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Annual report : National Institute of Environmental Health Sciences (Volume 1985) online

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were influencing the appearance of these peaks.

Uterine DMA synthesis patterns in the adult ovariectomized mouse show a
temporal bimodal pattern with the major peak in activity at 16 h with a
second at 24 h. This is earlier than the activity seen in the rat uterus
where the first peak is at 24 h. Another approach was used to investigate
whether the second peak was associated with uterine DNA synthesis. Low
doses of estriol given in two dose combinations at different times showed
synthesis response comparable to a single h injection of estradiol. Our
newest results have also indicated that the second peak is not unique to
the mouse uterus since studies in ovariectomized rats show two nuclear
receptor peaks at and 13-14 h. These findings are consistent with the
later timing of DNA synthesis in the rat uterus and a requirement for a
period of secondary stimulation. In order to evaluate the tissue respon-
siveness to estrogens in more detail and determine whether the two peaks of
DNA synthesis were occurring in different uterine cell types, we investi-
gated the DNA synthesis and mitogenic activity in these uterine cells by
thymidine autoradiography. Adult ovariectomized animals show estrogen
response, DMA synthesis and mitosis in epithelial cells but not the stroma.
Sexually immature animals showed a different pattern where DMA synthesis
and mitosis were observed in both epithelium and stroma. By using dif-
ferent age animals, we could demonstrate that stromal' mi togenesis is lost
between day 28-35 of development and coincides with sexual maturity.
Animals which were ovariectomized on day 16 did not show the development of
stromal responsiveness but rather gave an adult pattern. These experiments
indicate that this tissue responsiveness may be influenced by the ovary.


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not ho
a n i ma 1
si ve g
and tr
1 eve! s
1 evel s
mul ati

d tissue res
d developmen
mised in tre
rmonally res
s was examin
roup have si
s suggest th
es. By 2-4
eated groups
are seen in
s were low a
1 levels wer
terization o
ties, sedime
Is. Endocri
in an exper
as the DES
on of uterin
animals is n

ponsiveness can be induced by estrogen exposu
tally with DES were used since the uteri are
ated mice. To understand why uteri in some a
ponsive, the concentration of estrogen recept
ed. Results have shown that animals in the n
gnificantly lower levels of estrogen receptor
at at 1 month of age there is no pattern of r
months, differences in receptor levels betwee
are noticeable; and by 6 months, significant
the DES group. Nuclear receptor assays in t
nd showed no appreciable differences, indicat
e not due to differential accumulation. Bioc
f the receptor in the DES group showed simila
ntation values, and nuclear translocation cap
ne manipulation using ovariectomy and adrenal
imental animal with similarly lower uterine r
animal. However, hormone responsiveness meas
e weight, progesterone receptor, and DNA synt
ot compromised as it is in DES-exposed animal

re. Mice
functi onal ly
nimals were
ors in those

eceptor di f-
n control
ly lower
hese same
ing that the
hemi cal
r bindi ng
acity as
ectomy can
ured as sti-
hesis in

Protein synthesis increases dramatically in the uterus after estrogen stim-
ulation, and identification of estrogen responsive proteins are being
sought. However, it is not known whether any of these proteins are unique
to one cell type. This is particularly interesting since only the epithe-
lial cell shows estrogen responsiveness with DNA synthesis and mitosis.
This problem was approached by protein labeling experiments using C 3 5 S]
methionine and two dimensional gel electrophoresis which have illustrated
several proteins (32,000 - 54,000 mw range) in uterine tissue from estroge-
nized animals. Non-enzymatic separation of the three uterine tissue com-
partments have indicated that some of these proteins are unique to one cell
type. Preliminary results of computer analysis of these gels show that the
epithelial and stromal tissue compartments exhibit significant differences
in protein patterns. Estrogen stimulates high molecular weight proteins in
epithelial cells, while in the stroma, low molecular weight components are
most prevalent. Proteins from the epithelial compartment also show signif-
icant isoelectric charge trails suggesting the possibility of modifications
in protein structure. Certain uterine proteins on the gels are being
identified by antibody localization. Recent studies have suggested the
possibility that steroid receptors may be phosphoryl ated proteins. Two
dimensional gel electrophoresis was used to analyze estrogen receptor which
was partially purified from mouse uteri by steroid affinity chromatography.
Preparations were identified by silver staining as well as blotting on to
nitrocellulose paper. Preparations were immunoblotted with 125 1 estrogen
receptor antibody. Fluorographs of these blots produced 2 spots at 70,000
mw at two different isoelectric points (pi 7.4 and 6.4). These two recep-
tor specific spots may represent the nuclear and cytosolic forms of the
receptor and possibly the more acidic component may be a phosphoryl ated
form of the estrogen receptor.


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When the estrogen receptor is activated it has an increased affinity for
DNA. This property is believed to explain its nuclear localization and
interactive nature with the genone. Earlier results with the DES compounds
have indicated weak biological activity even with significant nuclear
receptor levels. It is possible that the nuclear receptor complexes pro-
duced by these compounds are not properly activated for stimulation of
responses. Plans are to analyze receptor complexes bound to DES compounds
for their ability to bind to DNA cellulose. A competitive displacement
assay with unlabeled DES compound receptor complexes will be developed.
These compounds will also be synthesized in radioactive form in order to
directly evaluate the interaction. Such information should help determine
what chemical structure of the ligand is involved with receptor activation.
Estradiol receptor complexes bind preferentially to oligonucleotides with
oligo dT having the greatest affinity. It will be possible, particularly
with radioactive compounds, to measure receptor binding with the oligo-
nucleotides to see if differences may be observed which suggest that par-
ticular ligand receptor complexes show a binding selectivity. Another plan
is to test different DES isomer receptor complexes for their ability to
bind nuclear acceptor protein (NAP) extracted from target tissue chromatin.
It would be expected these two approaches should be correlative since they
both involve analyzing the ability of estrogen receptor to interact with
various genomic components, although it will be interesting to see if some
complexes bind DNA but not chromatin sites or vice versa. If DES compound
receptor complexes do not bind as well as the DES complex, then this could
be an explanation for their poor biological activity.

Due to their chemical structures, the indenestrol compounds possess a
single chiral carbon atom and, therefore, exist individually as a racemic
mixture of enantiomers. A chiral ly-active HPLC column matrix will be used
to separate the individual enantiomers of I A. Receptor binding of the
individual IA enantiomers will be tested to see if one enantiomer binds
preferentially over the other. Enantiomeric receptor differences may also
be reflected in the nuclear receptor levels in which the active enantiomer
shows a translocation of receptor the same as DES while the other enan-
tiomer has weak activity. These studies with the individual enantiomers
would demonstrate estrogen receptor chiral recognition for nonsteroidal
compounds and may help explain the poor uterine responsiveness and nuclear
receptor occupancy of the indanyl compounds. Indenestrol A is an in vivo
and in vitro DES metabolite; these metabolites will be isolated and analyzed
to determine which enantiomer is produced. IA can be produced as an in
vitro metabolite by incubation of DES with horseradish peroxidase. ThTs in
vitro metabolite will be isolated to determine if the same enantiomeric form
is produced under both experimental conditions. Evaluation of the enan-
tiomeric forms will indicate if the in vivo metabol ism of DES to IA produces
an enantiomeric form which is hormonal ly active or inactive. The other
indenestrol isomers, IB and ethyl indenestrol (EI), will be isolated and
purified. These compounds differ from IA by having a different chiral car-
bon atom (IB), and EI has a chiral ethyl group rather than a methyl, as in
IA. Receptor binding and nuclear receptor interactions will be measured


Z01 ES 70065-09 LRDT

with these enantiomers as with the IA enantiomers. IB is more active than
IA in stimulating hormonal responses and nuclear receptor occupancy; it will
be interesting to assess if the IB enantiomers show receptor binding and
nuclear occupancy differences.

Ultimately, we plan

dense atom derivati

of these enantiomer

in combination with

structi ng a structu

their differential

an estrogen respons'

directly in vitro w

This test system ca

a nti hormonal activi

IA enantiomer or E-

than receptor bi ndi

probes for understa

to isolate the enantiomeric
ves (bromoacetoxy ) so that th
s can be deduced by x-ray cry

the receptor binding analysi
ral basis for the receptor li
in vivo properties, the DES c
ive cell culture system, such
nether these response differe
n also be used as another mea
ties of these DES compounds.
\|> DES) appear to have much po
ng affinity and, therefore, w
nding steroid hormone mechani

forms of these compounds as
e absolute configurations
stal lography . These data
s should aid in con-
gand binding site. Due to
ompounds will be tested in

as MCF-7, to assess
nces can be demonstrated.
ns of testing any potential

Certain compounds (e.g.,
orer biological activity
ould be potentially good

In the reproductive tract, DES has been shown to stimulate a uterine
peroxidase enzyme activity. Interestingly, DES is a substrate for this
enzyme system producing a DES p-quinone intermediate which rearranges to a
Z ,Z-dienestrol product. This DES quinone is a highly reactive compound
which interacts irreversibly with DNA and protein. We plan to determine if
the receptor would interact with the DES quinone. Experimental conditions
will be established to support stability of the quinone in aqueous solu-
tion. Competitive binding assays and rate inhibition studies will be
utilized tc assess the interaction. Direct binding reactions of receptor
preparations with H-DES p-quinone will be used later to determine if irre-
versible binding is occurring. These studies would evaluate whether a
potential reactive intermediate of DES metabolism in target tissue (i.e.,
DES p-quinone) could interact with the estrogen receptor in an irreversible
manner. This type of binding could potentially produce a hormone receptor
complex in vivo which is an acute persistent stimulant of tissue responses
since the ligand could not dissociate from the receptor.

Studies will be con
Receptor isolated a
salt resistant bind
occurring at this 1
peak is related to
is postulated as th
transcription. As
binding during tiss
of the second peak.
DNA synthesis stimu
second peak should
attempt to characte
two different peaks
exposed and if thes
the second receptor

tinued to characterize the
t the times of both recepto
i ng to demonstrate tight ge
ater peak. Such resistant
genomic stimulation of tiss
e intranuclear site of DNA
a working hypothesis, we wi
ue stimulation and determin
If this second receptor e
lation, then it is expected
be associated with the matr
rize the chromatin by fract

to assess whether new or d
e findings may be an explan

peak .

second nuclear rec
r peaks wil 1 be an
nomic binding reac
sites are expected
ue responses. Nuc
synthesis and acti
11 analyze nuclear
e if it increases
vent is related to
that a major port
ix. Later studies
ionation technique
ifferent sites are
ation for the appe

eptor peak,
alyzed for

if the
lear matrix
ve gene

at the time

ion of the

woul d
s at the

arance of


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In the rodent, it is not certain during development when estrogen tissue
responsiveness occurs and whether the estrogen receptor is present and
involved in the mechanism of action. In order to evaluate whether DES
exposure and fetotoxicity of the reproductive tract is mediated through the
estrogen receptor, studies are also planned to determine if the estrogen
receptor is present in fetal reproductive tract tissue at the time (9-16
days) used for DES exposure. The levels of nuclear and cytosolic forms of
the estrogen receptor in fetal mouse reproductive tracts will be assayed.
These tissues will be analyzed by a biochemical microreceptor binding assay
as well as immunohistochemical procedures using a monoclonal antibody to
the estrogen receptor provided by Abbott Laboratories.

Protein and genomic markers are being sought as an endpoint of estrogen
uterine responsiveness especially related to tissue growth. Proteins will
be labeled and analyzed by two dimensional gel electrophoresis at early and
late periods of hormone stimulation at times just prior to and during DNA
synthesis. The analysis will be applied to samples from estradiol and
estriol treated animals where patterns should differ since estriol stimula-
tion induces all uterine responses but the later ones related to hyperpla-
sia. Gels will be analyzed by computer scanning and matched for detection
of proteins in the estradiol group not present with estriol. Similar anal-
yses will be performed after uterine stimulation with the DES compounds
since their varied biological activity should show differential patterns.
Future plans would be to isolate any unique spots and produce antibodies
which could be used for assays of the material as well as probes for a
uterine cDNA library being developed in Dr. C. T. Teng's laboratory.

In conjunction with these protein gel studies, the uterine estrogen recep-
tor will be localized in tissue samples in 2-D gels by immunobl otting and
reaction with labeled estrogen receptor monoclonal antibody. Multiple
forms of the receptor, which have already been demonstrated, will be tested
by 32 P labeling to see if they represent a phosphoryl ated form.
Experimentally, this will require making enriched or partially purified
preparations of estrogen receptor by ammonium sulfate precipitation,
heparin-sepharose chromatography and/or DES-di thi opropyl sepharose affinity
column. Once these different receptor forms are determined, preparations
will be made from tissue fractions at different points of stimulation in
order to evaluate the level of phosphoryl ated and unphosphoryl ated forms.
Such findings will allow the correlation of phosphoryl ated receptor forms
to tissue stimulation. Mechanistically, EGF and its receptor specifically
phosphoryl ate tyrosine residues of intracellular proteins. Attempts will
be made to correlate uterine EGF actions, estrogen activity, and their spe-
cificity by determining whether EGF is involved in phosphoryl ating the
estrogen receptor.


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Quarmby, V.E. Fox-Davies, C, and Korach, K.S.: Estrogen action in the
mouse uterus: the influence to the neuroendocrine/adrenal axis.
Endocrinology 114: 108-115, 1984.

Quarmby, V.E., and Korach, K.S.: The influence of 17 3-estradiol on pat-
terns of cell division in the uterus. Endocrinology 114: 694-702, 1984.

Luster, M.I., Boorman, 6. A., Hayes, H.T., Dean, J.H., Hong, L., Pfeifer, R.,
Korach, K.S. and Rhodes, L.: Environmental Estrogens and Their Effects on
Immune Responses. In Mullen P.W. (Ed.): Mato Advance Study Institute
Series . New York, Plenum Press, 1984, pp. 37-46.

Duax, W.L., Swenson, D.C., Stroug, P.D., Korach, K.S., McLachlan, J. A., and
Metzler, M.: Molecular structures of metabolites and analogues of
diethyl stilbestrol and their relationship to receptor binding and biologi-
cal activity. Mol. Pharmacol . 26: 520-525, 1984.

McLachlan, J. A., Korach, K.S., Newbold, R.R., and Degen, G.H.:

Di ethyl stil bestrol and other estrogens in the environment. Fund. Appl .

Tox. 4: 686-691, 1984.

Quarmby, V.E., and Korach, K.S.: Differential regulation of protein
synthesis by estradiol in uterine component tissues. Endocrinology 115:
687-697, 1984.

Luster, M.I., Boorman, G.A., Korach, K.S., Dieter, M.P., and Hong, L.:
Myelotoxicity resulting from exogenous estrogens: evidence for biomodal
mechanism of action. Int. J. Immune Pharmacol . 6: 287-297, 1984.

Luster, M.I., Hayes, H.T., Korach, K.S., Tucker, A.W., Dean, J.H.,
Greenlee, W.F., and Boorman, G.A.: Estrogen immunosuppression is regulated
through estrogen responses in the thymus. J. Immunol . 133: 110-116, 1984.

Thomas, J. A., Korach, K.S., and McLachlan, J. A. (Eds.): Toxicology of the
Endocrine System , Raven Press, N.Y., 1985.

Korach, K.S., and Quarmby, V.E.: Morphological, Physiological and and
Biochemical Aspects of Female Reproduction. In Dixon, R.L. (Ed.): Target
Organ Toxicity: Gonads (Reproductive and Genetic Toxicity) . New York,
Raven Press (In Press).

Korach, K.S., and McLachlan, J. A.: The role of the estrogen receptor in
diethyl stilbestrol toxicity. Proceedings of the International Congress of
Toxicology. Archives of Toxicology (In Press).

Duax, W.L., Griffin, J.F., Weeks, CM., and Korach, K.S.: Molecular
Conformation, Receptor Binding and Hormone Action of Natural and Synthetic
Estrogens and Antiestrogens. In McKinney, J.D. (Ed.): Environmental Health
Perspectives - Structure Activity Correlations in Mechanism Studies in
Predictive Toxicology (In Press).


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Parker, C.E., Levy, L.A., Smith, R.W., Yamaguchi, K. Gaskell, S.J., and
Korach K.S.: Separation and detection of enantiomers of stilbestrol analo-
gues by combined liquid chromatography /therrnospray mass pectrometry. J.
Chromatog . (In Press).

Korach, K.S., Levy, L.A., and Sarver, P. J.: Stereochemical Analysis of
Stilbene Estrogens: Receptor Binding and Hormone Responsiveness. In
McLachlan J. A. (Ed.): Estrogens in the Environment II. Influences on
Development . New York, Elsevier/North Holland (In Press).

Korach, K.S., Fox-Davies, C, Quarmby, V.E., and Swaisgood, M.H.:
Diethy lsti lbestrol metabolites and analogs: biochemical probes for induc-
tions of differential uterine estrogen responses. J. Biol. Chem . (In
Press ).

Isenhower, W.D., Jr., Newbold, R.R., Cefalo, R.C., Korach, K.S., and
McLachlan, J. A.: Absence of estrogenic activity in some drugs commonly
used during pregnancy. Biol. Res. Preg. Perinatal (In Press).




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October 1, 1984 to September 30, 1985

TITLE OF PROJECT (80 characters or less Title must fit on one line between the borders.)

Molecular Mechanism of Steroid Hormone in Sex Organ Development

PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator ) (Name, title, laboratory, and Institute affiliation)

PI : Christina T. Teng





Visiting Associate




Head, Devel . Endo.




Research Chemist

and Pharm. Section




I ahnratnr y nf Rpprndnrt.i vp and DpvpI opmental Toxicolo gy


Dpv pI Fndocri no! og y and P harmacolo



NTFH^ NTH, R P <;parrh THa.iglp Park, North Carolina 27709








□ (a) Human subjects □ (b) Human tissues [^ (c) Neither

□ (a1) Minors

□ (a2) Interviews

SUMMARY OF WORK (Use standard unreduced type Do not exceed the space provided )

The mouse uterus has provided a system for study of estrogen action since it
contains estrogen receptors and depends on estrogen stimulation for maintenance
of physiological functions. We have previously identified an estrogen-
stimulated mouse uterine secretory protein (Mr - 70 x 10 3 ) by in vrtro
i5 S-methionine labeling experiments. Currently, the HPLC puriTTed /OK protein
was further characterized. Total amino acid analysis did not show an unusual
composition. The 70K protein is a glycoprotein with an apparent asparagine-
1 inked carbohydrate moiety. Individual sugar analysis revealed a single car-
bohydrate chain which contains sialic acid, galatose, mannose, fucose, and
glucosamine. The NH 2 terminus of the 70K protein was cleaved with cyanogen
bromide, and the resulting fragments were separated by HPLC. Two of the
fragments yielded amino acid sequence. We have obtained the 32 mer synthetic
ol igonucleotides according to the amino acid sequence which will be used as the
probe to select bacteria clones containing the cDNA insert coding for the 70K
protein mRNA. Rabbit polyclonal antibody raised against the purified 70K pro-
tein demonstrated specificity for the 70K protein by " Western Blot " analysis.
The uterine 70K protein was induced by estrogen but not by testosterone or pro-
gesterone. Slot blot analysis and the immuno-enzyme-1 inked method was used to
examine the tissue distribution of the 70K protein. Tissues such as lung,
brain, spleen, ovary, kidney, liver, muscle and intestine of estrogen-treated
mice did not have measurable amounts of 70K protein. Only uterine and vaginal
tissue gave positive reactions.


PHS 6040 (Rev 1/84)

GPO 91 4-9 I

Z01 ES 70067-02 LRDT


Nature of Problem

One of the most interesting and yet poorly understood problems in develop-
mental biology is the expression of sets of hormone-regulated genes in
tissue specific ways. For example, the vitellogenin genes are transcrip-
tionally regulated by estrogen in chicken liver cells and, yet, are totally
unresponsive to the same stimulation in the oviduct despite the presence of
functional estrogen receptors which mediated the expression of the oval-
bumin gene. Although the gene sequences are the same in all tissue, dif-
ferential expression of the gene seems associated with the modification of
gene structure. Transcripti onal ly-acti ve genes are largely confined to
chromatin domains in which the ONA structure is in a relatively open con-
figuration. Modulation of chromatin structure of a specific gene to this
open configuration has been observed during the process of development
stimulation by hormones, and integration of virus. The DNA methylation
pattern of an active gene is altered from that of the inactive gene and
sequential changes in DNA methylation pattern have been observed during
development. Whether a hormone-regulated gene(s) acquires the capability
to respond to hormone through such modification of gene structure during
development needs to be investigated. An androgen and an estrogen-
regulated gene in the genital tract of the male and female mouse, respec-
tively, will be used as a model system for such studies. The morphological
and physiological changes of the mouse genital tract during normal and
abnormal development have been wel 1 -studied. In addition, the availability
of many inbred mouse strains and the wide range of their susceptibility to
steroid-hormone induced abnormal development and cancer has been reported.
With this model system, we are able to gain more insight into the problem
of gene expression and development as well as how prenatal toxicity will
affect future gene expression. Some specific questions can be asked, such
as: What gene sequences are required for hormone regulated expression?
What are the events associated with differentiation-dependent changes of
the chromatin structure? How does prenatal toxicity effect the ability of
the genes to synthesize its product? Is there any DNA rearrangement, dele-
tion or amplification which occurs in and around genes due to prenatal DES
or exposure to other carcinogens.


The research goal of this laboratory is to understand the expression of a

Online LibraryNational Institute of Environmental Health ScienceAnnual report : National Institute of Environmental Health Sciences (Volume 1985) → online text (page 52 of 114)