and disorders of sex differentiation in male newborn
Sultan (1-2), Françoise Paris (1-2), Claire Jeandel (1) and Béatrice
Unité d'Endocrinologie Pédiatrique, Service de Pédiatrie
I, Hôpital A. de Villeneuve, Montpellier, France
(2) Unité INSERM U-439, Pathologie Moléculaire des Récepteurs
et Service d'Hormonologie Hopital Lapeyronie, Montpellier, France
the past twenty years, the documented increase in the disorders of male sexual
differentiation, such as hypospadias, cryptorchidism, and micropenis, has led
to the suspicion that environmental chemicals are detrimental to normal male
genital development in utero. Male sexual differentiation is critically dependent
on the normal action of androgens, and unbalanced androgen/estrogen ratios can
Environmental xenoestrogens (such as herbicides, pesticides, PCBs, plasticizers,
and polystyrenes) that mimic estrogens or environmental antiandrogens (such
as polyaromatic hydrocarbons, linuron, vinclozolin and pp'DDE) that disturb
endocrine balance, cause demasculinizing effects in the male fetus. These environmental
chemicals are often referred to as endocrine disruptors: they are thought to
mimic endogenous estrogens by entering the cell, binding to the receptor and
activating transcription, they may also antagonize normal androgen action.
We have established numerous cell lines to assess the estrogenicity and antiandrogenicity
of compounds found in the environment and to identify new products present in
wastewater effluents that are able to disrupt endocrine functions. Several cell
lines responding to estrogens have been obtained in our group, including cells
with different enzymatic equipment and cells expressing chimeric receptor or
natural estrogen receptors a and b. These cell lines have proved to be useful
for assessing the biological activity of pesticides, fungicides and the chemicals
found in plastic or discarded in the environment.
In order to generate a powerful tool for the investigation of androgen action
and the rapid screening of potential antagonists, we developed a new stable
prostatic cell line. The PALM cell line is an original cellular model to characterize
the response of hAR, and it provides an easy and rapid bioluminescent test to
identify new antagonists. We also developed a model based on a fusion protein
between the androgen receptor (AR) and the green fluorescent protein (GFP) to
study the intracellular dynamics of AR. The GFP-AR model was applied to define
the ability of several xenoestrogens and antiandrogens to inhibit the nuclear
transfer of AR.
The ubiquitous presence of endocrine disruptors in the environment and the increased
incidence of neonatal genital malformation support the hypothesis that disturbed
male sexual differentiation may in some cases be caused by increased exposure
to environmental xenoestrogens and/or antiandrogens.
is great concern that the incidence of congenital disorders of male sexual differentiation
is increasing. Several reports indicate an increase in the prevalence rates
of cryptorchidism, hypospadias and micropenis . It has been hypothesized
that the adverse trends in male sexual differentiation are related to environmental
xenoestrogens and/or antiandrogens, which may disrupt normal sex differentiation
during fetal life. In this short review, we summarize the secular trends in
the incidence of disorders of male sex differentiation, the occurrence of genital
abnormalities in the sons of women exposed to diethylstilbestrol during pregnancy,
and the adverse effects of prenatal estrogen and antiandrogen treatment in experimental
animals and in human male fetus. We also report the main environmental chemicals
with known estrogenic and/or antiandrogenic effects. Special attention is given
to the testing strategies for evaluation of estrogenic-like or antiandrogenic
activity of potential environmental disruptors .
data taken from registries indicate an increase in the prevalence of neonatal
cryptorchidism and hypospadias. For example, in England, the prevalence rate
of cryptorchidism has doubled within the last 25 years (1952: 1.4%; 1977: 2.9%).
During this same period, the incidence of hypospadias has significantly increased
in Europe (England, Hungary, France), as well as in the United States. This
increasing trend in abnormalities of male sex differentiation raises the question
of whether they are caused by environmental endocrine disruptors during pregnancy
Effects of DES
effects of diethylstilbestrol (DES) provide an unfortunate model of how a potent
estrogenic chemical prescribed during gestation can alter fetal sex differentiation
in human. The occurrence of genital abnormalities in the sons of women exposed
to DES during pregnancy is noteworthy: 20.8% of the males exposed to DES in
utero had epididymal cysts (vs 4.9% in controls), 4.4% had hypospadias (vs 1.1%
in controls), 11.4% presented with cryptorchidism and hypoplastic testes (vs
2.1% in controls), and 1.5% had micropenis (vs 0% in controls) .
set of data emphasizes the sensitivity of fetal external genitalia to excess
synthetic estrogen exposure.
The adverse effects of DES have been extensively studied in experimental animals
. After DES exposure in pregnant mice, male offspring exhibited micropenis,
hypospadias, and cryptorchidism along with underdevelopment of the vas deferens,
epididymis and seminal vesicles. These defects are similar to those of DES-exposed
human male fetus .
Effects of antiandrogens
exposure of male fetus to antiandrogen treatment similarly results in an undervirilized
or female external phenotype.
It has been clearly demonstrated that these deleterious effects of antiandrogens
depend on the dose and the chemical structure of the substance-but mainly on
the timing of exposure: the first trimester of gestation is the most sensitive
period in terms of fetal sex differentiation.
Both xenoestrogenic and antiandrogenic substances can disrupt the synthesis,
transport and metabolism of androgen. Most environmental antiandrogenic agents
antagonize androgen action within the target cell by competing with the androgen
receptor (AR) and inducing a conformational change of the AR or by reducing
transcriptional activation of target genes at the crucial period. Whereas chemical
exposure may be transient, some of the effects are irreversible .
The mechanisms of action of endocrine disruptors within an androgen target cell
are presented in Fig. 1.
In conclusion, three sets of evidence: secular trends in the incidence of disorders
of male sexual differentiation, the occurrence of genital abnormalities in the
sons of women exposed to DES during pregnancy, and the adverse effects of prenatal
estrogen/antiandrogen treatment in experimental animals, have pushed several
authors to advance the hypothesis that fetal exposure to xenoestrogens and/or
antiandrogens may account for the reported chronological changes in the incidence
of disorders of male sexual differentiation.
Effects of xenoestrogens
on their interactions with the ER-binding sites, environmental xenoestrogens
are a diverse group of chemicals (Tab. 1).
For those which preferentially bind to the ERb receptor, one may speculate that
subsequent down-regulation of the AR is involved in the development of urogenital
malformation during fetal life (J-A. Gustafsson, personal communication). It
should be mentioned that several environmental estrogens are antiandrogens-Vinclozolin,
a fungicide, and 44'-DDE, for example-inhibit AR mediated gene activation .
The Eurogen program, supported by the European Community, has recently been
implemented: the overall study objective is to determine whether there is an
association between environmental factors in the prenatal period and the development
of disorders of male sex differentiation. Prospective epidemiological studies
of genital malformation will be conducted in birth cohorts from Europe (Denmark,
Finland, Spain, England, and France), Japan and the United States, as these
countries are expected to have significant differences in the prevalence of
urogenital malformations. In a simultaneous case-control study, diet, drug usage,
exposure to chemicals during pregnancy, and lifestyle parameters will be evaluated
as possible causal factors in neonatal genital malformations.
Recombinant receptor reporter gene assay
receptor-reporter gene bioassays to evaluate the estrogenic/antiestrogenic,
androgenic/antiandrogenic activities of environmental chemicals and to identify
new products present in food and water are also important.
Simple cell models that express a gene under the control of defined promoters
responding to specific drugs and that produce a signal easy to quantitate are
of great interest for rapid screening of the biological effects of artificial
or natural compounds. In integrated systems, these cell models are very useful
to study the synergy or antagonism of different substances and, in the field
of environmental research, they are excellent tools to identify compounds able
to disrupt endocrine functions.
We have established numerous cell lines using a technology based on the bioluminescent
gene reporter assay. Analysis and selection of stable transfectants were simplified
using a low-light imaging system. We have used these systems to evaluate the
biological activity of compounds found in the environment and to identify new
products present in wastewater effluents.
5.1. Stable bioluminescent cells responding to estrogens
cell lines responding to estrogens have been obtained, including cells with
different enzymatic equpment and cells expressing chimeric receptors or natural
estrogen receptor a or b (9, 10). These cell lines could not only be used by
pharmaceutical companies, but they would also be helpful for monitoring the
biological activity of pesticides and chemicals found in plastic or discarded
in the environment.
The detection limit of these bioassays is lower than 10-12M estradiol, and high-throughput
screening could be performed using a 96-well microplate format.
The estrogenic activity of detergents (nonylphenols), plasticizers (bisphenol
A, phtalates) and pesticides (DDE products) was characterized with our reporter
cell lines. Using HELN ERa cell line, derived from Hela cell line, we observed
a luciferase activity induced by these chemicals tested at concentrations above
10-8M (Fig. 2). Similar results were obtained on the ERb cell line (results
On the contrary, phytoestrogens which exhibited a biphasic activation were more
potent at low concentrations (10-100 nM) on the ERb than in the ERa cell line
(Fig. 3). At high concentrations (1-10 mM), the estrogenic potency was similar
on the ERa and the ERb cell lines but was greater than that induced by estradiol.
Cells expressing the androgen receptor and a bioluminescent reporter geneIn
order to generate a powerful tool for the investigation of androgen action and
the rapid screening of novel agonists and antagonists, we developed a new stable
prostatic cell line . A line of androgen receptor (AR)-deficient PC-3 cells
was stably transfected with a human AR (hAR) expression vector and the reporter
gene MMTV-luciferase. It was characterized by its response to androgens and
antiandrogens, as reflected by the expression of measured luciferase.
The PC-3 cells were transfected with pSG5-puro-hAR and pMMTV-neo-Luc. Twenty-five
days after the initiation of double selection, clones that expressed luciferase
were identified by monitoring the chemiluminescence emanating from inducible
colonies in the presence of androgen.
Numerous neomycin-resistant and puromycin-resistant clones were selected as
luminescent. A number of these expressed functional hAR as shown by DHT induction.
One highly inducible clone was selected and named PALM, for PC-3-Androgen receptor-Luciferase-MMTV.
The androgen concentrations required to induce half-maximal luciferase gene
expression were 3 x 10-11 M for R 1881, 2 x 10-10 M for DHT and 3 x 10-9 M for
testosterone. The three agonists had the same maximal activity at 10-6M and
the fold induction was equal to 20. These results were better than those obtained
with the transiently tranfected PC-3 cell line.
The PALM cell line is a new and original cellular model to characterize the
response of hAR, and it provides an easy and rapid bioluminescent test to characterize
new agonists or antagonists. Moreover, no cellular damage occurs with the use
of a simple luminescence buffer and the androgen effect can thus be quantified
at different times within the same cells.
Different xenoestrogens, pesticides, herbicides and fungicides, were tested
alone or in presence of 0.1nM or 0.1 µM R 1881. None of them presented
androgenic activities. Maximum values obtained with 0.1 µM R 1881 were
not inhibited with higher concentrations of tested chemicals. Their antiandrogenic
activities are reported in Fig. 4. A summary of estrogen-like activity as well
as the antiandrogenic activity of the tested environmental disruptors is presented
in Tab. 2.
Cells expressing GFP-AR
analysis of the subcellular localization of the steroid receptors has usually
been performed by immunocytochemistry. It is generally acknowledged that the
estrogen receptor (ER) and the progesterone receptor (PR) are predominantly
nuclear, with a continuous shuttle between the nucleus and cytoplasm. The intracellular
localization of the mineralocorticoid receptor (MR), the glucocorticoid receptor
(GR) and the AR are more controversial. Depending on the immunostaining protocol,
these receptors have been described as being either in the cytoplasm or in the
nucleus in absence of ligand, and exclusively in the nucleus after incubation
with ligand. These techniques require the fixation and permeabilization of cells,
which can lead to artefacts in the pattern of subcellular localization. Moreover,
the AR can be in different states, i.e., associated with the heat shock proteins
in an unliganded form or associated with DNA or transcription factors in the
liganded form. The accessibility of the epitope to antibodies may vary for these
different forms and this could induce artefactual results in the immunostaining.
Having considered all the limits of immunocytochemistry, we developed a model
using a chimera of AR fused to the green fluorescent protein (GFP) . This
fluorescent reporter permitted the visualization of the AR in living transfected
We first verified that the fusion protein (GFP-AR) conserved the functional
characteristics of AR. We demonstrated the advantages of this GFP-AR tool versus
immunodetection. The intracellular dynamics of AR were evaluated and quantified
in living cells, which suggested some applications of the GFP-AR model, such
as antiandrogen screening and androgen insensitivity study. An example of inhibition
of nuclear trafficking is reported in Fig. 5.
Using this method, it was possible to select new compounds able to bind to the
androgen receptor but unable to trigger the translocation to the nucleus. Unlike
classical antiandrogens, these compounds do not exhibit low agonist activity
even at high concentration.
Besides the classical in vivo tests to identify chemical with endocrine disrupting
activity, such as the uterine weight bioassay, the sex accessory gland weight
or the induction of developmental malformations in offspring, we plan to implant
bioluminescent cell lines in nude mouse in order to evaluate in vivo the biological
consequences of environmental estrogens and antiestrogens.
conclusion, the systematic screening of environmental chemicals
and the chemicals present in human foods and water
is needed to identify putative causal agents and to assess their ability to
disrupt the endocrine system.
The EDSTAC is considering a screening battery to detect (anti)estrogenic and
(anti)androgenic activities using in vitro assays (Tier 1).
The battery should detect receptor-mediated effects:
Chemicals that test positive in Tier 1 should be labeled as potential
endocrine disruptors and subjected to in vivo testing (Tier 2).
There is an urgent need for prospective multicenter studies to
describe the epidemiological trend in newborn male congenital malformations.
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1: Potential action of endocrine disruptors in an androgen target cell.
1 competition for the LBD, 2 conformation change of AR, 3 nuclear transfer ,
4 DNA binding and transcriptional activation
2: Induction of luciferase activity by xenoestrogens in the HeLa cell line,
stably transfected with the reporter plasmid ERE-Luciferase and the expression
vector ERa. Results are expressed as a percentage of luciferase activity measured
The 100% value represents the value obtained in presence of E210-8 M.
3: Induction of luciferase activity by phytoestrogens in the HeLa cells stably
transfected with the reporter plasmid ERE-Luciferase and the expression vector
ERa or ERb. HEaLN and HEbLN cells were treated for 24 h with estradiol, genistein
and daidsein. Results are expressed as a percentage of luciferase activity measured
per well. The 100% value represents the value obtained in presence of E210-8
4: Detection of antiandrogenic activities of xenoestrogens with the PALM cell
Cells were treated with various concentrations of chemicals (1 nM to 10 µM)
with 0.1 nM R 1881. Luciferase activities were expressed relative to AR activities
with 0.1 nM R 1881 which was set at 100%.
5: COS-7 cells were transiently transfected with the plasmid coding the fusion
protein between the green fluorescent protein and the androgen receptor (GFP-AR).
After 48 hours, cells were incubated for 3 hours in presence of R1881 (1 nM),
bicalutamide (1 µM) alone or in competition with R1881. Cells were observed
and recorded directly with an epifluorescence microscopy coupled to a CCD camera.
Chemical products tested for estrogenic and antiandrogenic activities by stable