J. Endocrinol. Invest. 2: 83, 1979

SHORT COMMUNICATION

Human placental ~5-3,8-hydroxysteroid dehydrogenase, ~4,5isomerase: in vitro effect of dehydroepiandrosterone and allylestrenol

P. Scirpa, D. Mango, and A. Bompiani Department of Obstetrics and Gynecology, Catholic University, Roma, Italy

ABSTRACT. The in vitro effects of dehydroepiandrosterone (DHA) and allylestrenol on human placental ~5-3J3-hydroxysteroid dehydrogenase, ~ 4,5 isomerase activity (~5-3J3-HSDH) was in­vestigated by incubation of subcellular fractions with [4_14C] pregnenolone. It has been found that DHA inhibits the ~5-3J3-HSDH activity up to -81% at 5x10-5M concentration while allylestrenol seems to De able to lightly stimulate the ~5-3J3-HSDH activity with a maximum effect <+ 15-26%) at 5x10-7M concentration. The data concerning allylestrenol, seem of particular interest as this com­pound is used as a progestative drug during pregnancy, if one consider the inhibitory effect exert­ed by several natural and synthetic steroids on the ~5-3J3-HSDH activity.

INTRODUCTION It is well established that A5-3J3-hydroxysteroid dehy­drogenase, A 4,5 isomerase (A 5.3_,6-H SO H) system is es­sential for the biosynthesIs of estrogens and progeste­rone during human pregnancy. Because of the lack of these enzymes in the fetal compartment, the conver­sion of pregnenolone (A5_P) to progesterone (A4_P) and dehydroepiandrosterone (DHA) to androstendione have to take mainly place in the placenta (1). Recently two placental isoenzymes converting DHA and A5_p have been described (2, 3). These enzymes seem to be equally distributed between mithocondrial and mi­crosomal fractions (3). Inhibitory effects of endogenous unconJugated steroids on placental A5-3J3-HSDH have been described (3, 4) and interpreted as a potential mechanism regulating the estrogens biosynthesis (4). Similar inhibition on placental A5-3J3-HSDH is effected by several synthetic steroids (2, 5). In this work we have investigated, by in vitro incuba­tions of human placental fractions (800 x g superna­tant) with radioactive substrates, the possibility that A 5_ 3J3-HSDH activity could be influenced by DHA and aliy­lestreno)1, which was described to be able to stimulate the placental steroid metabolism (6-8).

1 17a·allyl-17/l-hydroxy-oestr·4-ene (Gestanon. Organon NV. Oss. The Nether­

lands).

Key-words. Dehydroepiandrosterone, allylestrenol, human placenta, b.~-3fJ­

HSDH. progesterone. pregnenolone.

Correspondence: Dr. Paolo Scirpa, Department of Obstetrics and Gynecology, Catholic University, Largo Gemelli, 8, 00168 Roma, Italy.

Received June 20, 1978; accepted December 10, 1978.

83

MATERIALS AND METHODS General reagents of analytical grade were used and were obtained from Merck (Darmstadt, F.R.G.), Carlo Erba (Milan, Italy) and Boheringer (Mannheim, F.R.G,). [4_14C] Pregnenolone (SA 53 mCi/mmol)was obtained from the Radiochemical Centre (Amersham, U.K,) and was purified before use by thin layer chromato­graphy (TLC) on silicagel G in cyclohexane: ethyl ace­tate (7 : 3). Other steroids were obtained from Makor Chemicals Ltd, (Jerusalem, Israel). The preparation of placental fractions was carried out at 4 C, using placentas obtained immediately after de­livery. Placental tissue was washed, free from mem­branes and homogenized in a Waring Blendor. The determination of the placental A5-3J3-HSDH activi­ty was performed on 800 x g supernatant fractions of placental tissue homogenized in 0.05 M phosphate buffer, pH 7.0, containing 0.25 M sucrose, The placen­tal preparation (equivalent to 8.0g of wet tissue) was in­cubated in duplicate at 37 C in Dubnoff metabolic shaker for 20 min with [4_14C] pregnenolone (320 nmols, 40,000 dpm) in the presence of NAD and air, The total volume of the incubation mixture was 6.3 ml. DHA and allylestrenol were added to the incubation medium in 0.1 ml of ethanol. At the end of the incuba­tion period each sample was extracted with diethyl ether (5 ml x 3) and taken to dryness. A5_P and A4_p presentinthe extract were separated by TLConsilicagel 60F-254 (Merck, Darmstadt, F.R.G.) in the system ben­zene : ethanol (95:5). The enzyme activity was deter­mined by measuring the transformation of [4_14C] preg­nenolone in [4_14C]' progesterone. Radioactivity asso­ciated with the steroids was measured in a liquid scin-

P. Scirpa, D. Mango, and A. Bompiani

Table 1 - "In vitro" effect of DHA on the conversion of l:J..5_p to l:J..4_p in the human placenta.

nmols of l:J..4_p formed per 9 wt in 20 min

+OHA

control 5 x 10-6 M 2.5 x 10-5 M 5 x 10-5 M

13.89 12.60 4.53 2.54 (-9.2%) (-67.3%) (-81.7%)

tillation spectrometer, with a counting efficiency of 90.5% for 14C. The 6 5-3tJ-HSDH activity was ex­pressed as nmols of 6 4-P formed per gram of wet tissue in 20 min. The results were not correctM for the experimental losses. Recovery of 6 5-P and 6 4-P through the whole procedure was 71.0% and 94.0% re­spectively. The rate of the conversion of 6 5-P to 6 4-P was linear for the first ten minutes; the maximum was reached after fifteen minutes. The Michaelis constant of the reaction was found to be 5x1 0-5 M.

RESULTS 6 5-3tJ-HSDH·activity in normal placentas

6 5-3tJ-HSDH activity was measured according to technique described under "Materials and Methods" in a group of 1 0 normal placentas. The mean value result­ed 13.18 nmols of 6 4 -P/g wt/20 min (range: 10.36-15.49).

"In vitro" effect of DHA and allylestrenol on placental 6 5-3tJ-HSDH As it may be seen in Table 1, DHA addedtothe incuba­tion medium at concentrations varying between 5x1 era M and 5x1 0 -5 M, inhibits the placental 6 5-3tJ-HSDH

activity, when 6 5-P is used as substrate. The maximum percentage inhibiton (-81.7%) was found with a DHA concentration of 5x10-5M.

Table 2 shows the influence of allylestrenol added to the incubation medium in 3 series of experiments, car­ried out on three different placentas (A, S, and C), in concentrations varying between 5x1 0-5 M and 5x1 (}-9M.

The placental 6 5-3tJ-HSDH resulted more active re­spect to the control values at every concentration used and the maximum percentage increase (15.1-26.5%) was found in the presence of allylestrenol 5x10-7M.

DISCUSSION Our results on the "in vitro" enzymatic conversion of 6 5-P to 6 4-P by the human placental 800 x g superna­tant fraction and the inhibition effected by DHA on this activity are similar to those of other authors (2, 3, 4, 5, 9).ln fact the 6 5-3tJ-HSDH placental activity is under to a product inhibition mechanism exerted by a series of naturally occuring steroids, whose inhibitory power is strictly structure-depending, being maximum for the 6 4-3 oxo-products of the reaction (9) and relatively mi­nor for the 6 5-3tJ-hydroxy alternative substrates. As far as DHA inhibition is concerned, it is possible that, in our experimental conditions, this steroid competes with 6 5-P either for the enzymatic activity (65-313-HSDH), or for NAD cofactor, to be converted to androstendione.

The structural difference between allylestrenol and these compounds may explain the lack of inhibition of this steroid in the in vitro placental production of A4-P. The experimental data on the effect of allylestrenol on 6 5-3tJ-HSDH, let us infer for a non-inhibition of the in vi­tro placental conversion of 6 5-P to 6 4 -P. It is also pos­sible, from our data, that allylestrenol might exert a slight stimulation on this conversion.

These results are in agreement with the studies of other

Table 2 - "In vitro" effect of allylestrenol on the conversion of l:J..5_p to l:J..4_p in three series of experiments, carried out on three different human placentas (A, B, C).

nmols of l:J..4_p formed per 9 wt in 20 min

control + allylestrenol

5x10-5M 5x1O-'M 5x10-8 M 5x10-9 M

A 11.72 12.95 13.49 13.14 13.04 (+10.4%) (+15.1 %) (+12.1%) ( +11.3%)

B 11.89 12.94 15.05 12.98 ( +8.8%) ( +26.5%) (+9.1 %)

C 12.02 13.32 14.43 13.57 (+10.8%) ( +20.0%) ( +12.8%)

84

authors (6-8), who reported the ability of allylestren-01 to stimulate the placental steroid metabolism. The data concerning allylestrenol, considering the in­hibitory effect exerted by several natural and synthetic steroids on the A5-3J3-HSDH activity, seem particulary interesting as far as this compound is used as a pro­gestative drug during pregnancy.

REFERENCES 1. Diczfalusy E.

Steroid metabolism in the foeto-placental unit. Excerpta Med. Int. Congr. Ser. 183: 65, 1968.

2. Goldman AS., Sheth K. Inhibitors of human placental C 19 and C 21 3 l3-hydro­xysteroid dehydrogenase. Biochem. Biophys. Acta 315: 233, 1973.

3. Ferre F., Breuiller M., Cedard L., Duchesne M.J. Saintot M., Descomps B., Crastes de Paulet A Human placental b.5-313-hydroxysteroid dehydrogenase activity. Steroids 26:551, 1975.

4. Townsley J.D. Inhibition of placental 313-hydroxy-steroid dehydro­genase by naturally occurring steroids.

85

DHA, allylestrenol and A5-3J3-HSDH

Acta Endocrinol. (Kbh.) 79: 740, 1975.

5. Depp R., Cox D.w., Pion R.J., Conrad S.H., Heinrichs W.L. Inhibition of the pregnenolone b.5-313- hydroxysteroid dehydrogenase-b.5 ,. isomerase systems of human pla­centa and corpus luteum of pregnancy. Gynecol. ·Invest. 4: 106, 1973.

6. Szontagh F.E., Sas M., Traub A., Kovacs L., Bardoczy A, Szereday Z. The influence of different norsteroids on the hormone excretion and on the hystomorphologic pattern in the trofoblast in early pregnancy. Gynecologia 156: 369, 1963.

7. Szontagh F.E.. Sas M. Recherches sur I'influence de I'allyloestrenol sur la secretion placentaire des steroides. Grenoble Medico-chirurgical 10: 5, 1965.

8. Barlas P., Adelung K., Rohner R., Weimer A., Keller E., Schindler E. The effect of drugs on the steroid metabolism of the normal placenta.

Acta Endocrinol. (Kbh.) (Suppl.) 184: 35, 1974.

9. Wiener M., Allen S.H.G. Inhibition of placental steroid synthesis by steroid metabolites. Steroids 12: 261, 1968.