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The role of catecholestrogens in placental steroidogenesis
3354
427
THE ROLE OF CATECHOLESTROGENS IN PLACENTAL STEROIDOGENESIS Eytan R. Barnea and Hasan N. Fakih* Yale University, School of Medicine, Department of Obstetrics and Gynecology New Haven, Connecticut
Received 7-22-85 ABSTRACT effect of The the catecholestrogen, 2-hydroxyestrone (2-OHEl) ,on placental steroidogenesis was studied by incubating 2-OHEl with placental explants for 24 hours and measuring the output of estradiol (E2) and progesterone (P4). 2-OHEl stimulated the accumulation of E2 and P4 in the media. This effect was inhibited by the a-adrenergic blocker, phenoxybenxamine, and the 8-adrenergic blocker, propranolol. We conclude that 2-OHEl affects placental steroidogenesis and that this effect could possibly be mediated through adrenergic receptors.
INTRODUCTION Barnea and Naftolin (1) have shown that the placenta contains a high activity of estrogen hydroxylase, the enzyme involved in the formation of catecholestrogens from estrogens.
This might
explain the 2-20 fold elevation of catecholestrogen blood levels observed in pregnancy (6,lO). The effect of catecholestrogens on human placental steroidogenesis has not been studied before. this study, we estrone, a
attempt to identify the effects of
catecholestrogen, on
In
2-hydroxy-
placental steroidogenesis, by
measuring the output of estradiol (E2) and progesterone (P4) in placental explants incubated with the catecholestrogen.
EXPERIMENTAL Placental tissues were obtained from six healthy subjects following elective cesarean section at term, without labor. Small placental fragments were dissected out in a sterile fashion and placed in a large excess of 0.9% NaCl solution to remove all blood. Subsequently, tiSSUeS were rinsed again in Dulbecco's Modified Eagle Medium (MEM) containing 0.5% antibacterial solution (5000 units Penicillin and 5000 mcg Streptomycin) and 0.5% bovine Volume 45, Number 5
May 1985
S
TEEOXDl
serum albumin. Incubations were carried out by placing approximately 300 mg placental tissue per dish in 2 mL MEM media with or without test compounds at 37'C in a gas mixture of 95% air and 5% CO for 24 hours. Following incubation, dishes were placed on ice an8 the media were stored at -2O'C until assayed. The placental tissue was also saved and was used for protein estimation. A progressive decrease in glucose concentration in the culture media during the first 24 hours of culture was used as an indicator for the viability of the explant culture system. Chemicals: Unlabeled 2-hydroxyestrone (2-OHEl) was obtained from Steraloids (Wilton, NH) and purified to homogeneity by highperformance liquid chromatography. The purified steroid was stored in a desiccator at 0-4Oc. 2-OHEl was dissolved in ethanol containing 0.1% ascorbic acid, and was diluted in the MEM media and immediately added to the cultures. Norepinephrine (NE), epinephrine (E), propranolol and phenoxybenzamine were of high analytical quality. MEM media were purchased from Dulbecco Co. (NY). Radioimmunoassay: The steroids, estradiol (E2) and progesterone (P4), in the media were measured using standard RIA techniques (9). The assay was validated by using internal reference standards. Inter- and intra-assay variability were, respectively, 10.8% and 9.7% for E2 and 11.3% and 10.2% for P4. The cross reactivity of 2-OHEl with the E2 assay was less than 1%. The protein concentration of placental tissue was measured according to the method of Lowry et al (11). Data are expressed as pg of EZ/mg placental protein, az Kj of Pl/mg placental protein. Statistical analysis: Statistical significance of differences between mean values were analyzed by one-way analysis of variance (ANOVA) and Student's t test.
RESULTS The results reported here are those of single experiments with five replicate dishes in each condition.
The trend observed
was the same in all the experiments performed. significantly stimulated culture media (Fig.1).
the
accumulation of
Dose response
2-Hydroxyestrone E2 and
studies showed
P4 in the no signif-
S
B
A
Figure 1:
429
TEEOIDI
Effect of 2I$ydroxyestqne (2-OHEl) in concentrations between 10 M and 10 M on E2(A) and P4(B) accumulation in the media. Mean 2 SD. *P < 0.001 *P < 0.001 (B) (A) **P < 0.001 **p < 0.009
icant difference in this stimulation in doses between 10B8M and 10s5M (Fig-l). To study whether this effect is mediated through adrenergic receptors, the adrenergic receptor blockers phenoxybenzamine, an 3
and ~1 2 adrenergic receptor blocker, or propranolol, a S
adrenergic
receptor
blocker, were
added to the
1
and B
2
culture media.
Both phenoxybenzamine and propranolol in concentrations of 10-5M significantly inhibited the effects observed for 2-h.ydroxyestrone alone (Fig.2).
10B5M Phenoxybenzamine and 10B5M propranolol,when
added alone, did not have any significant effect on E2 or P4 accumulation in the culture media (data not shown).
S
430
WPBILOXD1
B
Control
Fi.gure 2:
Z-OHE, 16%
2-OHE, + wnmy b lo-5 Y
lU6H + popon
Control
2-OHE, !dSY
100M
2-OHE, + ph.noxyb lc3Y
IO+, + p,opron 10-s M
Effect of lo-5M 2-hydroxyestrone (2-OIiEl)on E2 (A) and P4 B) release and the modulation of this eff ct -S -!i by 10 M phenoxybenzamine (phenoxyb.) and 10 M propranolol (propran.). Mean + SD. *P 5 0.001 *P = 0.02 (A) (B) **p = 0.004 **P = 0.025 ***p = 0.002 ***p = 0.04
DISCUSSION In
this
study we
2_hydroxyestrone, placental
in
have
shown that
physiologic
steroidogenesis.
the
concentrations
Incubating
2-OHEl
explants for 24h resulted in about two-fold concentrations of E2 and P4
catecholestrogen,
measured by RIA
can
with
affect
placental
increase in the
techniques (Fig.1).
The effect of catecholestrogens on placental steroidogenesis could be mediated through interaction with estrogen receptors or adrenergic
receptors,
or
through
an
unknown
mechanism.
S
WDEOIDI
Catecholestrogens are weak estrogens that can act as estrogen agonists or antagonists (1). The affinity of cytoplasmic estrogen receptors
for
2-OHEl
is
very
low
cytoplasmic estrogen receptors in
(2), and the
the
human
presence
placenta
is
of not
established (4). On the other hand, the human placenta is a rich source
of
adrenergic
B-adrenergic receptors
binding assays
(7).
receptors
were
also
(5,14), and
identified
recently
using
a-2
radioligand
Moreover, catecholestrogens were shown to
bind to adrenergic receptors in the brain
(13), and Inaba and
Kamate (8) and Paden -et al (12) have shown that catecholestrogens in
pharmacologic
doses
were
able
to
significantly
inhibit
in vitro. catecholamine receptor binding in brain tissue --
Caritis
et al (3) have reported that the B-sympathomimetics, isoproterenol -and terbutaline, could stimulate the accumulation of progesterone in human placental explant media. These facts, with the finding that the a-adrenergic blocker phenoxybenzamine and
the
B-adrenergic blocker propranolol, in
concentrations of 1Q05M, could inhibit the stimulatory effect of 2-hydroxyestrone
on
E2
and
P4
release
might
imply
that
2-hydroxyestrone exerts its effect on placental steroidogenesis through the adrenergic receptors. Whether catecholestrogens exert their effects on placental mechanism
is
steroidogenesis
still possible.
To
via
another unknown
further elucidate the exact
mechanism of action of catecholestrogens, experiments utilizing radio-labeled adrenergic receptor agonists or antagonists are in order.
432
m
TPD=OXDI
NOMENCLATURE 2-OHEl E2 P4 *
:
:
2-hydroxyestrone, 2, 3-dihydroxy-1,3,5 (lo)-estratrien - 17 - one estradiol, 1,3,5 176- diol (10) - estratriene -3, progesterone, 4 - pregnene - 3, 20-dione
To whom correspondence should be addressed. Address: Medical University of South Carolina Department of Obstetrics and Gynecology Division of Reproductive Endocrinology Charleston, South Carolina 29425-2233,
REFERENCES 1.
2. 3. 4. 5. 6. 7. 8. 9.
10. 11. 12. 13. 14.
Barnea, E.R.,and Naftolin, F., Proc. 65th Annual Meeting Endocrine Sot., San Antonio, TX, 635 (1983). Barnea, E.R., MacLusky, N.J., and Naftolin, F., Steroids 41, 643 (1983). Caritis, S.N., Zeleznik, A.J., Am. J. Obstet. Gynecol. 138, 677 (1980). Coulan, B.C., and Spelsberg, T.C., Trophoblast Research I, 249 (1984). 1583 (1983). Falkay, G., and Kovacs, L., Life Sci. 2, Fishman, J., and Dixon, D., Biochemistry 6, 1683 (1967). Olive, T.G., Dev. Gardey-Levassort, C., Ventura, M.A., Pharmacol. Therap. I, 85 (1984). Inaba, M., Kamate, K., J. Steroid Biochem. 11, 1491 (1979). Methods of Hormone Jaffe, B.M., and Behnnan, H.R., in : Radioimmunoassay (Jaffe, C.M., and Behrman, H.R., Editors), 2nd ed. New York: Academic (1979). Kono, S., Brandon, D., Merriam, G.R., Loriaux, D.L., and Lipsett, M.B., Steroids 2, 463 (1980). Lowry, O.H., Rosenbrough, N.J., Farr, A.L., and Randall, R.J., J. Biol. Chem. 193, 265 (1951). Paden, C.M., McEwen, B.S., Fishman, J., Snyder, L., DeGroff, F ., J. Neurochem. 2, 512 (1982). Schaeffer, J.M., and Hsueh, A.J., Am. J. Obstet. Gynecol. 138, 677 (1980). Shocken, D.D., Caron, M.G., Lefkowitz, R.J., J. Clin. Endocrinol. Metab. so, 1082 (1980).
427
THE ROLE OF CATECHOLESTROGENS IN PLACENTAL STEROIDOGENESIS Eytan R. Barnea and Hasan N. Fakih* Yale University, School of Medicine, Department of Obstetrics and Gynecology New Haven, Connecticut
Received 7-22-85 ABSTRACT effect of The the catecholestrogen, 2-hydroxyestrone (2-OHEl) ,on placental steroidogenesis was studied by incubating 2-OHEl with placental explants for 24 hours and measuring the output of estradiol (E2) and progesterone (P4). 2-OHEl stimulated the accumulation of E2 and P4 in the media. This effect was inhibited by the a-adrenergic blocker, phenoxybenxamine, and the 8-adrenergic blocker, propranolol. We conclude that 2-OHEl affects placental steroidogenesis and that this effect could possibly be mediated through adrenergic receptors.
INTRODUCTION Barnea and Naftolin (1) have shown that the placenta contains a high activity of estrogen hydroxylase, the enzyme involved in the formation of catecholestrogens from estrogens.
This might
explain the 2-20 fold elevation of catecholestrogen blood levels observed in pregnancy (6,lO). The effect of catecholestrogens on human placental steroidogenesis has not been studied before. this study, we estrone, a
attempt to identify the effects of
catecholestrogen, on
In
2-hydroxy-
placental steroidogenesis, by
measuring the output of estradiol (E2) and progesterone (P4) in placental explants incubated with the catecholestrogen.
EXPERIMENTAL Placental tissues were obtained from six healthy subjects following elective cesarean section at term, without labor. Small placental fragments were dissected out in a sterile fashion and placed in a large excess of 0.9% NaCl solution to remove all blood. Subsequently, tiSSUeS were rinsed again in Dulbecco's Modified Eagle Medium (MEM) containing 0.5% antibacterial solution (5000 units Penicillin and 5000 mcg Streptomycin) and 0.5% bovine Volume 45, Number 5
May 1985
S
TEEOXDl
serum albumin. Incubations were carried out by placing approximately 300 mg placental tissue per dish in 2 mL MEM media with or without test compounds at 37'C in a gas mixture of 95% air and 5% CO for 24 hours. Following incubation, dishes were placed on ice an8 the media were stored at -2O'C until assayed. The placental tissue was also saved and was used for protein estimation. A progressive decrease in glucose concentration in the culture media during the first 24 hours of culture was used as an indicator for the viability of the explant culture system. Chemicals: Unlabeled 2-hydroxyestrone (2-OHEl) was obtained from Steraloids (Wilton, NH) and purified to homogeneity by highperformance liquid chromatography. The purified steroid was stored in a desiccator at 0-4Oc. 2-OHEl was dissolved in ethanol containing 0.1% ascorbic acid, and was diluted in the MEM media and immediately added to the cultures. Norepinephrine (NE), epinephrine (E), propranolol and phenoxybenzamine were of high analytical quality. MEM media were purchased from Dulbecco Co. (NY). Radioimmunoassay: The steroids, estradiol (E2) and progesterone (P4), in the media were measured using standard RIA techniques (9). The assay was validated by using internal reference standards. Inter- and intra-assay variability were, respectively, 10.8% and 9.7% for E2 and 11.3% and 10.2% for P4. The cross reactivity of 2-OHEl with the E2 assay was less than 1%. The protein concentration of placental tissue was measured according to the method of Lowry et al (11). Data are expressed as pg of EZ/mg placental protein, az Kj of Pl/mg placental protein. Statistical analysis: Statistical significance of differences between mean values were analyzed by one-way analysis of variance (ANOVA) and Student's t test.
RESULTS The results reported here are those of single experiments with five replicate dishes in each condition.
The trend observed
was the same in all the experiments performed. significantly stimulated culture media (Fig.1).
the
accumulation of
Dose response
2-Hydroxyestrone E2 and
studies showed
P4 in the no signif-
S
B
A
Figure 1:
429
TEEOIDI
Effect of 2I$ydroxyestqne (2-OHEl) in concentrations between 10 M and 10 M on E2(A) and P4(B) accumulation in the media. Mean 2 SD. *P < 0.001 *P < 0.001 (B) (A) **P < 0.001 **p < 0.009
icant difference in this stimulation in doses between 10B8M and 10s5M (Fig-l). To study whether this effect is mediated through adrenergic receptors, the adrenergic receptor blockers phenoxybenzamine, an 3
and ~1 2 adrenergic receptor blocker, or propranolol, a S
adrenergic
receptor
blocker, were
added to the
1
and B
2
culture media.
Both phenoxybenzamine and propranolol in concentrations of 10-5M significantly inhibited the effects observed for 2-h.ydroxyestrone alone (Fig.2).
10B5M Phenoxybenzamine and 10B5M propranolol,when
added alone, did not have any significant effect on E2 or P4 accumulation in the culture media (data not shown).
S
430
WPBILOXD1
B
Control
Fi.gure 2:
Z-OHE, 16%
2-OHE, + wnmy b lo-5 Y
lU6H + popon
Control
2-OHE, !dSY
100M
2-OHE, + ph.noxyb lc3Y
IO+, + p,opron 10-s M
Effect of lo-5M 2-hydroxyestrone (2-OIiEl)on E2 (A) and P4 B) release and the modulation of this eff ct -S -!i by 10 M phenoxybenzamine (phenoxyb.) and 10 M propranolol (propran.). Mean + SD. *P 5 0.001 *P = 0.02 (A) (B) **p = 0.004 **P = 0.025 ***p = 0.002 ***p = 0.04
DISCUSSION In
this
study we
2_hydroxyestrone, placental
in
have
shown that
physiologic
steroidogenesis.
the
concentrations
Incubating
2-OHEl
explants for 24h resulted in about two-fold concentrations of E2 and P4
catecholestrogen,
measured by RIA
can
with
affect
placental
increase in the
techniques (Fig.1).
The effect of catecholestrogens on placental steroidogenesis could be mediated through interaction with estrogen receptors or adrenergic
receptors,
or
through
an
unknown
mechanism.
S
WDEOIDI
Catecholestrogens are weak estrogens that can act as estrogen agonists or antagonists (1). The affinity of cytoplasmic estrogen receptors
for
2-OHEl
is
very
low
cytoplasmic estrogen receptors in
(2), and the
the
human
presence
placenta
is
of not
established (4). On the other hand, the human placenta is a rich source
of
adrenergic
B-adrenergic receptors
binding assays
(7).
receptors
were
also
(5,14), and
identified
recently
using
a-2
radioligand
Moreover, catecholestrogens were shown to
bind to adrenergic receptors in the brain
(13), and Inaba and
Kamate (8) and Paden -et al (12) have shown that catecholestrogens in
pharmacologic
doses
were
able
to
significantly
inhibit
in vitro. catecholamine receptor binding in brain tissue --
Caritis
et al (3) have reported that the B-sympathomimetics, isoproterenol -and terbutaline, could stimulate the accumulation of progesterone in human placental explant media. These facts, with the finding that the a-adrenergic blocker phenoxybenzamine and
the
B-adrenergic blocker propranolol, in
concentrations of 1Q05M, could inhibit the stimulatory effect of 2-hydroxyestrone
on
E2
and
P4
release
might
imply
that
2-hydroxyestrone exerts its effect on placental steroidogenesis through the adrenergic receptors. Whether catecholestrogens exert their effects on placental mechanism
is
steroidogenesis
still possible.
To
via
another unknown
further elucidate the exact
mechanism of action of catecholestrogens, experiments utilizing radio-labeled adrenergic receptor agonists or antagonists are in order.
432
m
TPD=OXDI
NOMENCLATURE 2-OHEl E2 P4 *
:
:
2-hydroxyestrone, 2, 3-dihydroxy-1,3,5 (lo)-estratrien - 17 - one estradiol, 1,3,5 176- diol (10) - estratriene -3, progesterone, 4 - pregnene - 3, 20-dione
To whom correspondence should be addressed. Address: Medical University of South Carolina Department of Obstetrics and Gynecology Division of Reproductive Endocrinology Charleston, South Carolina 29425-2233,
REFERENCES 1.
2. 3. 4. 5. 6. 7. 8. 9.
10. 11. 12. 13. 14.
Barnea, E.R.,and Naftolin, F., Proc. 65th Annual Meeting Endocrine Sot., San Antonio, TX, 635 (1983). Barnea, E.R., MacLusky, N.J., and Naftolin, F., Steroids 41, 643 (1983). Caritis, S.N., Zeleznik, A.J., Am. J. Obstet. Gynecol. 138, 677 (1980). Coulan, B.C., and Spelsberg, T.C., Trophoblast Research I, 249 (1984). 1583 (1983). Falkay, G., and Kovacs, L., Life Sci. 2, Fishman, J., and Dixon, D., Biochemistry 6, 1683 (1967). Olive, T.G., Dev. Gardey-Levassort, C., Ventura, M.A., Pharmacol. Therap. I, 85 (1984). Inaba, M., Kamate, K., J. Steroid Biochem. 11, 1491 (1979). Methods of Hormone Jaffe, B.M., and Behnnan, H.R., in : Radioimmunoassay (Jaffe, C.M., and Behrman, H.R., Editors), 2nd ed. New York: Academic (1979). Kono, S., Brandon, D., Merriam, G.R., Loriaux, D.L., and Lipsett, M.B., Steroids 2, 463 (1980). Lowry, O.H., Rosenbrough, N.J., Farr, A.L., and Randall, R.J., J. Biol. Chem. 193, 265 (1951). Paden, C.M., McEwen, B.S., Fishman, J., Snyder, L., DeGroff, F ., J. Neurochem. 2, 512 (1982). Schaeffer, J.M., and Hsueh, A.J., Am. J. Obstet. Gynecol. 138, 677 (1980). Shocken, D.D., Caron, M.G., Lefkowitz, R.J., J. Clin. Endocrinol. Metab. so, 1082 (1980).