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Department of Cell Biology, The Vontz Center for Molecular Studies, University of Cincinnati, Cincinnati, Ohio 45267
Address all correspondence and requests for reprints to: Dr. Nira Ben-Jonathan, Department of Cell Biology, University of Cincinnati Medical School, 3125 Eden Avenue, Cincinnati, Ohio 45267. E-mail: nira.ben-jonathan{at}uc.edu
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Abstract |
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and
ERß were determined by RT-PCR. BPA and OP induced delayed, but
progressive, increases in serum PRL levels, up to 3-fold above control
levels, in both males and females. The low dose of either compound was
equally or more effective as the high dose in eliciting and sustaining
elevated serum PRL levels, namely hyperprolactinemia. In contrast, the
DES treatment resulted in a transient rise in serum PRL levels. BPA,
OP, and, to a lesser extent, DES increased the expression of both ER
and ERß in the anterior pituitary of males, but not females, whereas
the hypothalamic ERs were less responsive to these compounds. DES
treatment caused down-regulation of ER expression in the uterus and
up-regulation of ERß in the prostate, whereas BPA or OP was without
effect. In conclusion, exposure of newborn rats of either sex to
environmental estrogens results in delayed and sustained
hyperprolactinemia and differential alterations in ER expression in the
hypothalamus and pituitary. DES appears to target the lower
reproductive tract more effectively than the neuroendocrine system. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). BPA is composed
of two unsaturated phenolic rings that resemble diethylstilbestrol
(DES). BPA is a monomer of polycarbonate plastics and a constituent of
epoxy and polystyrene resins used in the food packaging industry and
dentistry (1, 2). The polymer bonds can hydrolyze at high
temperature and release BPA. Detectable amounts of BPA were found in
food cans (3), microwaved pizzas (4), and
human saliva after treatment with dental sealants (5). OP,
comprised of a single phenolic ring (Fig. 1), is a constituent of
alkylphenol polyethoxylates that are used as surfactants in detergents,
paints, and herbicides. Alkylphenols are degraded in treatment plants
to form stable products, OP and nonylphenol, which are hydrophobic and
can accumulate in sewage sludge (6). Within internal
organs of fish and birds, these compounds can reach a 10–100 times
higher concentration than in the environment and thus can pass through
the food chain to humans.
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Whereas gonadal steroids act as reversible regulatory agents in the adult, they function as organizational agents during fetal development. Inappropriate exposure of the developing fetus to exogenous estrogens can cause long-term deleterious effects. A case in point is the consequence of treating millions of pregnant women with DES in the 1950–1960s to prevent miscarriage. In utero exposure to DES resulted in lower fertility, reproductive tract anomalies, and increased incidence of vaginal adenocarcinoma in women and urogenital tract deformities, cryptorchidism, and reduced fertility in men (14). Subsequent work with laboratory animals, primarily rodents, established that prenatal or early postnatal exposure to DES induces numerous structural and functional abnormalities in estrogen-responsive tissues (15). Rodents are especially well suited for studying the developmental effects of xenoestrogens because they can be treated during the first few days of life, when their maturity level resembles that of a second trimester human embryo. Recent reports reveal that prenatal exposure of mice to BPA advanced the onset of puberty in females (16) and caused prostate enlargement in males (17), whereas exposure of newborn rats to OP caused disruption of estrous cyclicity (18).
The pituitary lactotroph is an established target of estrogen.
Estrogens can affect PRL release by acting directly on the lactotrophs
or indirectly on the hypothalamic dopaminergic system as well as on a
variety of PRL secretagogues of hypothalamic or pituitary origin
(19). As the neuroendocrine system that regulates PRL is
immature at birth, it was of interest to determine whether neonatal
exposure to xenoestrogens induces prolonged alterations in PRL release.
Another question was whether xenoestrogens affect the expression of ERs
in the neuroendocrine axis. Indeed, OP and BPA bind to ER and ERß
(7), both of which are expressed in the rat anterior
pituitary (AP) (20) and medial basal hypothalamus (MBH)
(21). In addition, two truncated estrogen receptor
products, named TERP1 and TERP2, are unique to the pituitary
(22) and are up-regulated by estrogens (20, 23).
Our specific objective was to compare the effects of treating newborn male and female rats with BPA, OP, and DES on plasma PRL levels and ER expression in the AP and MBH before puberty. For comparison, ER expression in the uterus and prostate was also determined. F344 rats were exclusively used because of their recognized sensitivity to xenoestrogens (10, 24).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Experimental paradigm
A flow diagram of the experimental protocol is shown in Fig. 2. All treatments started on postnatal
day 1. On days 1–5 of life, each pup received a sc injection of either
50 µl tocopherol-stripped corn oil (control) or solutions of BPA
(Aldrich, Milwaukee, WI), OP (Aldrich), or DES (Sigma, St.
Louis, MO) in corn oil. Two doses of BPA and OP (100 and 500 µg/day)
and a single dose of DES (5 µg/day) were used. Each treatment group
included 8–10 male or female pups.
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Determination of serum PRL by RIA
A modified PRL RIA was employed (25), using the
NIDDK kit with RP-3 rat PRL as a reference preparation. Briefly, serum
aliquots (10–20 µl), diluted in 100 µl PBS containing 0.1% BSA,
were incubated with 50 µl antirat PRL antibody (1:40,000) and 50 µl
iodinated rat PRL in 96-well plates. After a 2-day incubation at 4 C,
the PRL-antibody complexes were precipitated by the addition of 50 µl
protein A, followed by centrifugation at 3,000 x g for
10 min. The supernatants were aspirated, and the pellets were dissolved
in 20 µl 0.1 N NaOH. After adding 200 µl
scintillation fluid (Microscint 20, Packard Instruments, Downers Grove,
IL), radioactivity was determined in a Packard TopCount. The limit of
sensitivity of the assay was 100 pg/well.
Analysis of ER expression by RT-PCR
Total RNA was isolated from each tissue using Tri-Reagent
(Life Technologies, Inc., Gaithersburg, MD), and 5 µg
were reversed transcribed using Superscript II reverse transcriptase
and random hexamers (Life Technologies, Inc.) as
previously described (20). Optimal PCR conditions for
quantitation were established for each set of primers by varying the
cycle number, annealing temperature, and RNA concentrations. Final
conditions for all estrogen receptors were as follows: 1) ER, 200 ng
RNA amplified at 58 C for 26 cycles; 2) ERß, 200 ng RNA amplified at
60 C for 35 cycles; and 3) TERP, 500 ng amplified at 55 C for 28
cycles. Each PCR reaction also contained primers for ribosomal protein
L19 (L19), which served as an internal control.
Primer sequences and expected product sizes were as follows: ER:
sense primer, 5'-GCTCCA ATTCTGACAATCGAC-3'; antisense primer,
5'-TTTCGTATCCCGCCTTTCATC-3' (expected size of 308 bp); ERß: sense
primer, 5'-AACCTCAA AAGAGTCCTTGGTGTG-3'; antisense primer,
5'-AACACTTGCGAAGTCGGCAG-3' (expected size of 327 bp); TERP: sense
primer, 5'-GCTTGTTGAACAGCGACCAG-3'; antisense primer, 5'-CTTGT
CCAGGACTCGGTGG-3' (expected size of 366 bp for TERP 1 and 432 bp for
TERP 2); L19 for ER: sense primer, 5'-AGTATGCTTAGGCTACAGAAG-3';
antisense primer, 5'-TTCCTTGGTCTTAGACCTGCG-3' (expected product size of
500 bp); and L19 for ERß and TERP: sense primer,
5'-CGAAATCGCCAATGCCAACTC-3'; antisense primer, 5'-TGCTCC
ATGAGAATCCGCTTG-3' (expected product size of 333 bp).
The PCR products were separated on a 1.5% agarose gel stained with
ethidium bromide and analyzed by scanning densitometry (Chemi Doc,
Bio-Rad Laboratories, Inc., Hercules, CA). A
representative example of PCR optimization for ER, using anterior
pituitary RNA from female pups, is shown in Fig. 3. Note the linear increase in the ODs of
both ER and L19 between 60 and 500 ng RNA at 26 cycles. A similar
linearity was established for each ER.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, serum PRL levels
in the controls increased from 10 ng/ml on day 15 to 22 ng/ml on day 20
and remained unchanged to day 30, whereas all females treated with BPA,
OP, or DES became hyperprolactinemic. Serum PRL levels progressively
increased from days 20 to 30 in response to treatment with BPA or OP.
The maximal rise in serum PRL levels (3-fold above controls;
P < 0.05) was observed in the OP-treated group.
Notably, the lower dose of OP (100 µg/pup·day) was as effective as
the higher dose (500 µg/pup·day) in stimulating and sustaining
elevated serum PRL levels. A similar response was observed in the
BPA-treated group (Fig. 4). On the other hand, circulating PRL levels
in the DES-treated group (5 µg/pup·day) were already elevated on
day 15 (P < 0.05) and peaked 3-fold above controls on
day 25 (P < 0.05), but declined to control levels on
day 30.
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). As in females, hyperprolactinemia was
observed in all males treated with BPA, OP, or DES. Peak serum PRL
levels (2- to 3-fold above control values) were reached on day 25 in
the groups treated with the lower dose of BPA and OP and remained
elevated on day 30. The higher doses of these compounds caused similar
or lower increases in serum PRL. Similar to females, newborn males
treated with DES had elevated serum PRL levels that peaked on day 25
(P < 0.05) and declined to control levels on day
30.
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expression in females (Fig. 6)
without a significant change in pituitary ER. DES, but not BPA
or OP, caused a 30% reduction (P < 0.05) in uterine
ER expression. In males, hypothalamic ER expression decreased in
response to DES only (P < 0.05), whereas that in the
AP increased in all treatment groups (Fig. 7). Maximal stimulation (2.5-fold above
controls; P < 0.05) was seen in the AP of OP-treated
males. Unlike its lack of effects in females, DES significantly
(P < 0.05) increased ER expression in the AP of
males.
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), but did not affect the
hypothalamus (data not shown). Among the three treatment groups, DES
was the least effective on pituitary ERß expression. Treatment with
DES resulted in more than a 2-fold (P < 0.05) increase
in ERß expression in the prostate (Fig. 8), whereas BPA and OP were
ineffective.
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, TERP messenger RNA was undetectable in
the AP of all samples tested, whereas under identical conditions both
isoforms of TERP were detected in pituitary tissue from adult,
estrogen-treated ovariectomized F344 rats.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ERß in
the male, but not the female, pituitary with no effect on the
hypothalamus. DES significantly decreased ER expression in the
uterus and increased ERß in the prostate, but, in general, had lesser
effects on receptor expression in the hypothalamo-pituitary axis.
There is substantial evidence that prenatal and neonatal exposure of
male and female rodents to DES, a very potent synthetic estrogen,
causes multiple disturbances in the reproductive tract (15, 26). Although increased PRL release is a well characterized
effect of estrogens, serum PRL levels in adult mice, neonatally treated
with DES, were unchanged (27, 28). These reports agree
with our observation that DES, at a dose similar to that used by others
(27, 28, 29, 30), causes only a transient rise in serum PRL levels
in prepubertal male and female rats. Despite having a 10,000-fold
higher binding affinity to the ER than the xenoestrogens, DES had only
modest effects on receptor expression in the neuroendocrine system.
On the other hand, it down-regulated ER expression in the
uterus, consistent with a previous report (31), and up-
regulated ERß expression in the prostate; neither BPA nor OP
affected ER expression in these tissues. This together with its
inability to sustain hyperprolactinemia suggest that DES targets the
lower reproductive tract more effectively than the neuroendocrine axis,
although the effect of yet a lower dose of DES has not been determined.
Nonetheless, it should be noted that hyperprolactinemia has not been
observed in either humans or experimental animals exposed to various
doses of DES in utero.
The issues of doses and binding affinities to the ERs are at the heart of the controversy regarding xenoestrogens. In the absence of information on the pharmacokinetics of these compounds, it is difficult to estimate the doses that simulate environmental exposure levels. A number of factors can enhance the in vivo effectiveness of xenoestrogens. These include prolonged storage in body fat (32), conversion to active metabolites (33), resistance to degradation, or low binding to serum proteins (17). There is an urgent need for developing methods for assessing the bioavailability of these compounds in both humans and experimental animals. Based on the knowledge that xenoestrogens have low binding affinity to the ER in vitro, many investigators have treated experimental animals with exceedingly high doses (34). However, high and low doses of estrogens often result in opposite effects (35, 36). Our study reveals that the low dose of either BPA or OP was equally or more effective as the high dose. Perhaps an even lower dose can elicit significant estrogenic responses.
The maturation of the PRL regulatory apparatus in rats is delayed until after birth (37, 38, 39). During the first few weeks of life, the number of lactotrophs (40), PRL gene expression (41), and circulating PRL levels (42, 43) progressively increase. Sexual dimorphism, e.g. higher PRL production and increased lactotroph responsiveness to secretagogues in females than in males, is established during puberty. To examine xenoestrogen-induced changes in serum PRL levels with a minimal exposure to endogenous steroids, the experiment was terminated on prepubertal day 30. Therefore, we do not know whether the xenoestrogen-induced hyperprolactinemia persists beyond this age and whether it affects the onset of puberty or fertility in either sex. These issues should be addressed in future experiments.
The mechanisms underlying the delayed and sustained hyperprolactinemia in prepubertal rats may be complex. It is unlikely that xenoestrogens remain in the circulation for 4 weeks and continuously stimulate the lactotrophs. The levels of endogenous estrogens, especially in prepubertal males, are probably too low to elicit such a response unless some target cells become hypersensitive to their action. We speculate that the observed hyperprolactinemia is caused by xenoestrogen-induced reprogramming effects in one or more components of the PRL regulatory system. These include decreased dopamine biosynthesis, decreased density of dopamine D2 receptors on the lactotrophs, increased number of lactotrophs, and/or increased production of certain PRL-regulating factors from the hypothalamus or pituitary. Studies focusing on possible xenoestrogen-induced alterations in these components are underway.
Our second objective was to determine whether neonatal exposure to
xenoestrogens elicits long-term changes in ER expression. Estrogens are
known to down-regulate their receptors in the uterus and up-regulate
those in the pituitary (22). Given the minute size of the
AP and MBH, the low level of ER expression in these tissues, and the
large number of samples, our approach for examining ER expression was
limited to RT-PCR. The data reveal gender differences in both the site
and the magnitude of the response to xenoestrogens. In females, BPA and
OP caused small increases in ER expression in the MBH, but not the
AP, and did not affect ERß expression in any tissue examined. In
males, these compounds increased both ER and ERß expression
in the pituitary without affecting the hypothalamus. Whereas these
results indicate a higher responsiveness of the male pituitary to
xenoestrogens, neither the identity of the target cells nor the
mechanisms underlying gender differences are well understood.
Our hypothesis is that premature exposure to xenoestrogens alters the lactotrophs themselves, their regulatory systems, or both. As is inherent to all endocrine systems, perturbations at one site trigger compensatory mechanisms aimed at minimizing the disturbance. Although this is true for the adult organism with a fully developed negative feedback system, this mechanism may be imperfect in the very young who are still in the processes of developing homeostasis. Additionally, because of the diversity of xenoestrogens and the existence of tissue-specific ER coactivators (44), each compound can exert different effects on the various estrogen-responsive cells. As is the case with the tissue-specific agonist/antagonist actions of tamoxifen (45), some xenoestrogens may even antagonize the action of endogenous estrogens at certain sites.
In summary, this is the first report of induction of hyperprolactinemia after early exposure of rats to environmental estrogens. These observations may have some implications to human hyperprolactinemia, especially if there is a human homolog of the sensitive F344 rat. Hyperprolactinemia is associated with infertility in women and impotence in men. As concluded from several prospective studies, there is no clear correlation between exposure to oral contraceptives and hyperprolactinemia (46, 47). Yet, it can be argued that the inclusion of progesterone in most oral contraceptives may have averted a distinct estrogenic effect. Certainly, the observed discrepancy between the effects of BPA or OP and DES on the neuroendocrine axis suggest dissimilarity of action of the various estrogenic compounds. At present, there is no information about whether exposure of pregnant women or infants to xenoestrogens increases serum PRL levels or affects fertility, nor is it known whether humans are sufficiently exposed to such compounds to warrant concern. Future research, using a variety of experimental models, should help resolve these issues.
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Acknowledgments |
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Footnotes |
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Received June 12, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ß. Endocrinology 138:863–870 and ß subtypes. Endocrinology 138:4022–4025 and
ß in the rat pituitary gland. Endocrinology 139:3976–3983 and -ß mRNA in the
rat central nervous system. J Comp Neurol 388:507–525[CrossRef][Medline]
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) BPA or OP or with 5
µg/day DES (
). Control rats (
) were injected with corn oil.
Blood was collected on the designated days and analyzed for PRL by RIA.
Each value is the mean ± SEM of 8–10 serum samples.
Note that the low dose of BPA or OP was as effective as the higher dose
in eliciting hyperprolactinemia.
