n t of n o n s a l male s e x ~ a l d i f f e r e n t i a t i o n , b e l i e v e d to be the a c t i v e l n t r a c e l l u l a : the urethral
s i n c e DHT i s
androgen c o n t r o l l i n g f u s i o n of
f o l d s and thus normal d e v e l o l m e n t of the p e n i s and
penile urethra (1),
C o n g e n i t a l absence of t h i s enzyue l e a d s to
I n c o m p l e t e f u s i o n of the u r e t h r a l hypospadias (2). teratogens,
is a critical
folds, clinically
recogrLtzed as
I n h l b l t l o n of 5 ~ - r e d u c t a s e by e n v l r o ~ n t a l
including s y n t h e t i c p r o g e s t o g e n s , hss been s u g g e s t e d t o
e x p l a l n an a p p a r e n t i n c r e a s e in the i n c i d e n c e of h y p o s p a d l a s In
Volume 43, Number I
~
.A- m ~ o • ~ m
J a m m r y 1984
industrialized countries
in the past decade (3-8), but the evident~,
for such a relationship is anecdotal and circumstantial. itself can inhibit ~cL-reductase
Progesterone
activity in vitro, but substituti,~:~ L:,,
the 17~i and 16,, positions may reduce this inhibitory effect (9). Studies in the rat and rabbit have shown variable degrees of incomplete mascullnlzation after prenatal exposure to high doses of progestational
compounds such as the 19-nor and 17:~-ethlnyl
derivatives of testosterone
(3,10,II).
Several retrospective s~udles
of hypospadlc boys have suggested an increased rate of maternal ingestion of progestogens during early pregnancy (12-16), with some correlation between the time of ingestion and the severity of the defect (12,13,17).
Conversely, however, several prospective studies
of women who ingested synthetic progestogens
In early pregnancy have
failed to show an increased risk of hypospadlas offspring
(17-22),
in their male
These negative epidemiologlcal
conclusive, however,
findings
may not be
in view of the hlgh incidence of spontaneous
hypospad ias Since further clinical studies are unlikely to resolve this problem, we chose to explore possible mechanisms for a teratogenlc effect in vitro.
We have studied the effect of five of t h e
progestational agents contained in contraceptive preparations On c,~_ reductase activity in human genital skin flbroblasts of normal and hypospadlc boys.
This experimental model, using mature slteospeciflc
flbroblasts, has b~en used widely in the study of androgen-~edlated events of male sexual differentiation.
~ T I ~ O X D ~
15
MATERIALS
E a g l e ' s minimum e s s e n t i a l medium, amino a c i d s , t r i c i n e c h l o r i d e and t r y p s i n were p u r c h a s e d from Grand I s l a n d B i o l o g i c a l Company. F o u r w e l l e d (60mm) L i n b r o d i s h e s and f e t a l c a l f serum were o b t a i n e d from Flow Laboratories, and plastic tissue culture dishes (60 -.- and I00 mm) from Fisher Scientific. (1,23H) Testosterone (SA-52 Ci/mmol) was purchased from New England Nuclear, and nonradioactive steroids from Steraloids Inc. The progestatlonal compounds uorethynodrel, norethludroue aud ethlsterone were purchased from Sigma Chemicals Inc. The pure active stereochemical d-isomer of norgestrel and t h e r a c e m i c m i x t u r e , d l - n o r g e s t r e l , were k i n d l y d o n a t e d by Wyeth L a b o r a t o r i e s , T o r o n t o , OQtarfo, Canada. P r e c o a t e d s i l i c a g e l - 25 UV254 t h i n l a y e r chromatography p l a t e s (20x20 cm) were from Brink.man I n d u s t r i e s . Calf thymus D~A ( t y p e I ) , b a k e r ' s y e a s t RNA ( t y p e I I I ) , b o v i n e p a n c r e a s e RNAase ( t y p e I A ) , e t h i d i u m bromide (2,7-diamino-10-ethyl-9-phenylphenauthridium bromide), fructose 6 - p h o s p h a t e , g l u c o s e - 6 - p h o s p h a t e d e h y d r o g e n a s e ( B a k e r ' s y e a s t type VII), NADP and NADPH were purchased from Sigma Chemicals Inc.
METHODS
Cell Culture G e n i t a l ( p r e p u t i a l ) s k i n b i o p s i e s ware o b t a i n e d from 5 normal newborn males a t t h e time of c i r c u m c i s i o n , from 4 boys age 1-12 y e a r s a t the time of h y p o s p a d i a s r e p a i r , from 2 boys see 2 months and 5 y e a r s w i t h p h i m o s i s a t the time of c i r c u m c i s i o n and from 1 normal a d u l t male age 25 y e a r s . The e x p l a n t s were c u l t u r e d i n E a g l e ' s m i n i m a l e s s e n t i a l medimn c o n t a i n i n g 20nM t r i c i n e c h l o r i d e , 24nM NaHC03, 15Z f e t a l c a l f serum, 1Z n o n e s s e n t i a l amino a c i d , p e n i c i l l i n 100 ~U/ml and s t r e p t o m y c i n 100 ~g/ml a t 37°C i u 5Z CO2, The f i b r o b l a s t s were grown to c o n f l u e n c y and t h e n t r e a t e d w i t h 0.05Z t r y p s i n b e f o r e s u b c u l t u r e ; t h e y were m a i n t a i n e d for 3-5 g e n e r a t i o n s i n t h e same medium ( e x c e p t w i t h 10Z f e t a l c a l f serum) which was changed t w i c e weekly and t h e n s t o r e d i n l i q u i d n i t r o g e n . C e l l s for t h i s s t u d y were used b e f o r e t h e e i g h t e e n t h g e n e r a t i o n . A l l e x p e r i m e u t s were performed on the 7 t h day a f t e r s u b c u l t u r e f o l l o w i n g 24 h o u r s i n medium w i t h o u t f e t a l c a l f serum. 5~-reductase activity B a s a l 5 ~ - r e d u c t a s e a c t i v i t y was measured i n q u a d r u p l i c a t e 60 mm wells using a modification of the method of Wilson (23). The monolayers were washed twice with medium (without fetal calf serum) and incubated with 3 ml of the same medium containing approximately 105 cpm 3H testosterone (1.75 pmoles/well) and 0.05 ~M unlabelled testosterone (150 pmoles/we11) for I hour at 37°C in 5% CO 2, I n c u b a t i o n e x p e r i m e n t s w i t h the p r o g e s t a t i o u a l s t e r o i d s , p r o g e s t e r o n e , n o r e t h y n o d r e l , n o r e t h i n d r o n e , e t h i s t e r o n e , d - n o r g e s t r e l and d l - n o r g e s t r e l were p e r f o r m e d i n p a r a l l e l i n d u p l i c a t e 60 ,w, w e l l s u n d e r i d e n t i c a l c o n d i t i o n s ( e a c h s t e r o i d a t 10 -~ M was d i s s o l v e d i n t h e i n c u b a t i o n medium c o n t a i n i n g a p p r o x i m a t e l y 105 cpm 3H t e s t o s t e r o n e and 0 . 0 5 ~M u n l a b a l l e d t e s t o s t e r o n e . ) Dose r e s p o n s e
ifi
I
qL~ ]m ~ . o
x ~
w
s t u d i - ~ (10 - g - i q ' ~ ~|) w~r~ I~rf,~rmed in a s i m i l a r [~ann,.r w i t ~ p r o g e s t e r o n e and n o r e t h L n d r n n e in 2 (-eL] ] I n e ~ . A f t e r I n c n b a t l o n , the ~,~.dtum was removed and | m| we:; ,~zt¢~r~.~ w~th 8 v e t . r u e s b e n z e n e . C a r r i e r s t e r o i d s (~ ';~ each t e s t ~ s t e r o . ~ : dlhydrotestosterono, a n d r o s t e n e d t o n ~ , 5 , ~ - a n d r , ) s t a n e d t n n e an(l ~ , a n d r o s t a n e d l o l ) v e r e added [~ ~ ~I ~)f the e x t r a c t . ~ h l c h wa~ d r i e d under nitrogen. The r e s i d u e ~a~ r e c o n s t i t u t e d in O. ~. r~l ~.Chunol .u~d a p p l l e d t o 2Ox20 c~ s i l i c a Rel t h i n l a y e r c h r o m a t o g r a p h y ,?tate.~ (4 channels). & f r e t one a~cent |~ 95:~ benzent : ~ t h a n o l . te~to~t,'r,~:;" (Rf 0 . 3 9 ) and a n d r o s t e n e d i o n e (Rf ~).65) were i d e n t i f i e d u n d e r !iV Light, u h i l e a n d r o s t a n e d i o l (R~- ~ . 3 L ) , d i h y d c ~ t e s t n ~ t e r o u e (!~f ~;:.:~7) and a n d r o s t a n e d i o n e (Rf 0 . 7 7 ) were Located i n m a r k e t c h a n , ~ l s (3 per p l a t e ) by s p r a y l n K w i t h 6c):'. II2SO4 and a l l n u l n g the v i s i b l , , c ~ l o . r ~ t,~ d e v e l o p at 37°C. P:ach zone ~ ; ~craped f r o n the ~)tat,; and i t ~ '!"! c o n t e n t d e t e r m i n e d by l l , l ~ i d ~ c | n t | t l a t i o n ~-,*unrt.R ~I~ ml ,~.~'.i PPO/O.O06~ POPOP Ln t o l u e n e ) . ~i~c~; ne,~.'li'~ihi .~ 3~i t o . l i t , ; wr, re t,~,Jn~ i n the a n d r o s t e n e d ~ o n e arid :~ndr~canedLnn,. ~r._,a~, the eo,'q ,'~f ~1~ c o u n t s i t e m t h e DHT and a n d r o s : ~ e d ~ o l ;~r~as wa~ ~,~¢,d to r ~ l c , a l a t , ~ ~ , reduced p r o d u c t , .~ subscra,~. I-i=~,~- (~n.-,,ba',,d ,,:,diu~ w i t h e , a t ,~.1!--) was t|.qed I~o c o r r e c t f ~ r g~,~--. ~ . ~ , ~ ; t t i r r.~d~|t'~|.~; .~ .;uhsI. t';J~./, a:~."
874~.. The cnPff|cien! ,)~ .~rlati,~:~ t()r p a i r e d Jnplic.'_tte ~.q:~;:'~ '.,a~ ÷4~'3~. . ~ J b s t r a t o , , t t l i ~ a t i ,n ::!'~dor I ~ ; = ! :,,,~ditlc~u'a r~nged ~rom 1"0o90~ f o r th+" 12 ('+:11 lin,=m. :.t.arimti,'.~] :~n+:i,/mlm of rile I n c u b a t i o n e x p e r i m e n t s w i t h the pr~geSt~ti<,n;~1 ~t~rr,ld~ ~,,;ls ~ r [ o r ~ . d ~L=.tu¢ [actorial a n a l y s i s of carlnr~r,- ,¢i t h o . t r e p l l c a t i on (one ol)s,~rvat i , . , per ~ e l l ) and the Tuk~v m , , ] t t D l , r:~nR*~ r o ~ t . DHA measu[etment A f t e r t n c t l h a t t o n , the mou,,lav,,rs were wash.d t h r e e time~ wtt|t 50mH T r t R - s a l i n e ( 4 n C ) ; a n d t h e n i . c u h a t e d f o r 5 m i n u t e s i n I n l N.O5~ t r y p s l n t,~ s a [ i n e at 37 °C. The c e l l s were h ~ r v e s t e d by trituration and then c e n t r | f u g e d at 80(') g f o r ~ mln a t 4°C. The p e l l e t was washed once with I mE S(~ mr4 T r t s - s a l t n e (dec), r e s u s p e n d e d In I ml d t s t i l l * d w a t e r (deC) and t h e n s u b j e c t e d to s o n i c d i s r u p t i o n (2x5 s e c b u r s t s u s i n g I / 4 i n c h n e e d l e p r o b e o f F i s h e r s o n i c d i s m e m b r a t o r Hodel 3 0 0 ) . DNA c o n t e n t was measured u s i n g ¢he e t h l d l u m b r o m i d e method o f LeRecq and P a n l e t t i (2/,) a d a p t e d f o r a u t o m a t e d [ l u o r o m e t r ¥ (2~) v l [ ~ a T e c h n i c o n a u t o a n s l y z e r syste,m. The c e l l s o n t c a t e ~as d i l u t e d wich 5 v o l u m e s 0 . I N T r i s - C 1 (20°C) and mixed in a p r o p o r t i o n i n g pump ( f l o w r a t e 2 . 5 m l / m l n ) v k t h an e q u a l volume nf 25 ~H e t h l d t u m bromide In O . l H 1 3 r i s - ¢ l ( f i n s 1 c o n c e n t r a t i o n ethtdium bro~de 5 ~g/ml). F l u o r e s c e n c e was d e t e r m i n e d u s i n g an excitation w a v e l e n g t h of 300 nm and an e m i s s i o n w a v e l e n g t h of 756 h a . The i n t e n s i t y o f f l u o r e s c e n c e was l i n e a r w i t h DNA s t a n d a r d o v e r range 1-6 ~ g / a l and w i t h s e r i a l d i l u t i o n s of c e l l s o n l c a t e , l~lckground f l u o r e s c e n c e was l e s s t h a n l ~ . Cross-reactlvlty w i t h IU~ ~ 8 s 2"~rX" P r e t r e a t u e n t o f t h e s o n t c a t e w i t h Rl~ose ( 5 0 p g / a l ) a t 37°C ~or t 5 - 6 0 rain o r h e p a r i n 10-100 ~g/ml 426) had no e f f e c t on the meesurred OHA c o n t e n t . 1nits-assay variability was + 1 . 9 ~ and i n t e r - a s s a y variability +6.|~. The memlured DUA c o n t e ~ t e o r r e X e t e d w i t h c e l l c o u n t by hemo--cytometer (rmO,gA, n - l O ) , The mean (+._SO) c e l l u l a r 1)HA
J~'5"
n ~0
x ~m
17
content was 14.7+2.18 pg,/cell (n=lO), while the mean DNA content of each incubation ~ulture wa~ 14+5 ~g/well (n=ll3). Toxicity studles Glucose ~hosphate tsomerase (GPI) actlvlt~ GPl activity was measured in duplicate in the cell sonlcates
(27). ii)
Cell Viabillt~ Cell viability was assessed in duplicate using the erythrosin B (1% erychrosln B in 0.9% NaCI) dye exclusion technique.
RESULTS Basal 5a-reductase
activity
ranged from 0 . 8 - 1 2 . 1
r e d u c e d p r o d u c t / ~ g DNA/hour ( T a b l e 1 ) . for quadruplicate was +24%.
The c o e f f i c e n t
e x p e r i m e n t s run i n p a r a l l e l
The 5 newborn c e l l
lines
pmoles 5 ~ of v a r i a t i o n
for these |2 cell
had s t g n l f i c a n t l y
llnes
lower activity
than the 7 older males (2.6+2.2 vs. 10.7+2.6 pmoles 5a-reduced product/vg DNA/hr, p
line during l
hour co-incubatlon with testosterone and either progesterone or one of the five progestatlonal compounds at lO-5 H (Table I).
The 2
neonatal cell lines with basal activity less than 2 pmoles/og DNA/hr were excluded from statistical analysis of the progestogen studies. In the remaining I0 cell lines, 5a-reductase activity was reduced to 4.9+_4.5% of basal levels in the presence of progesterone (Figure l). Similar concentrations of norethynodrel, ethisterone, dl-norgestrel and d - n o r g e s t r e l
had no s t a t i s t i c a l l y
significant
effect,
but
norethlndrone inhibited 5a-reductase activity to 50.0+19.0% (range 17-70Z) of basal levels.
These effects were observed in both the 6th
and lOth generations of two cell lines (nos. 8 and 12). There was no
18
~
.,a- 'm m , o
l: D
m
OF BASAL5ocREDUCTA~,~TtVITY
Nommmemm et~
F_ig_ure I - Effect of progesterone and 5 progestational compounds (10 -5 I{) on 5~-reductase activity in Cultured fibroblast monolayers derived from genital skin of iO subjects with basal 5~-reductase activity greater than 2 pmoles/~g DNA/hr. Each bar graph represents the mean (+SE) percent of basal enzyme activity for the I0 cell lines in the presence of the specified steroid (*p
difference in the effect of the steroids on the cell lines from the hypospadic boys compared to the cell lines from normal males. The inhibitory effect of both progesterone and norethindrone on ~-reductase
activity was dose-related over the range I0-9-~0 -5 M
with approximate ED50 values of IO-8 M and 5xlO -5 respectively (Figure 2).
To test whether the effects o f progesterone and
JTIBOXDm
21
enzyme activity at I0 -5 ,'I, bat the effect was one-tenth that of progesterone
at I0 -7 1I in this system.
The inhibitory e f f e c t
of n o r e t h t n d r o n e was a p p a r e n t o n l y a t
lO -5 M, and absent at lO -8 M. progestogens
have approxlmately
Although all of the 5 synthetic the same potency in other biologlcal
systems (29), thls effect of norethlndrone
appeared to be unique.
However, we observed no inhibitory effect of norethindrone lO -8 ~I, a concentration
similar to Chat found in human plasma
following an oral dose of I m g in most contraceptive plasma norethlndrone globulln (32,33),
at
norethlndrorm
preparations.
(30,31), the dosage used
Furthermore,
since 96Z of the
Is bound to albumln and sex hormone binding
the biologically
active free concentration would be
only about I0 -lO M. Thus, although norethlndrone
can inhibit 5a-reductase activity
in genital skln flbroblast monolayers
at hlgh concentrations
(10 -5 N),
It seems unllkely Chat direct inhibition of 5a-reductase by thls agent in vivo can explaln any observed teratogenlc effects of progestatlonal
compounds on the male genltal tract.
the results of our experiments,
On the basis of
however, we cannot exclude an indirect
effect of these steroids on 5a-reductase activity either via receptor-medlated
transcriptional
events or through direct inhibition
of testosterone binding to the androgen receptor.
AClOIOWLEDGENENTS T h i s work was s u p p o r t e d by g r a n t s from t h e N e d t c a l R e s e a r c h C o u n c i l of Canada and t h e S t . B o n i f a c e H o s p i t a l R e s e a r c h F o u n d a t i o n . D r . Dean i s a f e l l o w o f t h e N e d t c a l R e s e a r c h C o u n c i l o f Canada. The a u t h o r s a r e i n d e b t e d t o H r s . ¥ v e t t e F e r r y and H r . I q b a l R t y a z for their expert technical assistance, t o Hs. Emma G u l b i s f o r h e r
~
22
T . m ~m.O X n m
secretarial assistance, and to l~r. ~. Decter [or his cooperation obtaining surgical specimens.
~:
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In this paper, the term p~ogestoF, ens wJ~l reter to those synti~et~c compounds with both progestztional and andro~enlc propertLes. The term proges~ins will h~ used only to refer to pure proRestatio~al agents. Voight, W. and Hsia, S.I.... ]. BIOL. CHEM. 248,4280 (1973). Goldman, E.q. and Bongiovanni, A.M., ANN. NY=~ACAD. SCI. 142) 75~ (1967). Aarskog, D., Personal communication. Sweet, R.A., Schrott, H.G. and Curllnd, R., MAYO CLIN. PREC. 49, 52 ( |974). Aarskog, D., ACTA PEDIATR. SCAND. (SUPPL.) 203, I (1970). Lorber, C.A., Cassldy, S.B. and F~gel, E., FERTIL. AND BTERIL. 3!, 2| (1979). Neto, R.M., Castilla, E.E. and Pas, J.E., AM. J. t~D. GENET. lO, 5 (19s|). Czeizel, A., Toth, J. and Erodi0 E., tlUM. HERED. 29, 166 (1979). Hau, G., TERATOLOGY 24, 285 (198l). Janet|oh, D.T., Riper, J.H. and Glebatis, D.M., N. ENGL. J. ~ D . 29l, 697 (1974). Harlap, S., Prywes, R. and Davies, A.M., LANCET i, 682 (197~). Heinonen, O.P., Soln, E.D., Monson, R.R,, Hook, E.B, and Shapiro, S., N. ENGL. J. t~D. 296, 67 (1977). Rothman, K.J. and Toulk, C., N. ENGL. J. MED. 299) 522 (197~). Ishizuka, N., and Kauashima, CONGENITAL ANOMALIES 4, 5 (1964). Wilson, J . D . J . BIOL. CHEM. 250, 3498 (|975). Le Pacq, J.B. and Paolettl, C., ANAL. BIOCHEM. 17, foe (1966). Van Dyke, K. and Szustklewlcz, C., ANAL. BIOCHE~. 23, I09
(1968). 26. 27. 28.
Karsten, U. and Wollenberger, A., ANAL. BIOCHEM. 77, 464 (1977). Beutler, E., in: Red Cell Metabolism 2rid Edltlon, Grune and Stratton Inc., New york,' ( t 9 7 5 ) , P 40. Volght, W., Fernandez, E.P. and Nsia, S.L., J. BIOL. CHEI4~ 245, 5594 (1970).
29.
Whitehead, M . I . , Townsend, P , T . , F r y s e - D a v l e s , J . ,
Re.
and King, R.J.B., N. ENGL..I. MED. 305, 1599 (1981). Wnrr~n. R.I. and Fotherbv. K.. J. ENDOCRINOL. 62, 605 (1974).
Ryder, T~A.
aj 31. 32. 33.
.x, in iut o x ID ul
Crawford, F .E . , Back, D . J . , L ' e o r a e , H. and Breckenridge, A.M., BR. HED. 5. 282, 1829 (1981). Kumond, G.L., Lahteenaaki, P.L.A., Lahteenaaki, P. and Luukkainen, T . , J . STER. BIOCHEH. 17, 375, 1982. Jenkins, N. and Fotherby, K., J . STEg. BIOQIEH. 13:521 (1980). TRIVIAL NAHES
5 a - a n d r o s t a n e d t o l , 5a-androstane-3~,17 B d l o l 5~-androstanedtone, 5~-androstane-3, 17-dione androstenedione, 4-androstene-3, 17--dione 5 ~ - d l h y d r o t e s t o s t e r o n e ; 17 B hydtoxy-$a-androstan-3-one e t h i s t e r o n e , 17 ~-ethlnyl-17-hydroxT-4-androsten-3-one n o r g e s t r e l , 1 7 a - e t h l n y l - 13 B-ethyl- 17-hydroxT-4-gonen-3-one n o r e t h l n d r o n e , 17~-eth/ny1-17-hydroxy-4-estren-3-one n o r e t h y n o d r e l , 17a-ethinyl-17-hydroxy-5(lO)-estren-3-one p r o g e s t e r o n e , 4-pregnene-3,20-dione t e s t o s t e r o n e , 17 B-hydroxy-4-a~r os t e n - 3-one
23
EFFECT OF PROGESTERONf. AND 5 PROGESTATIONALSTEROIDS tl(~ -5 11) ON ~,-REDUCTAS£ ' ACTIVITY T.H CUL~'UI~D ¥ I I B R O B L A S T " H O N O L A ~ ~ S K ~ - O - F ~ 12 tIALE SUBJE~'Ts.
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DONOR c ~ v . ~ , "J . . . . . . . . . . . . . PHI~NOT~P! TION I
Basal enzyme a c t i v i t y is expressed as the m e a n +_SD ,)[ qundruplic~t,: a s s a y s . Enzyme ~ c t v i t y in the presence of the p r o g e s t e t i o n a l s t e r o i d s t s the mean of d u p l i c a t e ~saays. Cell l i n e s 1, 2 and 5 ~ere no~ ~ested with p r o g e s t e r o n e (denoted by d a s h e s ) . &bbre~la~inns .med iq the cable i n c l u d e : PROG, progesterone; HOBSTHYN, n o r e t h y n o d r e l ; HORETHIN, no~echtndrnne: ~T~[h~, ~ t h i s t e r o n e ; NORC, n n r g e s t r e l : ~ , ~ u b o r n ; N, normal: ~, h y p o s p e d i a s .
TABLE 1:
|
U
N
0
W
|
t~ d~
13
THE EFFECT OF FIVE SYNT~TIC PROGESTATIONAL OONPOUNDS ON 5a-R.gDUCTASK ACTIVITY IN GENITAL SKIN FIBROBLAST HOHOI,kYSRS. H . J . l)ean and J . S . D . ~ t n t e r D e p a r t - ~ n t of P e d i a t r i c s ,
U n i v e r s i t y of Manitoba, Winnipeg, N a n l t o b a , Canada.
Received 9-20-83 ABSTKAL~ The s u g g e s t i o n has been mule t h a t p r e n a t a l e x p o s u r e to s y n t h e t i c p r o g e s t o g e n e c o n t r i b u t e s t o an i n c r e a s e d i n c i d e n c e of h y p o s p a d l u . One p o t e n t i a l n e c h a n i s n f o r such an e f f e c t ~Lght be i n h i b i t i o n of 5 a r e d u c t a s e , a key enzyme in norm .I s a l e s e x u a l d i f f e r e n t i a t i o n . We have e x s a t n e d the e f f e c t of p r o g e s t e r o n e and of f l v e s y n t h e t i c p r o s e s t o s e n s upon 5 a - r e d u c t a n e a c t i v i t y i n f l b r o b l n e t I o n o l a y e r s from 12 genltal s k l n cell lines o b t a i n e d f r o a n e r s a l newborn i n f a n t s , boys w l t h p h i m o s l s and h y p o s p a d i s s and a u o r m ~ a d u l t m a l e , B a s a l enz~ae a c t i v i t i e s ranged from 0 . 8 - 1 2 . 1 p l o l e s 5 a - r e d u c e d p r o d u c t / ~ S DNA/hour. P r o g e s t e r o n e and n o r e t h l n d r o n e i n h i b i t e d 5 a - r e d u c t a n e a c t i v i t y i n a d o s e dependent manner to • uu~isnm of 95Z and 50Z o f b e s a l l e v e l s r e s p e c t i v e l y a t 10- 5 H. S i a t l a r c o n c e n t r a t i o n s (10 - 5 N) of n o r e t h y n o d r e l , e t h i s t a r o n e , d l - n o r g e s t r a l and d - n o r g e s t r e l had l i t t l e or no e f f e c t . S t u d i e s of c e l l v i a b i l i t y showed t h a t t h e e f f e c t s of p r o g e s t e r o n e and n o r e t h t n d r o n e were s p e c i f i c f o r 5 ~ - r e d u c t a s e and n o t
non-specific toxic effects. INTRODUCTION The a t c r o s o a a l enzyme r e s p o n s i b l e f o r the c o n v e r s i o n of testosterone
to d i h y d r o t e s t o s t e r o n e (DHT), ~ - r e d u c t a s e ,
d e t e r a t n a n t of n o n s a l male s e x ~ a l d i f f e r e n t i a t i o n , b e l i e v e d to be the a c t i v e l n t r a c e l l u l a : the urethral
s i n c e DHT i s
androgen c o n t r o l l i n g f u s i o n of
f o l d s and thus normal d e v e l o l m e n t of the p e n i s and
penile urethra (1),
C o n g e n i t a l absence of t h i s enzyue l e a d s to
I n c o m p l e t e f u s i o n of the u r e t h r a l hypospadias (2). teratogens,
is a critical
folds, clinically
recogrLtzed as
I n h l b l t l o n of 5 ~ - r e d u c t a s e by e n v l r o ~ n t a l
including s y n t h e t i c p r o g e s t o g e n s , hss been s u g g e s t e d t o
e x p l a l n an a p p a r e n t i n c r e a s e in the i n c i d e n c e of h y p o s p a d l a s In
Volume 43, Number I
~
.A- m ~ o • ~ m
J a m m r y 1984
industrialized countries
in the past decade (3-8), but the evident~,
for such a relationship is anecdotal and circumstantial. itself can inhibit ~cL-reductase
Progesterone
activity in vitro, but substituti,~:~ L:,,
the 17~i and 16,, positions may reduce this inhibitory effect (9). Studies in the rat and rabbit have shown variable degrees of incomplete mascullnlzation after prenatal exposure to high doses of progestational
compounds such as the 19-nor and 17:~-ethlnyl
derivatives of testosterone
(3,10,II).
Several retrospective s~udles
of hypospadlc boys have suggested an increased rate of maternal ingestion of progestogens during early pregnancy (12-16), with some correlation between the time of ingestion and the severity of the defect (12,13,17).
Conversely, however, several prospective studies
of women who ingested synthetic progestogens
In early pregnancy have
failed to show an increased risk of hypospadlas offspring
(17-22),
in their male
These negative epidemiologlcal
conclusive, however,
findings
may not be
in view of the hlgh incidence of spontaneous
hypospad ias Since further clinical studies are unlikely to resolve this problem, we chose to explore possible mechanisms for a teratogenlc effect in vitro.
We have studied the effect of five of t h e
progestational agents contained in contraceptive preparations On c,~_ reductase activity in human genital skin flbroblasts of normal and hypospadlc boys.
This experimental model, using mature slteospeciflc
flbroblasts, has b~en used widely in the study of androgen-~edlated events of male sexual differentiation.
~ T I ~ O X D ~
15
MATERIALS
E a g l e ' s minimum e s s e n t i a l medium, amino a c i d s , t r i c i n e c h l o r i d e and t r y p s i n were p u r c h a s e d from Grand I s l a n d B i o l o g i c a l Company. F o u r w e l l e d (60mm) L i n b r o d i s h e s and f e t a l c a l f serum were o b t a i n e d from Flow Laboratories, and plastic tissue culture dishes (60 -.- and I00 mm) from Fisher Scientific. (1,23H) Testosterone (SA-52 Ci/mmol) was purchased from New England Nuclear, and nonradioactive steroids from Steraloids Inc. The progestatlonal compounds uorethynodrel, norethludroue aud ethlsterone were purchased from Sigma Chemicals Inc. The pure active stereochemical d-isomer of norgestrel and t h e r a c e m i c m i x t u r e , d l - n o r g e s t r e l , were k i n d l y d o n a t e d by Wyeth L a b o r a t o r i e s , T o r o n t o , OQtarfo, Canada. P r e c o a t e d s i l i c a g e l - 25 UV254 t h i n l a y e r chromatography p l a t e s (20x20 cm) were from Brink.man I n d u s t r i e s . Calf thymus D~A ( t y p e I ) , b a k e r ' s y e a s t RNA ( t y p e I I I ) , b o v i n e p a n c r e a s e RNAase ( t y p e I A ) , e t h i d i u m bromide (2,7-diamino-10-ethyl-9-phenylphenauthridium bromide), fructose 6 - p h o s p h a t e , g l u c o s e - 6 - p h o s p h a t e d e h y d r o g e n a s e ( B a k e r ' s y e a s t type VII), NADP and NADPH were purchased from Sigma Chemicals Inc.
METHODS
Cell Culture G e n i t a l ( p r e p u t i a l ) s k i n b i o p s i e s ware o b t a i n e d from 5 normal newborn males a t t h e time of c i r c u m c i s i o n , from 4 boys age 1-12 y e a r s a t the time of h y p o s p a d i a s r e p a i r , from 2 boys see 2 months and 5 y e a r s w i t h p h i m o s i s a t the time of c i r c u m c i s i o n and from 1 normal a d u l t male age 25 y e a r s . The e x p l a n t s were c u l t u r e d i n E a g l e ' s m i n i m a l e s s e n t i a l medimn c o n t a i n i n g 20nM t r i c i n e c h l o r i d e , 24nM NaHC03, 15Z f e t a l c a l f serum, 1Z n o n e s s e n t i a l amino a c i d , p e n i c i l l i n 100 ~U/ml and s t r e p t o m y c i n 100 ~g/ml a t 37°C i u 5Z CO2, The f i b r o b l a s t s were grown to c o n f l u e n c y and t h e n t r e a t e d w i t h 0.05Z t r y p s i n b e f o r e s u b c u l t u r e ; t h e y were m a i n t a i n e d for 3-5 g e n e r a t i o n s i n t h e same medium ( e x c e p t w i t h 10Z f e t a l c a l f serum) which was changed t w i c e weekly and t h e n s t o r e d i n l i q u i d n i t r o g e n . C e l l s for t h i s s t u d y were used b e f o r e t h e e i g h t e e n t h g e n e r a t i o n . A l l e x p e r i m e u t s were performed on the 7 t h day a f t e r s u b c u l t u r e f o l l o w i n g 24 h o u r s i n medium w i t h o u t f e t a l c a l f serum. 5~-reductase activity B a s a l 5 ~ - r e d u c t a s e a c t i v i t y was measured i n q u a d r u p l i c a t e 60 mm wells using a modification of the method of Wilson (23). The monolayers were washed twice with medium (without fetal calf serum) and incubated with 3 ml of the same medium containing approximately 105 cpm 3H testosterone (1.75 pmoles/well) and 0.05 ~M unlabelled testosterone (150 pmoles/we11) for I hour at 37°C in 5% CO 2, I n c u b a t i o n e x p e r i m e n t s w i t h the p r o g e s t a t i o u a l s t e r o i d s , p r o g e s t e r o n e , n o r e t h y n o d r e l , n o r e t h i n d r o n e , e t h i s t e r o n e , d - n o r g e s t r e l and d l - n o r g e s t r e l were p e r f o r m e d i n p a r a l l e l i n d u p l i c a t e 60 ,w, w e l l s u n d e r i d e n t i c a l c o n d i t i o n s ( e a c h s t e r o i d a t 10 -~ M was d i s s o l v e d i n t h e i n c u b a t i o n medium c o n t a i n i n g a p p r o x i m a t e l y 105 cpm 3H t e s t o s t e r o n e and 0 . 0 5 ~M u n l a b a l l e d t e s t o s t e r o n e . ) Dose r e s p o n s e
ifi
I
qL~ ]m ~ . o
x ~
w
s t u d i - ~ (10 - g - i q ' ~ ~|) w~r~ I~rf,~rmed in a s i m i l a r [~ann,.r w i t ~ p r o g e s t e r o n e and n o r e t h L n d r n n e in 2 (-eL] ] I n e ~ . A f t e r I n c n b a t l o n , the ~,~.dtum was removed and | m| we:; ,~zt¢~r~.~ w~th 8 v e t . r u e s b e n z e n e . C a r r i e r s t e r o i d s (~ ';~ each t e s t ~ s t e r o . ~ : dlhydrotestosterono, a n d r o s t e n e d t o n ~ , 5 , ~ - a n d r , ) s t a n e d t n n e an(l ~ , a n d r o s t a n e d l o l ) v e r e added [~ ~ ~I ~)f the e x t r a c t . ~ h l c h wa~ d r i e d under nitrogen. The r e s i d u e ~a~ r e c o n s t i t u t e d in O. ~. r~l ~.Chunol .u~d a p p l l e d t o 2Ox20 c~ s i l i c a Rel t h i n l a y e r c h r o m a t o g r a p h y ,?tate.~ (4 channels). & f r e t one a~cent |~ 95:~ benzent : ~ t h a n o l . te~to~t,'r,~:;" (Rf 0 . 3 9 ) and a n d r o s t e n e d i o n e (Rf ~).65) were i d e n t i f i e d u n d e r !iV Light, u h i l e a n d r o s t a n e d i o l (R~- ~ . 3 L ) , d i h y d c ~ t e s t n ~ t e r o u e (!~f ~;:.:~7) and a n d r o s t a n e d i o n e (Rf 0 . 7 7 ) were Located i n m a r k e t c h a n , ~ l s (3 per p l a t e ) by s p r a y l n K w i t h 6c):'. II2SO4 and a l l n u l n g the v i s i b l , , c ~ l o . r ~ t,~ d e v e l o p at 37°C. P:ach zone ~ ; ~craped f r o n the ~)tat,; and i t ~ '!"! c o n t e n t d e t e r m i n e d by l l , l ~ i d ~ c | n t | t l a t i o n ~-,*unrt.R ~I~ ml ,~.~'.i PPO/O.O06~ POPOP Ln t o l u e n e ) . ~i~c~; ne,~.'li'~ihi .~ 3~i t o . l i t , ; wr, re t,~,Jn~ i n the a n d r o s t e n e d ~ o n e arid :~ndr~canedLnn,. ~r._,a~, the eo,'q ,'~f ~1~ c o u n t s i t e m t h e DHT and a n d r o s : ~ e d ~ o l ;~r~as wa~ ~,~¢,d to r ~ l c , a l a t , ~ ~ , reduced p r o d u c t , .~ subscra,~. I-i=~,~- (~n.-,,ba',,d ,,:,diu~ w i t h e , a t ,~.1!--) was t|.qed I~o c o r r e c t f ~ r g~,~--. ~ . ~ , ~ ; t t i r r.~d~|t'~|.~; .~ .;uhsI. t';J~./, a:~."
874~.. The cnPff|cien! ,)~ .~rlati,~:~ t()r p a i r e d Jnplic.'_tte ~.q:~;:'~ '.,a~ ÷4~'3~. . ~ J b s t r a t o , , t t l i ~ a t i ,n ::!'~dor I ~ ; = ! :,,,~ditlc~u'a r~nged ~rom 1"0o90~ f o r th+" 12 ('+:11 lin,=m. :.t.arimti,'.~] :~n+:i,/mlm of rile I n c u b a t i o n e x p e r i m e n t s w i t h the pr~geSt~ti<,n;~1 ~t~rr,ld~ ~,,;ls ~ r [ o r ~ . d ~L=.tu¢ [actorial a n a l y s i s of carlnr~r,- ,¢i t h o . t r e p l l c a t i on (one ol)s,~rvat i , . , per ~ e l l ) and the Tuk~v m , , ] t t D l , r:~nR*~ r o ~ t . DHA measu[etment A f t e r t n c t l h a t t o n , the mou,,lav,,rs were wash.d t h r e e time~ wtt|t 50mH T r t R - s a l i n e ( 4 n C ) ; a n d t h e n i . c u h a t e d f o r 5 m i n u t e s i n I n l N.O5~ t r y p s l n t,~ s a [ i n e at 37 °C. The c e l l s were h ~ r v e s t e d by trituration and then c e n t r | f u g e d at 80(') g f o r ~ mln a t 4°C. The p e l l e t was washed once with I mE S(~ mr4 T r t s - s a l t n e (dec), r e s u s p e n d e d In I ml d t s t i l l * d w a t e r (deC) and t h e n s u b j e c t e d to s o n i c d i s r u p t i o n (2x5 s e c b u r s t s u s i n g I / 4 i n c h n e e d l e p r o b e o f F i s h e r s o n i c d i s m e m b r a t o r Hodel 3 0 0 ) . DNA c o n t e n t was measured u s i n g ¢he e t h l d l u m b r o m i d e method o f LeRecq and P a n l e t t i (2/,) a d a p t e d f o r a u t o m a t e d [ l u o r o m e t r ¥ (2~) v l [ ~ a T e c h n i c o n a u t o a n s l y z e r syste,m. The c e l l s o n t c a t e ~as d i l u t e d wich 5 v o l u m e s 0 . I N T r i s - C 1 (20°C) and mixed in a p r o p o r t i o n i n g pump ( f l o w r a t e 2 . 5 m l / m l n ) v k t h an e q u a l volume nf 25 ~H e t h l d t u m bromide In O . l H 1 3 r i s - ¢ l ( f i n s 1 c o n c e n t r a t i o n ethtdium bro~de 5 ~g/ml). F l u o r e s c e n c e was d e t e r m i n e d u s i n g an excitation w a v e l e n g t h of 300 nm and an e m i s s i o n w a v e l e n g t h of 756 h a . The i n t e n s i t y o f f l u o r e s c e n c e was l i n e a r w i t h DNA s t a n d a r d o v e r range 1-6 ~ g / a l and w i t h s e r i a l d i l u t i o n s of c e l l s o n l c a t e , l~lckground f l u o r e s c e n c e was l e s s t h a n l ~ . Cross-reactlvlty w i t h IU~ ~ 8 s 2"~rX" P r e t r e a t u e n t o f t h e s o n t c a t e w i t h Rl~ose ( 5 0 p g / a l ) a t 37°C ~or t 5 - 6 0 rain o r h e p a r i n 10-100 ~g/ml 426) had no e f f e c t on the meesurred OHA c o n t e n t . 1nits-assay variability was + 1 . 9 ~ and i n t e r - a s s a y variability +6.|~. The memlured DUA c o n t e ~ t e o r r e X e t e d w i t h c e l l c o u n t by hemo--cytometer (rmO,gA, n - l O ) , The mean (+._SO) c e l l u l a r 1)HA
J~'5"
n ~0
x ~m
17
content was 14.7+2.18 pg,/cell (n=lO), while the mean DNA content of each incubation ~ulture wa~ 14+5 ~g/well (n=ll3). Toxicity studles Glucose ~hosphate tsomerase (GPI) actlvlt~ GPl activity was measured in duplicate in the cell sonlcates
(27). ii)
Cell Viabillt~ Cell viability was assessed in duplicate using the erythrosin B (1% erychrosln B in 0.9% NaCI) dye exclusion technique.
RESULTS Basal 5a-reductase
activity
ranged from 0 . 8 - 1 2 . 1
r e d u c e d p r o d u c t / ~ g DNA/hour ( T a b l e 1 ) . for quadruplicate was +24%.
The c o e f f i c e n t
e x p e r i m e n t s run i n p a r a l l e l
The 5 newborn c e l l
lines
pmoles 5 ~ of v a r i a t i o n
for these |2 cell
had s t g n l f i c a n t l y
llnes
lower activity
than the 7 older males (2.6+2.2 vs. 10.7+2.6 pmoles 5a-reduced product/vg DNA/hr, p
line during l
hour co-incubatlon with testosterone and either progesterone or one of the five progestatlonal compounds at lO-5 H (Table I).
The 2
neonatal cell lines with basal activity less than 2 pmoles/og DNA/hr were excluded from statistical analysis of the progestogen studies. In the remaining I0 cell lines, 5a-reductase activity was reduced to 4.9+_4.5% of basal levels in the presence of progesterone (Figure l). Similar concentrations of norethynodrel, ethisterone, dl-norgestrel and d - n o r g e s t r e l
had no s t a t i s t i c a l l y
significant
effect,
but
norethlndrone inhibited 5a-reductase activity to 50.0+19.0% (range 17-70Z) of basal levels.
These effects were observed in both the 6th
and lOth generations of two cell lines (nos. 8 and 12). There was no
18
~
.,a- 'm m , o
l: D
m
OF BASAL5ocREDUCTA~,~TtVITY
Nommmemm et~
F_ig_ure I - Effect of progesterone and 5 progestational compounds (10 -5 I{) on 5~-reductase activity in Cultured fibroblast monolayers derived from genital skin of iO subjects with basal 5~-reductase activity greater than 2 pmoles/~g DNA/hr. Each bar graph represents the mean (+SE) percent of basal enzyme activity for the I0 cell lines in the presence of the specified steroid (*p
difference in the effect of the steroids on the cell lines from the hypospadic boys compared to the cell lines from normal males. The inhibitory effect of both progesterone and norethindrone on ~-reductase
activity was dose-related over the range I0-9-~0 -5 M
with approximate ED50 values of IO-8 M and 5xlO -5 respectively (Figure 2).
To test whether the effects o f progesterone and
JTIBOXDm
21
enzyme activity at I0 -5 ,'I, bat the effect was one-tenth that of progesterone
at I0 -7 1I in this system.
The inhibitory e f f e c t
of n o r e t h t n d r o n e was a p p a r e n t o n l y a t
lO -5 M, and absent at lO -8 M. progestogens
have approxlmately
Although all of the 5 synthetic the same potency in other biologlcal
systems (29), thls effect of norethlndrone
appeared to be unique.
However, we observed no inhibitory effect of norethindrone lO -8 ~I, a concentration
similar to Chat found in human plasma
following an oral dose of I m g in most contraceptive plasma norethlndrone globulln (32,33),
at
norethlndrorm
preparations.
(30,31), the dosage used
Furthermore,
since 96Z of the
Is bound to albumln and sex hormone binding
the biologically
active free concentration would be
only about I0 -lO M. Thus, although norethlndrone
can inhibit 5a-reductase activity
in genital skln flbroblast monolayers
at hlgh concentrations
(10 -5 N),
It seems unllkely Chat direct inhibition of 5a-reductase by thls agent in vivo can explaln any observed teratogenlc effects of progestatlonal
compounds on the male genltal tract.
the results of our experiments,
On the basis of
however, we cannot exclude an indirect
effect of these steroids on 5a-reductase activity either via receptor-medlated
transcriptional
events or through direct inhibition
of testosterone binding to the androgen receptor.
AClOIOWLEDGENENTS T h i s work was s u p p o r t e d by g r a n t s from t h e N e d t c a l R e s e a r c h C o u n c i l of Canada and t h e S t . B o n i f a c e H o s p i t a l R e s e a r c h F o u n d a t i o n . D r . Dean i s a f e l l o w o f t h e N e d t c a l R e s e a r c h C o u n c i l o f Canada. The a u t h o r s a r e i n d e b t e d t o H r s . ¥ v e t t e F e r r y and H r . I q b a l R t y a z for their expert technical assistance, t o Hs. Emma G u l b i s f o r h e r
~
22
T . m ~m.O X n m
secretarial assistance, and to l~r. ~. Decter [or his cooperation obtaining surgical specimens.
~:
REFERENCES I. 2.
6.
~Jilson, J.D., ANN. REV. PHYSIOL. 40, 279 (1978). Imperato-McGinley, J., Guerrero, L., Gautier, T. and Peterson. R,E., SCIENCE 184, 1213 (1974). Aarskog, D., N. ENGL. J. t4ED. 300. 75 (1979). Kallen, B. and Winberg, I., AcTA--PEDIATRIC SCAN]) SUPPL 293 (I~)82). Fyol Registry 1977 Department of Health and t~elfare Canada courtesy of Dr. P. Bannister 1979. Campbell, N., Newcombe, R.G. and Ueatherall. ;.A.C., BR. ~ID. ~.
7.
Bjerkedal,
3. 4. 5.
3, 52 (1973). T. and Bakketelg,
L.S.,
INT. J. EPII)EMIOL. 4. 3l
(197s). 8.
9. lO. I I. 12. |3. 14. 15. |6. 17. l~. |9. 20. 21. 22. 23. 24. 25.
In this paper, the term p~ogestoF, ens wJ~l reter to those synti~et~c compounds with both progestztional and andro~enlc propertLes. The term proges~ins will h~ used only to refer to pure proRestatio~al agents. Voight, W. and Hsia, S.I.... ]. BIOL. CHEM. 248,4280 (1973). Goldman, E.q. and Bongiovanni, A.M., ANN. NY=~ACAD. SCI. 142) 75~ (1967). Aarskog, D., Personal communication. Sweet, R.A., Schrott, H.G. and Curllnd, R., MAYO CLIN. PREC. 49, 52 ( |974). Aarskog, D., ACTA PEDIATR. SCAND. (SUPPL.) 203, I (1970). Lorber, C.A., Cassldy, S.B. and F~gel, E., FERTIL. AND BTERIL. 3!, 2| (1979). Neto, R.M., Castilla, E.E. and Pas, J.E., AM. J. t~D. GENET. lO, 5 (19s|). Czeizel, A., Toth, J. and Erodi0 E., tlUM. HERED. 29, 166 (1979). Hau, G., TERATOLOGY 24, 285 (198l). Janet|oh, D.T., Riper, J.H. and Glebatis, D.M., N. ENGL. J. ~ D . 29l, 697 (1974). Harlap, S., Prywes, R. and Davies, A.M., LANCET i, 682 (197~). Heinonen, O.P., Soln, E.D., Monson, R.R,, Hook, E.B, and Shapiro, S., N. ENGL. J. t~D. 296, 67 (1977). Rothman, K.J. and Toulk, C., N. ENGL. J. MED. 299) 522 (197~). Ishizuka, N., and Kauashima, CONGENITAL ANOMALIES 4, 5 (1964). Wilson, J . D . J . BIOL. CHEM. 250, 3498 (|975). Le Pacq, J.B. and Paolettl, C., ANAL. BIOCHEM. 17, foe (1966). Van Dyke, K. and Szustklewlcz, C., ANAL. BIOCHE~. 23, I09
(1968). 26. 27. 28.
Karsten, U. and Wollenberger, A., ANAL. BIOCHEM. 77, 464 (1977). Beutler, E., in: Red Cell Metabolism 2rid Edltlon, Grune and Stratton Inc., New york,' ( t 9 7 5 ) , P 40. Volght, W., Fernandez, E.P. and Nsia, S.L., J. BIOL. CHEI4~ 245, 5594 (1970).
29.
Whitehead, M . I . , Townsend, P , T . , F r y s e - D a v l e s , J . ,
Re.
and King, R.J.B., N. ENGL..I. MED. 305, 1599 (1981). Wnrr~n. R.I. and Fotherbv. K.. J. ENDOCRINOL. 62, 605 (1974).
Ryder, T~A.
aj 31. 32. 33.
.x, in iut o x ID ul
Crawford, F .E . , Back, D . J . , L ' e o r a e , H. and Breckenridge, A.M., BR. HED. 5. 282, 1829 (1981). Kumond, G.L., Lahteenaaki, P.L.A., Lahteenaaki, P. and Luukkainen, T . , J . STER. BIOCHEH. 17, 375, 1982. Jenkins, N. and Fotherby, K., J . STEg. BIOQIEH. 13:521 (1980). TRIVIAL NAHES
5 a - a n d r o s t a n e d t o l , 5a-androstane-3~,17 B d l o l 5~-androstanedtone, 5~-androstane-3, 17-dione androstenedione, 4-androstene-3, 17--dione 5 ~ - d l h y d r o t e s t o s t e r o n e ; 17 B hydtoxy-$a-androstan-3-one e t h i s t e r o n e , 17 ~-ethlnyl-17-hydroxT-4-androsten-3-one n o r g e s t r e l , 1 7 a - e t h l n y l - 13 B-ethyl- 17-hydroxT-4-gonen-3-one n o r e t h l n d r o n e , 17~-eth/ny1-17-hydroxy-4-estren-3-one n o r e t h y n o d r e l , 17a-ethinyl-17-hydroxy-5(lO)-estren-3-one p r o g e s t e r o n e , 4-pregnene-3,20-dione t e s t o s t e r o n e , 17 B-hydroxy-4-a~r os t e n - 3-one
23
EFFECT OF PROGESTERONf. AND 5 PROGESTATIONALSTEROIDS tl(~ -5 11) ON ~,-REDUCTAS£ ' ACTIVITY T.H CUL~'UI~D ¥ I I B R O B L A S T " H O N O L A ~ ~ S K ~ - O - F ~ 12 tIALE SUBJE~'Ts.
m
12
s. 9.
~.2
1
0..2
Nll
5
6 7.
n
NI lIB
AGE
3 4
2
l
~gLL
7 11 10 8 ~ !'e,
N H H
H: H
L7 14 9 10 7
H tl S N H
i).~
34','. 9.~
~
l~o?
,):
• .'~,.|
1.9
1;r..37o ..~ Li).~lZ.~
~.6
O.3
nt.?;t.A
5.1 p,.h 9.7
O.3
e.I. it
5.~I.0 m.
r) 0
8.9 2.(~ fl.0
3.,?
~.~,
2.5 7.", 8.2
I). | 0.9
4J,
;.__. . . . . .
7.~
5.':) II.h 8.7
4.3 O. 7 I.I :1.2 ~.2
~ ......
.
.
,
..
8.8 1 l ..-! 10,.2 12;.7 9;~
7.2 | .7 3.2 0 O. 8
~'~'~ I ~'z
•
6.9 l~.q 1').3 II),~, %;
5.8 ~1.'1 ~..~ t.7 t'). 7
~" -~'~6~T~'£~'~ ~'['V-x:d:'t p.~'l*~..'~ ~, reduced pr,~du~.~/. ~ [ ~ ; . . . . . . . . . . .
12.1+3.6
t.2.~l.0 0.8~0 .4
2. ~Tt. 1
6.34-0.9
DONOR c ~ v . ~ , "J . . . . . . . . . . . . . PHI~NOT~P! TION I
Basal enzyme a c t i v i t y is expressed as the m e a n +_SD ,)[ qundruplic~t,: a s s a y s . Enzyme ~ c t v i t y in the presence of the p r o g e s t e t i o n a l s t e r o i d s t s the mean of d u p l i c a t e ~saays. Cell l i n e s 1, 2 and 5 ~ere no~ ~ested with p r o g e s t e r o n e (denoted by d a s h e s ) . &bbre~la~inns .med iq the cable i n c l u d e : PROG, progesterone; HOBSTHYN, n o r e t h y n o d r e l ; HORETHIN, no~echtndrnne: ~T~[h~, ~ t h i s t e r o n e ; NORC, n n r g e s t r e l : ~ , ~ u b o r n ; N, normal: ~, h y p o s p e d i a s .
TABLE 1:
|
U
N
0
W
|
t~ d~