EFFECT OF ENDOCRINE DISRUPTORS ON THE MALE MOUSE
REPRODUCTIVE SYSTEM
BY
HEATHER N. SCHLESSER
DISSERTATION
Submitted in partial fulfillment of the requirements
for the degree of Doctor of Philosophy in VMS - Veterinary Biosciences
in the Graduate College of the
University of Illinois at Urbana-Champaign, 2009
Urbana, Illinois
Doctoral Committee:
Professor Paul S. Cooke, Chair
Professor Rex A. Hess
Associate Professor David Bunick
Associate Professor Marie-Claude C. Hofmann
Professor Janice Mary Bahr
ABSTRACT
The widespread use of soy protein-based infant formulas for human infant
nutrition has raised concerns regarding potential estrogenic effects of soy isoflavones on
normal development and adult function of the reproductive system in humans and
animals. The isoflavone genistein is the most estrogenically active molecule present in
soy. Human infants may also be exposed to other environmental estrogens. Bottle fed
infants are most likely exposed to bisphenol A. The xenoestrogen bisphenol A (BPA)
has a chemical structure similar to the potent synthetic estrogen diethylstilbestrol (DES),
and is found in plastic baby bottles. This study tested the hypothesis that neonatal
genistein and/or BPA exposure cause male reproductive tract defects. Male C57Bl/6
mice were orally treated from postnatal day 1 (PND 1) to PND 5 with a dose of genistein
(50 mg/kg) that resulted in physiological genistein serum levels or a dose of BPA equal
to the no observable adverse effect level (50mg/kg) for reproductive toxicity or a
combination of genistein and BPA at three different concentrations of BPA (50mg/kg
genistein; 0.5, 5, or 50 mg/kg of BPA; Gen/BPA0.5, Gen/BPA5, and Gen/BPA50
respectively). DES injected at 1ng/g was used as a positive control for assessment of
changes in male reproductive tract development. The endpoints chosen for the study
were germ cell proliferation, efferent duct epithelial cell height, anogenital distance, daily
sperm production, testis weight, and reproductive performance. Treatment with 50
mg/kg genistein did not alter any of the endpoints measured. Germ cell proliferation was
increased with BPA, Gen/BPA0.5, Gen/BPA5, and Gen/BPA50 treatment. Treatment
with BPA also resulted in an increase in daily sperm production at both postnatal day 35
and 110 The phenomenon of increased daily sperm production was also seen with the
combination of genistein and BPA at two of the three treatment doses (Gen/BPA0.5 and
Gen/BPA50). An increase in daily sperm production may be due to an increase in germ
cell proliferation that was observed at postnatal day five with BPA, Gen/BPA0.5, or
Gen/BPA50 treatment. Animals treated with BPA and the combination of genistein and
BPA did not have a significant increase in testis weights as a result of the increase in
daily sperm production. Treatment with three different doses of BPA in combination
with genistein resulted in a nonmonotonic dose response curve. Presence of a
ii
nonmonotonic dose response curve indicates that doses of BPA below the NOAEL may
cause effects and should be further investigated. The results of the current study indicate
that environmental estrogens, such as the soy phytoestrogen genistein at physiological
concentrations, did not cause adverse affects on the developing or adult male
reproductive tract. In contrast, BPA is capable of increasing germ cell proliferation
during development and increasing juvenile and adult daily sperm production. The
combination of genistein and BPA also was capable of increasing germ cell proliferation
and increasing daily sperm production. These results show that BPA is capable of
eliciting a response at a dose below the reported NOAEL when combined with other
environmental estrogens. Although the effects seen here may not be considered adverse,
the ability of a chemical to alter reproductive system development is noteworthy and
warrants further investigation.
iii
ACKNOWLEDGMENTS
I would like to thank my PhD advisor, Dr. Paul Cooke, for giving me a chance. I
would also like to thank my committee for allowing me the time to pursue a degree in
teacher education. My committee members, Drs. Janice Bahr, David Bunick, Rex Hess,
and Marie-Claude Hofmann, have been a great source of support throughout my project.
I would also like to thank my laboratory members for their support and suggestions
throughout my project. I am not sure I could have finished without their support.
Finally, I would like to thank my husband, who has proven to be the best sounding board
a person could ask for.
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TABLE OF CONTENTS
CHAPTER 1: REVIEW OF LITERATURE ................................................................. 1 1.1. ESTROGEN RECEPTOR........................................................................................ 1 1.1.A. Sources of Estrogen in the Male Reproductive Tract................................. 1 1.1.B. Expression of Estrogen Receptors in the Male Tract ................................. 4 1.1.C. Estrogen Actions in the Neonatal and Adult Male Reproductive Tract ... 9 1.2: GENISTEIN ........................................................................................................... 15 1.2.A. Properties of Genistein ................................................................................ 15 1.2.B. Binding Affinity ............................................................................................ 22 1.2.C. Reproductive System Effects....................................................................... 26 1.3: BISPHENOL A ...................................................................................................... 30 1.3.A. Properties of Bisphenol A ............................................................................ 30 1.3.B. Binding Affinity ............................................................................................ 36 1.3.C. Reproductive System Effects....................................................................... 38 CHAPTER 2: NEONATAL EXPOSURE TO ENDOCRINE DISRUPTORS ......... 43 2.1: INTRODUCTION ................................................................................................. 43 2.2: SPECIFIC AIMS AND HYPOTHESIS ................................................................. 46 2.3: MATERIALS AND METHODS ........................................................................... 49 2.3.A. Mice ............................................................................................................... 49 2.3.B. Chemicals ...................................................................................................... 49 2.3.C. Dosing ............................................................................................................ 50 2.3.D. Germ Cell Proliferation ............................................................................... 50 2.3.E. Efferent Ductule Epithelial Cell Height ..................................................... 51 2.3.F. Anogenital Distance ...................................................................................... 52 2.3.G. Daily Sperm Production .............................................................................. 52 2.3.H. Reproductive Performance ......................................................................... 52 2.3.I. Statistical Analysis ......................................................................................... 53 2.4: RESULTS .............................................................................................................. 53 2.4.A. Body weight .................................................................................................. 53 2.4.B. Germ Cell Proliferation ............................................................................... 54 2.4.C. Efferent Ductule Epithelial Cell Height ..................................................... 54 2.4.D. Anogenital Distance ..................................................................................... 55 2.4.E. Daily Sperm Production .............................................................................. 55 2.4.F. Testis Weight................................................................................................. 56 2.4.G. Reproductive Performance ......................................................................... 56 2.5: DISCUSSION ........................................................................................................ 80 CHAPTER 3: SUMMARY AND CONCLUSION ...................................................... 93 REFERENCES................................................................................................................ 95 CURRICULUM VITAE ............................................................................................... 117 v
CHAPTER 1: REVIEW OF LITERATURE
1.1. ESTROGEN RECEPTOR
1.1.A. Sources of Estrogen in the Male Reproductive Tract
Once thought to be primarily a female hormone contributing to female health and
fertility, estrogen is produced by males and plays an essential function in male fertility as
well (Korach et al. 1996; Hess et al. 1997; Luconi et al. 2002). There is a high
concentration of estrogen in the rete testis fluid of the rat (Free and Jaffe 1979) and
concentrations of estrogen in the testis, rete testis fluid, and testicular venous blood far
exceed the concentration in male serum in various species, indicating a testicular site of
estrogen synthesis (Hess 2000, 2003).
Estrogen biosynthesis is catalyzed by a microsomal member of the cytochrome
P450 superfamily, aromatase cytochrome P450 (Simpson et al. 1994). Aromatase
expression was first described in germ cells of the adult mouse testis (Nitta et al. 1993).
However, aromatase is expressed in the testis at all stages of development (O'Donnell et
al. 2001), and estrogen production has been reported in fetal testis (Weniger et al. 1993).
Leydig and Sertoli cells both contribute to estrogen production in the immature postnatal
(Tsai-Morris et al. 1985; Dorrington and Khan 1993) and adult testis (Carreau et al.
1999). Immature Sertoli cells show high levels of basal and FSH–stimulated aromatase
activity, which declines as the Sertoli cells cease division and commence maturation into
terminally differentiated cells (Dorrington and Khan 1993; O'Donnell et al. 2001).
Unlike Sertoli cells, which decrease estrogen production during maturation, Leydig cells
increase estrogen production as they mature (Tsai-Morris et al. 1985; Papadopoulos et al.
1
1986). Leydig cell aromatase is regulated by LH and T in adult cells (Genissel et al.
2001). Aromatase knockout mice are initially fertile but develop progressive infertility.
Aromatase knockout mice develop impaired spermatogenesis between 4.5 and 12 months
of age, with no alteration in gonadotropins and androgens (Robertson et al. 1999).
Aromatase knockout mice show an arrest at early spermatogenic stages, indicating that
local expression of aromatase is essential for spermatogenesis, and provides evidence for
a direct action of estrogen on male germ cell development and thus fertility (Robertson et
al. 1999). Aromatase is first detectable in pachytene spermatocytes and remains in germ
cells throughout spermiogenesis, becoming restricted to the elongated spermatid
cytoplasm and flagella (Nitta et al. 1993; Janulis et al. 1996; Janulis et al. 1996; Janulis et
al. 1998). P450 aromatase has been identified in rat sperm cytoplasmic droplets (Janulis
et al. 1996; Hess 2000), and human sperm flagella (Aquila et al. 2002). Localization of
aromatase to sperm indicates that sperm may provide a local source of estrogens in the
epididymis and in the female reproductive tract. Growth factors and cytokines have been
reported to influence germ cell aromatase expression, which is also stimulated by cAMP
(Bourguiba et al. 2003). The production of estrogen by the germ cells and secretion into
the seminiferous tubule fluid may be important for the function of the efferent ductules
and epididymis (Hess 2000, 2003).
Although estrogen biosynthesis by the testis is necessary for proper
spermatogenesis to occur, inappropriate exposure to estrogen can be deleterious to testis
development. Exposure to estrogen during the fetal and neonatal period can impact the
development and organization of the hypothalamic-pituitary-testis (HPT) axis (Cook et
al. 1998; Atanassova et al. 1999; O'Donnell et al. 2001). Neonatal exposure to even
2
weakly estrogenic substances can elevate FSH levels during puberty in rats and cause
long-term changes in the testis (Atanassova et al. 2000). Exogenous estrogen exposure
has been shown to inhibit proliferation of precursor Leydig cells during the neonatal
period, thus altering steroidogenic capacity of adult testis (Abney 1999; O'Donnell et al.
2001). Neonatal exposure to diethylstilbestrol (DES) impairs testicular steroidogenesis in
adulthood via a mechanism which cannot be explained by affects on the HPT axis
(Atanassova et al. 1999). Exposure to DES during the neonatal Sertoli cell proliferative
period decreases Sertoli cell number (Sharpe et al. 1998). The affect of DES is thought to
be through a reduction of FSH, which is a mitogen for Sertoli cell proliferation. Fetal
exposure to estrogen does not appear to affect Sertoli cell proliferation in the rat (Cook et
al. 1998; Wistuba et al. 2003). Estrogen may also inhibit Sertoli cell maturation,
preventing premature Sertoli cell differentiation during the proliferative period. Neonatal
estrogen administration causes a maturational delay in Sertoli cells in rats (Sharpe et al.
1998). In the early postnatal period proliferation of rat gonocytes, which express ER
(Saunders et al. 1998), is stimulated by estradiol in vitro (Li et al. 1997). It has also been
shown that exposure to xeno- or phytoestrogens in utero up-regulates the expression of
platelet-derived growth factor (PDGF) receptors in gonocytes (Thuillier et al. 2003).
PDGF is known to stimulate gonocytes proliferation in vitro (Li et al. 1997). Other
studies using strong estrogens such as DES and estradiol show inhibition of gonocytes
numbers in cultured fetal rat testes (Lassurguere et al. 2003). These results indicate that
estrogen can have both inhibitory and stimulatory affects on gonocyte proliferation.
In the adult, maintenance of spermatogenesis is dependent on normal levels of
FSH, LH and T, and it is important to consider that estrogen is a component of the
3
negative feedback action on gonadotropin secretion at the level of the hypothalamus and
pituitary. Therefore high doses of estrogen administered to adult rats are expected to
decrease serum FSH, LH and T. However, low doses of estrogen are capable of
stimulating FSH (Jong et al. 1975).
1.1.B. Expression of Estrogen Receptors in the Male Tract
The actions of estrogen are mediated through estrogen receptors. ER and ER
are encoded by two different genes located on different chromosomes. Multiple RNA
splice variants for ER (Flouriot et al. 1998) and alternative splice variants (Gaskell et al.
2003; Aschim et al. 2004; Zhou et al. 2006) and polymorphisms (Aschim et al. 2005) for
ER have been reported for humans. Such a molecular diversity due to alternative
splicing mechanisms of the correspondent genes is also observed in rodents (Kuiper and
Gustafsson 1997; Petersen et al. 1998; Varayoud et al. 2005). Estrogen receptors are
members of the steroid receptor superfamily of ligand-activated transcription factors
(Evans 1988). Estrogen receptors reside within the nucleus of target cells in an inactive
state in the absence of ligand. Upon binding its ligand, 17-estradiol (E2), the receptor
undergoes an activating conformational change permitting it to interact with specific
cofactors and bind DNA response elements within target gene promoters (Beekman et al.
1993; Horwitz et al. 1996). The DNA-bound receptor-ligand complex is then capable of
either activating or repressing target gene transcription, depending on both the cell and
the promoter context. Estrogen receptors are known to play crucial roles in control of
reproduction and reproductive behavior and development of secondary sexual
characteristics in animals (Evans 1988). However, estrogen receptors are quite
4
promiscuous in ligand binding (Anstead et al. 1997; Blair et al. 2000), and many nonsteroidal compounds in the environment have been found to possess estrogenic activity
(Sohoni and Sumpter 1998; Akingbemi and Hardy 2001) as a result of binding to ER
and ER.
The estrogen receptor knockout mouse models have illustrated the importance of
estrogen in the male reproductive tract. The creation of these models shed light on the
idea that estrogen was not only a female hormone. The reproductive tract of males
homozygous for estrogen receptor alpha disruption (ERKO) develops normally during
the prenatal period. However, ERKO males display atrophy of the testes and
seminiferous tubules, tubule dysmorphogenesis, and reduced sperm counts as they age
(Lubahn et al. 1993; Eddy et al. 1996; Rissman et al. 1999). These males also appear to
be infertile (Lubahn et al. 1993). However, ERKO mice are infertile primarily due to
the malfunctioning of the efferent ductules of the epididymis (Hess et al. 1997).
Estrogen also functions through a second ER isoform, ERER is expressed at
high levels in estrogen-target tissues such as prostate (Weihua et al. 2001) and testis
(Makinen et al. 2001). Although ER mice are infertile, ER knockout mice are fertile
(Krege et al. 1998). These males exhibit normal fertility and sperm counts (Couse et al.
1999). The drastic difference in phenotype between the two ER knockout animals
illustrates the different roles these two receptors play. It is known that ER levels may
determine the cellular response to ER ligands (Meegan and Lloyd 2003). ER stability is
also influenced by specific ligands (Wijayaratne and McDonnell 2001), which may cause
the interaction of the receptor with tissue-specific coregulators (Nilsson et al. 2001) and
ER–associated proteins (Saceda et al. 1998) or with the proteasome degradation pathway
5
(Marsaud et al. 2003). Estrogen’s function varies depending on the isoform that is
activated. For example, in breast cancer cell lines, E2 in the presence of ER elicits
proliferation, but in the presence of ER, it inhibits proliferation (Strom et al. 2004). In
vitro studies show that homo- and heterodimers of ERand ER can form (Cowley et al.
1997; Pettersson et al. 1997), and it has been proposed that in cells expressing both
ERand ER, ER may act to modulate ER transcriptional activity and that the relative
expression of both subtypes is central to determining cellular responses to estrogenic
ligands (Hall and McDonnell 1999).
Knockout animals lacking both ER and  (ERKO) were also generated to
clarify the roles of each receptor in the physiology of estrogen target tissues. ERKO
males possess a grossly normal reproductive tract. However, they are infertile (Couse et
al. 1999). The testis of adult (2.5 to 7 months) ERKO males exhibited various stages
of spermatogenesis. However, the number of epididymal sperm was reduced by
approximately 80%, and the motility was reduced to 5% (Couse et al. 1999). The
phenotype of ER males is similar to that of ERKO males.
Both ER and ER are present in fetal rodent testis (Cooke, et al. 1991; Fisher et
al. 1997; Saunders et al. 1997; Saunders et al. 1998; van Pelt et al. 1999; Akingbemi
2005). Although ER has only been localized to the fetal Leydig cell (Fisher et al. 1997;
Jefferson et al. 2000), ER has been localized to the Sertoli cell, Leydig cell, and
gonocytes in rodents (Saunders et al. 1997; 1998; van Pelt et al. 1999) and humans
(Gaskell et al. 2003). Studies have shown that ER occurs in two isoforms within the
human testis, wild-type ER also known as ER1 and ER2 a splice variant which may
also act as a dominant negative receptor. Human fetal gonocytes contain mRNA for both
6
splice variants, but only ER2 protein is present in relatively high levels in these cells.
Expression of the ER2 isoform in gonocytes suggests there may be limited estrogen
action (Gaskell et al. 2003). In contrast, human fetal Sertoli cells, Leydig cells, and
peritubular cells appeared to contain both ER isoforms, suggesting that these cells are
capable of responding to estrogens (Gaskell et al. 2003).
In the postnatal rodent testis, ER continues to be expressed in Leydig cells
(Fisher et al. 1997). ER is still expressed in Leydig cells, immature Sertoli cells,
spermatogonia, and spermatocytes (Saunders et al. 1998; van Pelt et al. 1999). ER is
the predominant ER isoform found in the adult seminiferous epithelium of the rodent, and
is localized to Sertoli cells, spermatogonia, pachytene spermatocytes and round
spermatids (Fisher et al. 1997; Saunders et al. 1998) (Table 1). More recently Lucas et
al. (2008) showed that ER was present in the Sertoli cell of adult rodent testis. Analysis
of the human ER splice variants in the adult human testis shows that Sertoli cells show
strong immunostaining for ER2, but less intense staining for ER1. The ER2 isoform
also appeared more predominant, in comparison to ER1 in type B spermatogonia,
perhaps suggesting these cells are less responsive to estrogens (Saunders et al. 2002).
Both variants were present in type A spermatogonia and pachytene spermatocytes
whereas round spermatids appeared to have only ER1 protein (Saunders et al. 2002).
Expression of ER1 in round spermatids could be interpreted to mean that round
spermatids may be the germ cell type best able to respond to estrogens in the adult human
testis. ER expression is also present in mature sperm. Immunohistochemical detection of
ERs has been reported for human (Misao et al. 1997; Durkee et al. 1998) and rat sperm
7
(Saberwal et al. 2002). These studies showed distribution of receptors in both the head
and flagellum.
Cooke et al. (1991) determined that efferent ductules were the first site of
epithelial ER expression in the developing male mouse reproductive tract. Expression of
ER was present starting at gestational day 16. These researchers used steroid
autoradiography, which has the advantage of indicating a functional receptor capable of
binding estrogen. The efferent ductules contained 3-fold more silver grains per epithelial
cell than more distal parts of the epididymis. The greater amount of silver grains in the
efferent ductules indicates a higher concentration of ER in this location. The
concentration of estrogen receptors indicates that the efferent ductules of the male
reproductive tract may be sensitive to increased estrogen levels. Hess and colleagues
showed that the adult efferent ductules also express ER and ER with greater
expression of ER (Hess et al. 1997). Expression of estrogen receptor is not limited to
the testis and epididymis. In fact, both estrogen receptors have been localized in the
developing penis of a number of species, including humans (Crescioli et al. 2003;
Schultheiss et al. 2003; Dietrich et al. 2004), rodents (Jesmin et al. 2002; Jesmin et al.
2004) and rabbits (Srilatha and Adaikan 2004).
8
Table 1: Expression of estrogen receptors in postnatal and adult testicular cells (rodent and primate).
1.1.C. Estrogen Actions in the Neonatal and Adult Male Reproductive Tract
Recent evidence showing production of estrogen by the testis and localization of
both ER isoforms within the testis have led to the question of estrogen’s involvement in
testicular physiology, including spermatogenesis (Sharpe 1998; Couse. and Korach 1999;
O'Donnell et al. 2001; Hess 2003). Estrogen is produced locally within the testis,
suggesting a paracrine action on spermatogenesis. However, it is also possible for
circulating estrogens to impact testicular function through endocrine feedback
mechanisms, which would in turn regulate spermatogenesis. It is has been shown that
testis is responsive to 17-estradiol as early as 14.5 days post coitum (Montani et al.
2008). It is also known that early differentiation and morphogenesis of male reproductive
organs corresponds to the testosterone surge by fetal Leydig cells that occurs at about 12
weeks of gestation in humans (Williams-Ashman and Reddi 1991; Klonisch et al. 2004)
and at late gestation and early neonatal period in rodents (Ward and Weisz 1984; ElGehani. et al. 1998). Alterations in androgenic activity during the critical period of
9
differentiation, resulting from abnormalities in testosterone, 5-reductase, or androgen
receptor, cause altered development of internal and external genitalia, including
hypospadias and shorter penis (Gray et al. 2001; Sultan et al. 2001; Kim et al. 2002;
Foster and Harris 2005).
In the 1990s speculation arose concerning the affect of environmental estrogens
on male fertility. It was speculated that environmental estrogens may have deleterious
affects on male fertility if males were inappropriately exposed during gestation or
postnatally. It was hypothesized that exposure to estrogen could adversely affect
reproductive tract development leading to impaired reproductive function in adulthood
(Sharpe 1993; Sharpe and Skakkebaek 1993). Exposure to xeno- and phytoestrogens has
been hypothesized to be involved in a proposed decline in sperm counts in the latter half
of the twentieth century (Sharpe and Skakkebaek 1993; Auger et al. 1995; Toppari et al.
1996). However, environmental estrogens ability to affect human health and male
fertility has been greatly debated (Daston et al. 1997; Sonnenschein and Soto 1998).
Epidemiological studies have suggested links between inappropriate estrogen
exposure and higher frequency of reproductive abnormalities in men and wild animals
(Toppariet al. 1996; McLachlan 2001; Safe et al. 2001; Mosconi et al. 2002; Fisher 2004;
Vidaeff and Sever 2005; Storgaard et al. 2006). The affects of DES on development and
function of the male reproductive tract represent the potential consequences of
inappropriate estrogen exposure during the critical period of differentiation. DES was
administered to women from 1940 to 1970 to prevent miscarriages (Bibbo et al. 1977).
Male offspring of women exposed to DES during pregnancy have higher incidence of
epididymal cysts, cryptorchidism, hypospadias, and smaller penis (Gill et al. 1979; Swan
10
2000; Klip et al. 2002). Male offspring born to mothers exposed to DES in utero also
have a higher incidence of hypospadias, which implies a transgenerational affect (Klip et
al. 2002; Brouwers et al. 2006). Pregnant mothers with high intake of phytoestrogens as
a result of vegetarian diet are more likely to give birth to boys with hypospadias (North
and Golding 2000). Laboratory animals exposed neonatally to estrogen also develop
hypospadias (McLachlan et al. 1975; Kim et al. 2004; Newbold 2004). Neonatal
exposure to estrogen at low doses enlarges the adult prostate gland but higher doses have
the opposite effect (vom Saal et al. 1997; Gupta 2000; Putz et al. 2001; vom Saal and
Hughes 2005) and perinatal estrogen exposure predisposes the prostate gland to a
precancerous growth at adulthood by an epigenetic mechanism (Ho et al. 2006). Studies
have also reported an affect on male external genitalia due to exogenous estrogen
administration. These studies have shown that laboratory animals exposed neonatally to
estrogen have smaller penises (McLachlan et al. 1975; Zadina et al. 1979; Newbold
2004) and rabbits treated with bisphenol A had subtunical deposition of fat in the corpora
cavernosa of their penises (Moon et al. 2001; Goyal et al. 2005). Male mice treated with
0.2 g/pup per day of DES on alternate days from postnatal day 2 to 12 showed a
malformation of the os penis, significant reductions in penile length, diameter, and
weight, accumulation of fat cells in the corpora cavernosa penis, and significant
reductions in weight of the bulbospongiosus and levator ani muscles (Goyal et al. 2005).
Conversely, ERKO males treated with DES developed none of the above abnormalities.
Failure of ERKO males to develop effects from DES treatment indicates that ER is
necessary for mediating the harmful affects of neonatal DES exposure in the developing
penis (Goyal et al. 2007).
These studies indicate that prenatal and/or neonatal exposure
11
to estrogens can have permanent and even transgenerational, deleterious affects on the
development of male reproductive organs.
Neonatal estrogen exposure down-regulates androgen receptor level in male
reproductive organs (Prins and Birch 1995; McKinnell et al. 2001; Williams et al. 2001;
Woodham et al. 2003) and lowers plasma testosterone (Sharpe et al. 1998; Atanassova et
al. 2000). The neonatal concentration of intratesticular testosterone in rat pups treated
with DES for postnatal days 1-3 was reduced by 90% at postnatal days 5-8 (Goyal et al.
2005). The decrease in intratesticular testosterone at PND 5-8 correlates with the
developmental period when stromal cells start differentiation in the rat penis (Murakami
1986, 1987) suggesting a role for estrogen-induced lower androgen action in inducing
penile abnormalities by reprogramming stromal cell differentiation. These alterations in
androgenic activity during the critical period of differentiation also lead to the deformities
in male reproductive tract development which are seen in DES-treated animals and
human males born to mothers given DES. It has also been shown that testosterone, when
given with DES, is capable of preventing the abnormalities induced by DES (Rivas et al.
2003). Indicating testosterone may be protective against the affects of DES.
Gross morphological abnormalities are not the only defects seen in animals
treated with estrogen. Neonatal exposure to relatively low concentrations of DES have
been shown to negatively affect the development of Sertoli cells (Sharpe et al. 1998;
Atanassova et al. 1999). Although it has been argued that xeno- and phytoestrogens may
be involved in the decline in sperm counts in the latter half of the twentieth century, some
researchers have shown that estrogens may exert stimulatory affects on early germ cell
development in the testis of several animals (Kula 1988; Slowikowska-Hilczer and Kula
12
1994; Kula et al. 1996; Li et al. 1997; Miura et al. 1999). The mixed results of estrogen
exposure fuel the debate over environmental estrogens’ involvement in the decline of
male fertility.
The efferent ductules have the highest concentration of ER in the male
reproductive tract, indicating its sensitivity to estrogen stimulation. The efferent ductules
are a series of tubules that connect the rete testis to the epididymis. The efferent ductules
function to concentrate the sperm by absorbing 90% of the fluid leaving the testis (Hess
2000, 2003; Hess et al. 2001). The major cell types in the efferent ducts are nonciliated
and to a lesser extent ciliated cells, which chiefly function in absorption/ion secretion and
mixing of the luminal contents, respectively (Lee et al. 2001). Aquaporins expressed in
the efferent ductules mediate water transport out of the lumen, decreasing the water
content of the semen and thereby increasing sperm quality (Wellejus et al. 2008).
Interestingly similar defects are seen in the efferent ductule of males treated with
exogenous estrogens and ERKO males. Hess and colleagues conducted in vitro ligation
experiments and found the ability of the ERKO efferent ductules to reabsorb fluid was
greatly impaired (Hess et al. 1997). ERKO efferent ductules show numerous structural
differences compared to wild-type ductules, all of which are consistent with a decrease in
fluid reabsorption (Hess 2000, 2003; Hess et al. 2000). A molecular target of estrogen
action in the efferent ductules appears to be Na+/H+ exchanger-3, and the ER-dependent
expression of Na+/H+ exchanger-3 in ERKO mice is effected (Lee et al. 2001). Altered
Na+/H+ exchanger-3 expression has downstream affects on the expression of other
proteins central to fluid reabsorption (Zhou et al. 2001). Structural and functional
development of efferent ductules are susceptible to changes after estrogen exposure
13
during early life, and this has been associated with downregulation of aquaporin-1
expression in adult rats after neonatal exposure to DES (Fisher et al. 1998; Fisher et al.
1999). In contrast neonatal exposure to DES causes an upregulation in aquaporin-9
expression (Wellejus et al. 2008). Neonatal exposure to highly potent estrogenic
substances causes dilation of the rete testis and fluid accumulation, permanent reductions
in the water channel protein aquaporin-1, changes in the cell- and region-specific
expression of ER protein in the male reproductive tract and compromised
spermatogenesis in adulthood (Bibbo et al. 1977; Gill et al. 1977; 1979; Aceitero et al.
1998; Fisher et al. 1998; 1999; Atanassova et al. 2001). The upregulation of aquaporin-9
may be attributed to the widening of the efferent ductule lumen caused by inhibited fluid
reabsorption and increased intratubular pressure. These studies highlight the fact that the
development of the efferent ductules is a target for estrogen action, with either too little
or too much being detrimental to its function. The mechanisms by which estrogen
exposure and deficiency impair efferent ductule development and function is not clear,
but may relate to estrogen exposure changing patterns of androgen receptor and estrogen
receptor expression in the male reproductive tract. Changing patterns of androgen
receptor and estrogen receptor expression may interfere with the androgen: estrogen
balance, as well as the balance of ER subtypes expression, that is likely important in the
function of the efferent ductules. Rivas and colleagues (2002) showed that combined
administration of DES plus GnRH antagonist or flutamide induced significantly greater
distension/overgrowth of the rete testis and efferent ducts and a reduction in epithelial
cell height of the efferent ducts than did DES administration alone. The findings from
this study suggest that reduced androgen action sensitizes the reproductive tract to
14
estrogens, demonstrating that the balance in action between androgens and estrogens,
rather than their absolute levels, may be of fundamental importance in determining
normal or abnormal development of some regions of the male reproductive tract (Rivas et
al. 2002). Another explanation for the similar effects of neonatal estrogen exposure and
deficiency is that inappropriate estrogen exposure may lead to down-regulation of ERs,
resulting in an estrogen deficiency syndrome similar to that seen in ERKO mice.
Developmental exposure to DES has been shown to decrease the ER levels in rat uterus
in adulthood (Medlock et al. 1988; Medlock et al. 1991; Medlock et al. 1992). Similarly,
studies in testis suggest that neonatal DES exposure lead to down-regulation of ER and
AR, but an increase in ER (Tena-Sempere et al. 2000), perhaps suggesting a permanent
change in either estrogen responsiveness, or in estrogen-dependent gene expression.
1.2: GENISTEIN
1.2.A. Properties of Genistein
Phytoestrogens are classically defined as estrogenic compounds found in plants.
Genistein (4’, 5, 7 – trihydroxy-isoflavone) is one of the most abundant phytoestrogens.
It is present in many plants that are important components of human and animal diets
(Farnsworth et al. 1975; Setchell et al. 1984; Mazur and Adlercreutz 2000), and is the
most estrogenic compound found in soy (Montani et al. 2008).
Phytoestrogens bind to
the estrogen receptor and induce specific estrogen-responsive gene products.
Phytoestrogens can exert estrogenic affects on the central nervous system, induce estrus,
15
stimulate growth of the genital tract of female animals (Lieberman 1996) and stimulate
ER-positive breast cancer cell growth (Kurzer and Xu 1997).
Phytoestrogens are divided into three classes based on their chemical structure.
These three groups are isoflavonoids, lignans, and coumestans (Kurzer and Xu 1997;
Setchell 1998; Miniello et al. 2003). Genistein, daidzein, glycitein, biochanin A, and
formononetin comprise the isoflavonoids. Isoflavonoids are primarily found in soybeans
(Coward et al. 1993; Wang and Murphy 1994; Miniello et al. 2003). Soybeans contain
the isoflavonoids genistein and daidzein as inactive glycosides or as metabolically active
aglycones (Axelson et al. 1984; Kurzer and Xu 1997) (Figure 1). The malonyl and acetyl
glycosides are susceptible to heat and readily convert to the more stable -glycoside
(Barnes et al. 1994); therefore, depending on the extent of processing of the soybean, the
relative proportions of these conjugates can vary considerably among different soy-foods
(Coward et al. 1993; Wang and Murphy 1994; Coward et al. 1998). In soy-protein
formulas the isoflavonoids are present mainly as glycosides of genistein, which account
for more than 65% of the total isoflavones (Miniello et al. 2003). The systemic
bioavailability of genistein is much greater than that of daidzein and the bioavailability of
these phytoestrogens is greater when ingested as glycosides rather than the active
aglycones (Setchell et al. 2001). Gut microflora strongly affect the conversion of plant
precursors, such as glucosylated genistein, into active aglycone genistein (Axelson and
Setchell 1981; Setchell et al. 1984). The metabolic pathways of daidzein and genistein
catabolism in humans were originally proposed by Setchell and Adlercreutz (1988) and
has been expanded recently by Joannou et al (1995), based on the isoflavone metabolites
found in human urine (Figure 2). Daidzein is metabolized to dihydrodaidzein, which is
16
further metabolized to both equol and O-desmethylangolensin (O-DMA). Genistein is
transformed to dihydrogenistein and is further metabolized to 6-hydroxy -O- DMA
(Kurzer and Xu 1997). Isoflavone and lignan absorption and utilization require a series
of deconjugation and conjugation steps. Genistein absorption initially occurs passively
through diffusion, because the rate of absorption increases with the increased intake of
genistein (Sfakianos et al. 1997). Absorption is facilitated by hydrolysis of the sugar
moiety by human gut bacterial -glucosidases, gastric hydrochloric acid, and glucosidases in foods (Kelly et al. 1993). Cleavage of the carbohydrate portion releases
the biologically active aglycone which is a heterocyclic phenol structurally resembling
estrogens (Murkies et al. 1998) (Figure 3). Genistein is made of three rings: a phenolic
B-ring bound to the 3 position of the pyran C-ring, and adjacent to the C-ring is the
phenolic A ring. The 4’-hydroxy group on the B-ring in combination with the 7’hydroxy group are responsible for the estrogenicity of genistein, while the 5’-hydroxy
and 4-keto groups on the A/C ring are responsible for the growth inhibitory action of
genistein (Zava and Duwe 1997). These heterocyclic phenols are capable of binding both
ER and ERand sex-hormone binding globulins (Chen and Rogan 2004)Unlike E2
which has similar affinity for both ERs, genistein and daidzein have greater affinity for
ER than ER (Cooke et al. 2006). After absorption in the small intestine, isoflavones
and lignans are conjugated with glucuronic acid and sulfate by hepatic phase II enzymes
(UDP-glucuronosyltransferases and sulfotransferases). Like endogenous estrogens, these
conjugates are excreted through both urine and bile and undergo enterohepatic
circulation. After excretion into bile, conjugated isoflavones and lignans can be
deconjugated once again by gut bacteria. Deconjugation may promote reabsorption,
17
further metabolism, and degradation in the lower intestine (Setchell and Adlercreutz
1988; Xu et al. 1995). The biological affect exerted by these estrogen-like compounds
has been greatly debated.
Phytoestrogens may have beneficial affects on some types of cancer,
cardiovascular health, brain function, alcohol abuse, osteoporosis and menopausal
symptoms (Hirayama 1984; Tajima and Tominaga 1985; Lee et al. 1991; Goodman et al.
1997), but also can have adverse effects on reproductive systems (Kurzer and Xu 1997;
Bingham et al. 1998). The phytoestrogen coumestrol causes infertility in sheep, a
condition known as clover disease (Bennetts et al. 1946; Bennetts and Underwood 1951;
Braden et al. 1967; Shutt and Braden 1968), and daidzein significantly decreases
expression of ER and AR mRNAs in rat uteri (Bennetts and Underwood 1951; Diel et
al. 2000).
Asian populations which consume diets high in soy are estimated to ingest
between 30 and 50 mg/day of soy isoflavones (Adlercreutz et al. 1993; Kimira et al.
1998) equivalent to about 1.5 mg/kg/day of isoflavone. Ingestion of 1.5 mg/kg/day of
isoflavone results in a mean plasma concentration of genistein of 406.8 nmol/L (Uehar et
al. 2000). Infants consuming soy based formulas are exposed to a much higher
concentrations of genistein than adults on a soy containing diet. Leading infant formulas
such as Nursoy, Isomil, Alsoy, and Prosbee have isoflavone contents ranging from 32-47
mg of isoflavones/L (Setchell et al. 1997). It has been estimated that soy-protein infant
formulas contain 87 ±3 g/g of genistein (Irvine et al. 1998). Infants consume 6 – 11
mg/kg/day of isoflavones, which is greater than that consumed by adults on a high-soy
diet (1.5 mg/kg/day) (Le et al. 2004). Ingestion of 11 mg/kg/day of isoflavone results in
18
a plasma concentration of genistein of 2.53 mol/L (Setchell et al. 1998). The plasma
concentration of genistein in soy fed infants is 13,000 – 22,000-fold higher than
endogenous plasma estradiol concentrations in early life (Setchell et al. 1997; 1998). It is
also 16 times higher than plasma genistein concentrations found in Japanese adults.
Irvine et al. (1998) reported that infants as young as 4 weeks old can digest, absorb, and
excrete genistein and daidzein from soy-based formulas as effectively as adults
consuming soy products. It is also known that infants have a higher concentration of
genistein aglycone, or the active form of genistein (Chang et al. 2000; Doerge et al.
2002).
Soy-protein formula was first described in 1909 as a cow-milk substitute (Merritt
and Jenks 2004). Its use as a milk substitute for infants with cow-milk allergies was not
introduced until 1929. The first soy-protein formulas on the market are referred to as first
generation formulas. These formulas had an unpleasant caramel-like color and caused
mineral deficiencies and carbohydrate intolerance. A second generation formula was
developed in the 1960s and was based on a soy protein isolate of at least 90%. More
importantly, the distribution of nutrients in these soy protein formulas is quite similar to
that in cow-milk formulas (Miniello et al. 2003). Although these second generation soyprotein formulas are more nutritiously sound and have corrected some of the problems
with the first generation formulas, the formulas still contain plant-derived estrogens,
known as phytoestrogens. The presence of these phytoestrogens could have short- and/or
long-term affects on the development of the infants that receive them. In the United
States, it is estimated that 18 -25% of infants are given soy-protein formulas (Strom et al.
2001; Miniello et al. 2003). About 540,000 - 750,000 infants/year have consumed soy-
19
protein formulas in the United States. It is estimated that some 20,000,000 infants in the
United States have consumed soy infant formula since the 1960s (Strom et al. 2001).
The use of soy-infant formula was originally advocated or prescribed only for
infants exhibiting signs of intolerance to cow’s milk. However, it is sometimes used as
the breast milk substitute of choice, and is frequently used within the first months of life
(Polack et al. 1999). Although the soy isoflavones are weak estrogens, soy formula
contains so much of them that estimates of daily estrogenic dose per kilogram body
weight are equivalent to 0.01 to > 1 contraceptive pill per day, depending on the potency
estimate used (Klein 1998; Chen and Rogan 2004). Also, adults rarely exceed 25% of
calories from soy (Baird et al. 1995), however, infants fed soy formula get 100% of their
calories from soy. Concerns have been expressed as to whether or not the high
isoflavonoid phytoestrogen content of these soy formulas pose adverse affects to the
developing infant. It has been hypothesized to be harmful for development of the male
reproductive system and subsequent adult fertility (Chen and Rogan 2004). Soy
formulas are given during a developmental period when humans have low endogenous
estrogen concentrations It has previously been shown that isoflavonoid phytoestrogens
can exert a range of affects in animals such as birds, rodents, cheetahs, and sheep (Alpert
1976; Leopold et al. 1976; Setchell et al. 1987; Irvine et al. 1995; Whitten et al. 1995;
Whitten and Naftolin 1998). The high concentrations of isoflavonoid phytoestrogens are
likely to exert some biological effects in human infants.
20
Figure 1: Chemical structures of the isoflavones found in soybeans. Daidzein and genistein are also
present as acetylglucosides (6" - O - acetyldaidzin, 6"- O - acetlygenistin) and malonylglucosides (6"
- O - malonyldaidzin, 6" - O - malonylgenistin) (Kurzer, M. S. and Xu, X. 1997).
Figure 2: Proposed metabolic pathways for the catabolism of daidzein and genistein by human gut
bacteria. The formation of O-DMA from daidzein and 6'-hydroxy-O-DMA from genistein likely
involves several reduction reactions. The formation of equol from dihydrodaidzein may go through
reduction, dehydration, and further reduction reactions (Kurzer, M. S. and Xu, X. 1997).
21
Figure 3: Chemical structure of estradiol-17 and genistein.
1.2.B. Binding Affinity
Similarity between genistein and estrogen’s chemical structure and genistein’s
affinity for ER has lead to the concern that genistein can cause deleterious effects on
reproductive development and function in both humans and wildlife. To understand the
genistein effects in vivo, it is necessary to identify the interaction of genistein with
plasma sex steroid binding proteins. These proteins such as -fetoprotein (AFP) bind
particular steroids with high affinity and specificity and are believed to be important
modulators of endogenous steroid hormone action (Hammond 1995). If a large amount
of AFP is present this will reduce the amount of free estradiol to very low concentrations,
reducing its biological activity by restricting its availability to target cells. However,
binding of exogenous estrogens to AFP would reduce the biological activity of the
exogenous estrogen, while displacing biologically active estradiol. If the exogenous
estrogens are incapable of binding AFP this would allow their action on target cells,
while AFP sequesters the naturally present estradiol. AFP is found in the amniotic fluid
and in the blood of fetuses and neonates, it binds estradiol with high affinity and high
specificity (Payne and Katzenellenbogen 1979; Garreau et al. 1991). Some studies have
suggested that the binding of estradiol by AFP in the perinatal period is of critical
importance in preventing early estrogen exposure from inducing inappropriate sexual
22
differentiation of the brain of the female (Milligan et al. 1998). However, a study
conducted by Milligan and colleagues showed that genistein does not bind AFP (Milligan
et al. 1998). Genistein’s inability to bind AFP indicates that genistein may be capable of
binding ER at a time when AFP is binding endogenous estrogen.
Immediately after birth hormonal imprinting takes place. Hormonal imprinting
occurs when the hormone first begins signaling through its target receptor. Hormonal
imprinting is necessary for the normal maturation of the receptor – signal transduction
system. Although each receptor is sensitive to imprinting, the steroid receptor
superfamily is especially sensitive in the perinatal critical period (Csaba 2000). During
the perinatal critical period if genistein is present and binds the receptor instead of the
target ligand E2 faulty imprinting occurs. Faulty imprinting results in life-long
consequences that can result in abnormalities at the receptor, morphological,
biochemical, genetic and behavioral levels in adult animals (Bern et al. 1973; Bern et al.
1975; Iguchi 1992; Nelson et al. 1994; Karabelyos and Csaba 1998).
Similarities in genistein’s chemical structure to E2allows it to bind both estrogen
receptors (Kuiper et al. 1998). However, because the chemical structure of genistein is
not identical to E2 the binding is not identical. Although E2 has similar binding affinity
for both estrogen receptors, genistein has been shown to bind ER with greater affinity
(Cooke et al. 2006). Genistein has been reported to bind the ER in cytosol preparations
of sheep uteri with relative binding affinities of 0.9% of E2 (Shutt and Cox 1972).
Genistein has also demonstrated binding to cytoplasmic ER present in the pituitary gland
and the hypothalamus of the ewe (Molteni et al. 1995).
23
Antiestrogenic affects of phytoestrogens have also been observed. It has been
proposed that phytoestrogens may be able to compete effectively with endogenous
mammalian estrogens for binding to the ER and prevent estrogen- stimulated growth in
mammals (Adlercreutz et al. 1995). Binding of phytoestrogens to the ER instead of
endogenous estrogens may also result in interference with the release of gonadotropins
and interruption of the feedback-regulating system of the HPT axis. There are some data
supporting the idea that genistein can bind ER instead of endogenous estrogen. Genistein
and coumestrol have competitively suppressed the binding of [3H]estradiol to ER when
added to rat and human mammary tumor tissue (Verdeal et al. 1980).
A study conducted by van Lipzig and collegues (2004) to predict the ligand
binding affinity and orientation of xenoestrogens to the estrogen receptor determined that
the energetically favorable orientation of genistein in the ER is different from its
orientation in the crystal structure of ERas proposed by Pike et al. (1999). These
authors proposed that the difference in genistein’s binding affinity for the two estrogen
receptors could account for genistein being an agonist for ER and only a partial agonist
for ER Pike and colleagues identified the structure of the ligand-binding domain of
ER in the presence of genistein. The overall structure of the ER-ligand binding
domain is very similar to that previously reported for ER (Brzozowski et al. 1997).
Genistein was found to be completely buried within the hydrophobic core of the protein
and binds in a manner similarly to E2. However, in the ER – genistein complex, the AF2 helix does not adopt the distinct ‘agonist’ position, but instead, lies in a similar
orientation to that induced by ER antagonists. Failure of the AF-2 helix to adopt the
distinct agonist position after genistein binding to ER is consistent with genistein’s
24
partial agonist character in ER (Pike et al. 1999). Genistein is not limited to binding
only the ER. Genistein is capable of binding other cellular proteins.
It is possible that tyrosine phosphorylation plays an important role for cell
proliferation and cell differentiation. Protein tyrosine kinases (PTKs) are oncogene
products that catalyze phosphorylation of their own tyrosine residues as well as those in
other proteins (Hunter and Cooper 1985). Isoflavones, especially genistein have been
shown to inhibit cellular PTKs (Chang and Geahlen 1992; Boutin 1994). In vitro
experiments conducted by Akiyama et al., (1987) with the epidermal growth factor
receptor in intact A431 cells, show that genistein inhibits the receptor tyrosine
autophosphorylation and tyrosine phosphorylation of both natural and artificial
compounds. PTK inhibition has been proposed to explain the growth-inhibiting affects
of genistein in a number of different human cancer cells. Genistein has been shown to
induce terminal differentiation and inhibit proliferation in a dose-dependent manner in
human leukemia and melanoma cells (Constantinou et al. 1990; Constantinou and
Huberman 1995). However, genistein inhibits tyrosine kinases only at
supraphysiological concentrations. These concentrations, typically 100 mol/L, are
many times greater than what is obtained through consumption of dietary genistein.
DNA topoisomerases are enzymes that catalyze topologic changes in DNA and
are required for DNA replication (Champe and Harvey 1987; Granner 1990). Genistein
has been shown to inhibit both topoisomerase I and II activity (Okura et al. 1988;
Markovits et al. 1989; Constantinou et al. 1990; Kiguchi et al. 1990). Genistein has also
been shown to inhibit the relaxation of pBR322 (supercoiled) DNA catalyzed by
topoisomerase II at concentrations greater than 7.4 M (Okura et al. 1988). Genistein is
25
able to induce topoisomerase II – mediated double-strand breaks, which suggests that
genistein may stabilize the topoisomerase II – DNA complex, allowing the breaks to
occur but preventing them from being resealed (Constantinou et al. 1990).
Genistein’s estrogenic affects either as an estrogen agonist or antagonist have
been hypothesized to have an effect on the male reproductive system development when
consumed during the developmentally critical period.
1.2.C. Reproductive System Effects
Fetal testes are responsive to genistein as well as E2 by gestational day 14.5
(Montani et al. 2008) which makes them a target for gestational and neonatal exposure to
genistein. Genistein’s affects on male reproductive tract development have been
extensively studied using animal models. However, data from human infant exposure to
soy-based formulas is lacking. Current research to study the affects of genistein in
animals employs two main methods of genistein administration: maternal administration,
or direct injection of the pups. Both of these methods do not properly mimic the form of
exposure seen in human infants. Human exposure to genistein is primarily through the
diet, (e.g,. soy infant formula). A neonatal mouse receives 100% of their nutrients from
the mother’s milk. Previous studies have shown that exposure of the mother to genistein
in the diet has limited lactational transfer to the pups (Lewis et al. 2003; Doerge et al.
2006). For example pregnant dams exposed to 16 mg/kg of genistein orally during
lactation had a serum circulating concentration of genistein of 1.8 g/mL but the
concentration found in the milk was only 0.04 g/mL which is 45 times less than
circulating concentrations in the dam (Doerge et al. 2006). The inefficient transfer of
26
genistein through the milk makes maternal dosing inefficient and necessitates the dam be
dosed with toxic concentrations to achieve a concentration in the milk that mimics human
exposure concentrations.
Human studies on soy formulas have mainly focused on nutritional status, with
only one on long-term safety. The long-term study was conducted by Strom et al. (2001).
In the study conducted by Strom and colleagues, they interviewed 811 subjects in their
twenties or early thirties who, as infants, were given soy formula (120 males and 128
females) or cow-milk formula (295 males and 268 females). They determined that there
was no difference in the answers to general questions about health and reproduction
between the two treatment groups. They did find that women fed soy infant formula
reported a longer duration of menstrual bleeding (about 8 hours) and greater discomfort.
These researchers also noted that individuals given soy infant formula reported more use
of asthma or allergy drugs and a greater tendency for sedentary activities.
Studies conducted using animal models tend to show more drastic effects of
genistein treatment. Studies conducted by Newbold et al ( 2001) reported that outbred
neonatal CD-1 mice treated by subcutaneous (sc) injections with genistein (50
mg/kg/day) on postnatal days 1 – 5 had prevalence of 35% of uterine adenocarcinoma at
18 months of age. The same lab (Jefferson et al. 2002), also reported an increased
incidence of multi-oocyte follicles in females treated by sc injections on postnatal days 1
– 5 with 10, or 100 mg/pup/day (~ 5, and 50 mg/kg/day). Other studies have also shown
an advanced mean day of vaginal opening by as many as 4 days (Lewis et al. 2003).
Advanced vaginal opening indicates an accelerated rate of puberty onset. Affects of
genistein treatment are not exclusive to females. A feeding study in twin marmoset
27
monkeys showed that the twin fed with soy formula for ~ 8 hours each weekday and 2
hours on weekends from postnatal days 4-5 until postnatal days 35-45 had low plasma
testosterone concentrations (1.3 ± 2.1 ng/ml) compared with the twin that was fed cowmilk formula (2.8 ± 3.9 ng/ml) (Sharpe et al. 2002). No difference in testis weight was
noted in these twins. However, the twin fed soy infant formula had higher Leydig cell
counts. Similar results have also been seen in rodent models. Time-bred Long-Evans
dams were fed diets containing 0, 5, 50, 500, or 1000 ppm of soy isoflavones from
gestational day 12 until weaning at PND 21. Males from the highest exposure group
exhibited elevated serum concentrations of sex steroid hormones androsterone at PND 21
and testosterone at PND 90, although steroidogenesis was decreased in individual Leydig
cells (Akingbemi et al. 2007).
In vitro studies have also shown that genistein is capable of promoting sperm
capacitation, acrosome reaction, and fertilizing ability in mice. However, in vivo studies
in mice do not support this in vitro data. Fielden and colleagues (2003) dosed C57BL/6
dams with genistein from gestational day 12 to postnatal day 20. Offspring from these
dams were assessed for testicular growth, sperm count and motility, and sperm fertilizing
ability in vitro. These researchers found no significant treatment related affect on male
offspring body weight, anogenital distance, seminal vesicle weight, or testis weight.
They also found no significant affect on sperm count, the percent of motile sperm or the
number of motile sperm at any age. However, they did find that male offspring from
dams treated with the highest dose of genistein (10 mg/kg/d) had a significantly increased
in vitro fertilizing ability. They found that these males had a 17% increase in fertilizing
ability on PND 105 and 315 (Fielden et al. 2003). Wisniewski et al. (2003) observed
28
demasculinization of the reproductive system in rat pups after in utero and lactational
exposure to genistein (0, 5, 300 ppm from gestational day 1 – PND 21). These animals
had smaller testis size, fewer animals with preputial separation at postnatal day 40, lower
plasma testosterone concentrations (3.72 ± 0.55, 1.76 ± 0.31, 2.23 ± 0.42 ng/ml in 0, 5,
and 300 ppm, respectively). In addition, fewer males were capable of ejaculation at PND
70. These researchers did note however, that sperm counts were not significantly altered
between any of the groups. It has also been shown that testes from male ICR mice that
were exposed to genistein (1000 g/mouse/day), starting 1 day after birth for 5 days, had
a decrease in the mRNA level of ER  and AR (Adachi et al. 2004). In another study,
dosing neonatal rats by subcutaneous injection with 4 mg/kg/day of genistein from PND
2 – 12, researchers found a minor but significant decrease in epithelial cell height of the
efferent ducts at day 18. Decrease in efferent duct epithelial cell height was transient and
was not apparent by PND 25. Treatment with genistein did not affect the expression of
AQP-1 or rete testis morphology (Fisher et al. 1999). In another study using neonatal rats
to look at the effects of genistein on the physiology and morphology of the hypothalamus
and pituitary, rats were injected with 100 g – 1000 g from PND 1 -10. These animals
were then castrated at PND 21. Males exposed to 100 g of genistein showed increased
pituitary response to GnRH. The males exposed to 1000 g of genistein showed
decreased tonic LH or attenuated GnRH- stimulated LH secretion. Genistein treatment
did not have an affect on the volume of the sexually dimorphic nucleus of the preoptic
area (Faber and Hughes 1991). Prenatal exposure of genistein has been shown to
influence sexual differentiation (Kurzer and Xu 1997). Neonatal subcutaneous injections
of 10 – g genistein to rat pups was associated with increased pituitary response to
29
gonadotropin releasing hormone, increasing genistein was associated with decreasing LH
secretion on postnatal days 1 – 10 (Faber and Hughes 1993).
Genistein is not the only estrogenic chemical infants are exposed to during a
developmentally critical stage of their lives. Soy-protein formulas are typically
administered to infants in plastic baby bottles which may not be as innocuous as once
thought. It is known that these plastic baby bottles and the containers which store the
soy-protein formula contain the xenoestrogen bisphenol A.
1.3: BISPHENOL A
1.3.A. Properties of Bisphenol A
Bisphenol A (BPA; 4, 4’-isopropylidenediphenol) is a product of industrial
society produced in large quantities worldwide. The amount of BPA produced is greater
than 3.7 million metric tons/year and estimated to increase by 6 to 7% each year
(Anonymous 2005). Based on these estimates the amount of BPA production currently
(2008) is between 4.41 million metric tons and 4.53 million metric tons per year. BPA is
synthesized by the combination of two equivalents of phenol with one equivalent of
acetone. BPA was first synthesized in 1891 by A.P. Dianin (Vandenberg et al. 2009).
Industrial production of BPA uses an acid-catalyzed condensation reaction to minimize
by-products. BPA is the key building block used in the manufacture of two types of
polymers used in food contact articles, specifically, polycarbonate polymers and epoxybased enamels and coatings. Polycarbonate plastic, first made in 1952, is a light-weight
high performance plastic. Polycabonate plastic possesses several qualities that make it a
30
favorable choice for many uses. Its durability, shatter-resistance and heat-resistance
make it an ideal choice for tableware as well as reusable bottles and food storage
containers that can be conveniently used in the refrigerator and microwave (Biles et al.
1997). Epoxy polymers are resistant to solvents and can bond to a variety of substrates,
especially metals (Biles et al. 1997). These properties make epoxy resins a popular
choice for use in enamel coatings on the food contact surfaces of metal food and
beverage cans. Many of the epoxy resins are synthesized by the condensation of BPA
with epichlorhydrin to create BPA diglycidyl ether (Vandenberg et al. 2009). Bisphenol
A plastics and resins are used in the manufacture of milk and food containers, baby
formula bottles, water carboys (Biles et al. 1997), the interior lining of food cans
(Brotons et al. 1995), and dental resins and composites (Olea et al. 1996; Staples et al.
1998). These food contact substances have been regulated for many years pursuant to
regulations published in Title 21 of the Code of Federal Regulations (CFR).
Polycarbonate (PC) polymers are regulated in 21 CFR 177.1580. Epoxy-based enamels
and coatings are regulated in 21 CFR 175.300 (b) (3) (viii), 21 CFR 177.1440 and 21CFR
177.2280 (FDA 2008).
Polymeric reactions do not go entirely to completion, which allows BPA to leach
from these materials during use of the product. The ester bond linking BPA molecules in
polycarbonate and resins is subject to hydrolysis, resulting in leaching of BPA monomer
even from new polycarbonate into water at room temperature (Howdeshell et al. 2003).
The rate of leaching due to hydrolysis of the ester bonds increases as temperature
increases in response to acidic or basic conditions (Carey 2003). Polycarbonate is
marketed as being highly durable, which prolongs its use in consumer households.
31
However, as polycarbonate begins to show signs of wear, the rate of leaching can
increase over 1000-fold relative to the rate of leaching from new products (Howdeshell et
al. 2003). Many studies have been conducted showing the ability of BPA to leach from
these materials under normal usage conditions (Brotons et al. 1995; Biles et al. 1997;
Biles et al. 1997).
Bisphenol A has been shown to migrate from epoxy can coatings into infant
formula liquid concentrates. A study conducted by Biles et al. (Biles et al. 1997) of all
the major manufacturer’s formulas showed a range of BPA in the infant formula
concentrates of 0.1 ppb – 13.2 ppb. The amount actually consumed would be less
however, because manufacturer directions call for a 1:1 (V/V) dilution of the concentrate
with water. The study conducted by Biles and colleagues shows that BPA recoveries are
higher in soy-based infant formulas than milk-based infant formulas (104% vs. 86%)
(Biles et al. 1997). The results from the study conducted by Biles and colleagues indicate
that infants fed soy-based infant formulas would receive a higher amount of BPA than
infants fed cow’s milk-based infant formulas. In an FDA report by Biles and colleagues
(Biles et al. 1997) the amount of BPA in a variety of polycarbonate baby bottles was
determined after mimicking conditions of repeated use, and typical use. These
researchers found that BPA residues varied widely in the different polycarbonate baby
bottles tested (7.4 – 46.6 g/g). They found that with repeated use there was an initial
high migration rate of BPA (4.9%), but the migration decreased with repeated use and
leveled out with each subsequent interval in the experiment (1.7%, 0.9%, 0.9%). These
researchers also tested the migration of BPA after conditions that would mimic typical
use. They found that the migration amount, when converted by using the internal surface
32
area in contact with the food (249 cm2) when a bottle is completely filled (250 mL), was
equivalent to 2ng/g in formula (Biles et al. 1997). These conditions are probably more
likely to simulate the actual use of such articles in the home and show that such usage is
likely to result in transferring little if any BPA into food. Actual migration of BPA from
reusable items may exceed the available residual levels because additional BPA can be
formed through polymer hydrolysis with repeated heating operations with aqueous foods
(Lieng-Huang 1965; Gardner 1979; Gachter and Muller 1990).
The FDA found that the small amount of BPA that migrated into food from the
use of PC-based polymers and BPA-based epoxy coatings result in a cumulative daily
intake for adults of 11 g/ person/ day. The cumulative daily intake of BPA for adults
was estimated based on: 1) the migration level of BPA into food, or into food-simulating
solvents, under the most severe conditions of use (i.e. time and temperature), and 2)
information on the types of food contacted, the fraction of the diet that would come into
contact with that type of food contact material, and whether the finished food contact
article would be intended for single or repeated use. The FDA’s evaluation also
considered that the use of can enamels in infant formula packaging and the use of PC
baby bottles results in an estimated daily intake of 7 g/infant/day (FDA 2008). Other
researchers have estimated that an infant aged 1 to 2 months would ingest 0.035 mg/day
of BPA and an infant aged 4 to 6 months would ingest 0.05 mg/d of BPA (Willhite et al.
2008). A directive of the European Union has established a specific migration limit in
food of 3 mg BPA/kg food (Brotons et al. 1995). At the time the migration limit was set
by the European Union, BPA was of little concern and thus given a higher tolerance
limit. However, concerns about BPA’s toxicity have been heightened because BPA was
33
shown to be estrogenic (Krishnan et al. 1993), and have a chemical structure similar to
that of the potent synthetic estrogen DES (Roy et al. 1997; Schonfelder et al. 2002).
BPA has been detected in virtually everyone in the United States, Europe, and
Asia who were examined, always within the range of approximately 0.1 – 10 ng/ml in
both blood and tissue, and within the same range in urine after conjugation (vom Saal
2006). BPA is rapidly metabolized suggesting that continuous human exposure to this
ubiquitous chemical must be occurring throughout the world. Findings published by the
CDC from the 2003 – 2004 National Health and Nutrition Examination Survey revealed
that 93% of people in the United States had detectable concentrations of BPA in their
urine. Surprisingly, concentrations in children were significantly higher than
concentrations in adolescents, who had higher concentrations than adults (Calafat et al.
2008). Human exposure to BPA occurs through multiple routes. However, oral exposure
is considered the major route of exposure. Oral exposure occurs due to the leaching of
BPA from polycarbonate containers and from the plastic linings of cans containing food
and beverages. BPA is also detected in indoor air primarily associated with dust, which
indicates exposure can occur through inhalation. BPA is also found in streams and rivers,
and leaches from landfills, suggesting that BPA is a common contaminant of water used
for drinking and bathing (Taylor et al. 2008).
The Children’s Total Exposure to Persistent Pesticides and Other Persistent
Organic Pollutants study conducted in 2000 and 2001 in the states of North Carolina and
Ohio, investigated the exposures of 257 preschool children and their primary caregivers
to a variety of anthropogenic organic chemicals that were likely to be found at low
concentrations in their everyday environments (Wilson et al. 2004). In the study
34
conducted by Wilson and collegues (2004) BPA was detected in > 50% of indoor air,
hand wipe, solid food, and liquid food samples, and the route of exposure was
predominantly through dietary ingestion (99%).
BPA is absorbed from the gut where it is conjugated by enterocytes and
hepatocytes to its glucuronide. BPA glucuronide is the major BPA metabolite and has
little or no estrogenic activity (Volkel et al. 2002). BPA is conjugated in the liver by an
isoform of UDP-glucuronosyltransferase namely UGT2B1 (Yokota et al. 1999). BPA is
also conjugated to BPA sulfate by phenol sulfotransferases found in the liver (Suiko et al.
2000; Ye et al. 2005); sulfation of BPA abolishes its estrogenetic activity (Shimizu et al.
2002). It is not yet known what proportion of BPA is metabolized to BPA glucuonide
and BPA sulfate. BPA achieves maximum circulating concentrations within 12 minutes
of ingestion, and its elimination half life is 21 hours (Yoo et al. 2000; Yoo et al. 2001).
BPA is excreted primarily in the urine in adult humans and feces in adult rodents
(Vandenberg et al. 2007). Between birth and weaning in rodents, there is typically about
a 10-fold increase in the rate at which chemicals such as BPA and DES are metabolized
to inactive conjugates (Fischer and Weissinger 1972; Matsumoto et al. 2002). However,
it has also been shown that testosterone reduces the metabolism of BPA in rats (Shibata
et al. 2002; Takeuchi et al. 2006). Not all BPA ingested is conjugated by the liver (Zalko
et al. 2003). In rodents, conjugated BPA is deconjugated by glucuronidases in the lower
intestine and colon (Sakamoto et al. 2002).
At a young age infants may be exposed to both BPA and genistein in high
quantities. The purpose of the current study is to determine if exposure to these estrogens
35
individually and concurrently can cause temporary and/or permanent alterations to the
male reproductive system.
1.3.B. Binding Affinity
Sir Charles Edward Dodds reported that BPA mimicked the activity of the
hormone estradiol in 1936 (Dodds and Lawson 1936), making BPA one of the first
chemicals discovered to possess estrogenic properties. Dodds also noted that BPA has a
chemical structure similar to that of the estrogenic drug DES, which was synthesized by
Dodds a few years after the initial report concerning BPAs estrogenic properties (Dodds
and Lawson 1936). Concerns about BPAs estrogenic properties stem from the fact that it
has similar chemical structure to DES. It is feared that BPA may be capable of inducing
reproductive changes similar to those induced in male offspring from mothers treated
with DES. These concerns have been substantiated by the finding that the toxicity of
BPA mainly affects developmental processes and the reproductive system (Miao et al.
2008).
The endocrine disrupting potential of BPA is carried out through its ability to bind
both nuclear ER and  (Krishnan et al. 1993; Soto et al. 1997; Kuiper. et al. 1998) and
plasma membrane-bound estrogen receptors (Wozniak et al. 2005). However, BPA
displays a 10-fold higher binding affinity for ER than ER(Gould et al. 1998; Kuiper et
al. 1998; Welshons et al. 2006). BPA is generally classified as a weak estrogen because
it was found to be about 1,000 - 10,000 times less potent than E2 (Krishnan et al. 1993,
Kuiper et al. 1998; Sohoni and Sumpter 1998; Welshons et al. 2006). However, results
from recent studies have revealed a variety of pathways through which BPA is capable of
36
eliciting cellular responses at very low concentrations (Welshons et al. 2006). Several
membrane steroid receptors have been described, including a membrane-bound form of
ER (mER) that is similar but not identical to the nuclear ER (Powell et al. 2001;
Watson et al. 2007) and a transmembrane ER called G protein-coupled receptor 30
(GPR30) (Thomas and Dong 2006). BPA has been shown to bind to both mER and
GPR30, and studies have determined that these membrane-bound receptors are capable of
nongenomic steroid actions (Watson et al. 2005; Thomas and Dong 2006; Watson et al.
2007). Information regarding BPA’s ability to bind membrane-bound receptors has led
some researchers to suggest that BPA may have the same efficacy and potency as 
(Welshons et al. 2003; Watson et al. 2005; Wetherill et al. 2007). It is known that when
estrodiol binds ER, the ER regulates the rate of gene transcription through its association
with coregulators. However, when BPA binds ER a unique conformation of ER is
produced altering ER’s interaction with coregulators.
There is some evidence that BPA binds to the thyroid hormone receptor, acting as
a thyroid hormone antagonist by preventing the binding of T3. One study found the
affinity of BPA for the thyroid hormone receptor several fold lower than its affinity for
the ERs (Moriyama et al. 2002). However, other studies have been unable to duplicate
these results, finding that BPA does not competitively inhibit the binding of labeled T3 to
the thyroid hormone receptor or induce thyroid hormone-dependent production of growth
hormone in GH3 cells (Kitamura et al. 2002; Kitamura et al. 2005).
The antiandrogenic properties of BPA are still disputed. Using a competitive
binding assay with labeled dihyroxytestosterone and a yeast reporter assay, antiandogenic
activity of BPA was detected in the 10-5 to 10-7 M range, making BPA as potent as the
37
antiandrogen flutamide (Sohoni and Sumpter 1998). However other studies have not
been able to demonstrate any antagonistic activity (Gaido et al. 2000; Sun et al. 2006;
Kruger et al. 2008).
BPA is also capable of binding the aryl hydrocarbon receptor (AhR) (Kruger et al.
2008), a ligand-dependent transcription factor present in almost every tissue. AhR is
thought to be activated through many chemicals with diverse structures and may mediate
the toxic and/ or biological affects of these chemicals. Although AhR has been
implicated in several signal transduction pathways, the effects of BPA binding to AhR
remain unknown at this time. AhR can cross-talk with other receptors including ERs and
the androgen receptor, indicating endocrine-related endpoints may be affected by its
activation (Pocar et al. 2005).
It has been found that many estrogenic endocrine disrupting chemicals, including
BPA, show reduced binding to plasma proteins and thus exhibit an elevated free
concentration in blood relative to estradiol (Nagel et al. 1997; Nagel et al. 1999).
Chemicals that can bypass the barriers created by plasma binding proteins (which
regulate the free fraction of natural or manmade estrogens) have a greater biological
activity and thus pose a greater hazard than would be predicted if this was not taken into
account (vom Saal et al. 2005).
1.3.C. Reproductive System Effects
It was discovered in 1936 that bisphenol A could stimulate the growth of the
rodent uterus, which is an indication of estrogen action (Dodds and Lawson 1936).
38
Although the estrogenic action of BPA has been available for many years, recent
concerns for a potential relationship between BPA and negative trends in human health
have spurred research on BPA. The human health concerns include increases in
abnormal penile/urethra development in males, early sexual maturation in females, an
increase in neurobehavioral problems such as attention deficit hyperactivity disorder and
autism, an increase in childhood and adult obesity and type 2 diabetes, a regional
decrease in sperm count, and an increase in hormonally mediated cancers, such as
prostate and breast cancers (vom Saal et al. 2007). A recent study showed a correlation
between urinary BPA concentrations and the risk of heart disease, type 2 diabetes, and
abnormal liver enzymes (Lang et al. 2008).
Bisphenol A has been tested in male fertility studies in laboratory animals. Some
studies have reported no affects on the reproductive function of male offspring following
maternal exposure (Ashby et al. 1999; Cagen et al. 1999), others have reported that
exposure to low doses of BPA causes reproductive toxic effects (Nagel et al. 1997; vom
Saal et al. 1998). The reported reproductive toxic effects include increases in murine
prostate gland weight at 6 months of age after exposure in utero to low dose
concentrations (2 and 20 g/kg/day) (Nagel et al. 1997), and reduced sperm efficiency
(daily sperm production per gram of testes) in a subset of BPA animals from which
enlarge prostates had been observed (vom Saal et al. 1998). Another study has shown
that doses of BPA between 0.2 and 20 g/kg/day given to rats and mice resulted in a
significant decrease in daily sperm production and fertility (vom Saal et al. 1998; AlHiyasat et al. 2002; Chitra et al. 2003). Some researchers have seen transient affects of
injection of pups with BPA. Atanassova (Atanassova et al. 2000) treated rats neonatally
39
with a subcutaneous injection of 0.5 mg bisphenol A or vehicle from days 2 – 12, while
maintained on a standard soy-containing diet. Testis weight, seminiferous tubule lumen
formation, the germ cell apoptotic index (apoptotic/viable germ cell nuclear volume), and
spermatocyte nuclear volume per unit Sertoli cell nuclear volume were used to
characterize pubertal spermatogenesis. Treatment with BPA significantly increased
spermatocytes nuclear volume per unit Sertoli cell and some of the other aspects of
pubertal spermatogenesis, such as advanced lumen formation. In general, plasma FSH
concentrations in the treatment groups changed in parallel to the spermatogenic changes
(reduced when pubertal spermatogenesis retarded, increased when pubertal
spermatogenesis advanced). By day 25, although the changes in FSH concentrations
largely persisted, all of the stimulatory affects were no longer evident. In adulthood
animals treated with BPA had normal or increased testis weights and exhibited
reasonably normal mating/fertility. These mixed results do not allow for a clear
understanding of the effects of BPA treatment.
Recently regulatory agencies in the United States and the European Union have
decided that BPA was safe at current levels of human exposure. Their decision was
based on a review of all studies conducted using Good Laboratory Practices (GLP). GLP
is a federal rule for conducting research on the health affects or safety testing of drugs or
chemicals submitted by private research companies for regulatory purposes. The two
studies that were identified by both the U.S. FDA and the European Food Safety
Authority as central to the decision to declare BPA as safe at current human exposure
levels were Tyl el al. (2002; 2008). Tyl et. al., ( 2002; 2008) determined the noobservable-adverse-effect level (NOAEL) of BPA by conducting generational studies in
40
both mice and rats. In the mouse study, these researchers determined that in CD-1-inbred
mice the NOAEL dose for reproductive toxicity is 50 mg/kg/day, and the NOAEL for
systemic toxicity is 5 mg/kg/day.
The U.S. EPA calculated a reference dose (RfD) of 50 g/kg/day for BPA from
the lowest-observable-adverse-effect level using a safety factor of 1000. Three safety
factors of 10-fold each were applied to account for the following: human risk estimated
from animal studies; variability within human populations; and extrapolation for
subchronic to chronic exposures (Welshons et al. 2003). A reference dose is typically
calculated using the NOAEL, but the reference dose for BPA was calculated using the
LOAEL because a NOAEL had not been determined and adverse responses were
detected even at the lowest dose administered (Welshons et al. 2003).
For many years toxicologists have relied on the principle that “the dose makes the
poison,” implying that higher doses were expected to cause greater harm. Thus, effects
that are not seen at high doses are not expected at low doses. The threshold model is
used most often for risk assessment of noncarcinogens (Welshons et al. 2003). The
threshold model identifies a “safe” dose by assessing different doses of a chemical until
the NOAEL is determined. In contrast to the threshold model, multiple studies have
found that some hormones exhibit a biphasic dose response for different endpoints
(Welshons et al. 2003; Vandenberg et al. 2006). The U-shaped and inverted U-shaped
dose response curves are considered “nonmonotonic.” U-shaped nonmonotonic dose
responses curves indicate high responses at low and at high concentrations of exposure,
whereas inverted U-shaped curves indicate greatest response at intermediate doses
(Conolly and Lutz 2004).
41
Much controversy exists around the correct NOAEL dose of BPA. It has been
speculated by some researchers that BPA exhibits a nonmonotonic dose response curve,
indicating that high and low doses of BPA have a different affect than medium doses.
These researchers argue that the dose chosen as the NOAEL by the EPA may be too large
because doses below the NOAEL were not tested in the study (Willhite et al. 2008).
42
CHAPTER 2: NEONATAL EXPOSURE TO ENDOCRINE DISRUPTORS
2.1: INTRODUCTION
Genistein is an isoflavone found in high concentrations in soybeans and soyprotein foods and possesses weak estrogenic activity and thus may alter male
reproductive development and/or function. The relative binding affinity of genistein to
ER has previously been reported as 0.9% of E2 (Shutt and Cox 1972). Plasma genistein
concentrations in 4-month-old human infants fed soy infant formula are 2.53 M, a value
significantly higher than in infants fed either cow’s milk (0.01 M) or human breast milk
(0.01 M).
Male reproductive organs including the testis are vulnerable to estrogenic
compounds. Neonatal exposure to exogenous steroid hormones such as DES, which is
well known as a potent synthetic estrogen, reportedly causes marked atrophy of the
testes, testicular carcinoma, and uterine adenocarcinoma (Arai et al. 1983; Newbold et al.
1984; 2002; Visser et al. 1998; McLachlan et al. 2001).
Bisphenol A is an estrogenic endocrine-disrupting monomer used in the
manufacture of polycarbonate plastic products, including many types of food and
beverage containers such as baby bottles. BPA is also used in the manufacture of epoxy
resins that line metal food and beverage cans such as infant formula cans. It has been
known since 1935 that BPA possesses estrogenic properties and has a similar chemical
structure to DES. Incomplete polymerization during the manufacture of polycarbonate
plastics and epoxy resins allows BPA to leach (Brotons et al. 1995; Biles et al. 1997;
Biles et al. 1997; Carey 2003; Howdeshell et al. 2003). Concerns over the use of BPA
43
have increased with the detection of BPA in human fetal umbilical cord blood
(Schonfelder et al. 2002) within the range known to cause increases in prostate gland
weight in rats and mice (Nagel et al. 1997).
The discrepancies between the high concentrations of endocrine disruptors that
are needed to elicit effects in laboratory assays and their low concentrations in the
environment have lent credence to the belief that risks to human health or wildlife are
negligible (Safe 1995). However, a matter that complicates studying the affect of
endocrine disruptors is the reality that individuals are exposed to multiple endocrine
disruptors at one time. The low concentrations of endocrine disruptors in the
environment and in human tissues have fueled the expectation that synergisms between
estrogenic chemicals may exist (Kortenkamp and Altenburger 1998). Although the
successful demonstration of synergism will help to alleviate some of these beliefs of the
negligible affect of endocrine disruptors, combinations of estrogenic compounds have not
been adequately studied. It is possible that combinations of estrogenic compounds can
result in effects from synergism to antagonism. Synergism or antagonism are generally
identified as outcomes that exceed, or fall short of, responses expected from additive
interactions of the individual mixture components. A widely used method of predicting
additive effects is based on the expectation that the effects of a mixture should be the
arithmetic sum of the effects of its individual components. If we look at a human infant
being fed soy-infant formula we find that in vivo BPA is competing for binding to ERs
with the endogenous estrogen, E2 and with genistein from soy infant formula. BPA has a
relative binding affinity for ER of 0.008% (Blair et al. 2000), whereas Genistein’s
relative binding affinity is 0.9% (Shutt and Cox 1972). Accordingly, if equal
44
concentrations were available, the assumed order of binding to the ERs would be E2,
genistein, and then BPA. The consequences of concurrent consumption of genistein and
BPA on the development of the male reproductive tract is not known.
The goals of the present study were to:
1. Determine whether neonatal exposure of mouse pups to serum concentrations
of genistein present in soy-fed infants produces immediate and/or long-term
male reproductive effects. It has previously been determined that dosing with
50 mg/kg genistein in 24% soy infant formula results in a serum concentration
of genistein equal to that seen in serum of human infants consuming soy
infant formula (Cimafranca et al. 2009).
2. Determine whether neonatal exposure of mouse pups to the BPA noobservable-adverse-effect-level produces immediate and/or long-term male
reproductive effects.
3. Identify the affects genistein and BPA would have on the male reproductive
system. Genistein was administered at levels that produced physiologically
relevant serum concentrations. BPA was administered in three different
doses.
I hypothesized that exposure to weakly estrogenic chemicals, such as genistein and BPA,
will cause male reproductive tract defects.
45
2.2: SPECIFIC AIMS AND HYPOTHESIS
Specific Aim 1: Determine if oral administration of genistein, at levels that produce
serum concentrations in mouse pups equal to that seen in human infants consuming soy
infant formula, has an affect on the male reproductive system.
Many studies on neonatal exposure to genistein have previously been conducted.
Previous studies have used two main routes of administration: 1. dosing dams, either
orally or by injection; 2. direct injection of the pups. These two routes of administration
do not adequately depict the exposure of human infants to genistein from soy infant
formula. Pups from dams that were dosed with genistein are exposed through lactational
transfer. Doerge and colleagues documented that genistein transfer through the milk
(Doerge et al. 2006) is an inefficient way of transferring genistein. Dosing dams with
500 ppm genistein resulted in total genistein concentrations ranging from 0.022 – 0.053
M in the serum of pups. If we assume a linear increase of serum genistein
concentrations with an increase in genistein consumption, it is estimated that 23,868 ppm
– 57,500 ppm genistein would need to be consumed by a dam to result in serum
concentrations in the pups that mimic human infant exposure from soy infant formula.
Therefore, it is impossible to dose a dam with high enough levels of genistein to obtain
physiological serum concentrations in the pups. Direct injection of the pups with
genistein bypasses first-pass metabolism. First-pass metabolism is necessary because the
liver metabolizes the ingested compound delivering only a small amount to the
circulatory system. Therefore, bypassing first-pass metabolism may result in the animal
being exposed to hyperphysiological concentrations of genistein. In the present study,
46
animals received an oral dose of genistein that resulted in serum concentrations equal to
that seen in 4-month-old human infants receiving a diet of 100% soy-protein formula
(2.53 mol/L) (Cimafranca et al. 2009). The physiological relevance of the dose of
genistein and the route of administration makes the current model to study the affects of
genistein on the male reproductive system a physiological one. I hypothesized that
exposure to the weak estrogen genistein, at levels mimicking physiological levels seen in
human infants consuming a diet of soy infant formula, will cause male reproductive tract
defects. The endpoints chosen for study were germ cell proliferation, efferent duct
epithelial cell height, anogenital distance, daily sperm production, testis weight, and
reproductive performance of male C57BL/6 mice.
Specific Aim 2: Determine if oral administration of Bisphenol A (BPA) has an affect on
the male reproductive system.
Administration of the estrogenic compound BPA is thought to have adverse
effects on development and reproduction. Previously the no-observable-adverse-effectlevel (NOAEL) for BPA has been estimated as 50 mg/kg/day, through maternal dosing
(Tyl et al. 2002; Tyl et al. 2008). I hypothesized that oral administration of 50 mg/kg/day
of BPA will cause male reproductive tract defects. The endpoints chosen for study were
germ cell proliferation, efferent duct epithelial cell height, anogenital distance, daily
sperm production, testis weight, and reproductive performance of male C57BL/6 mice.
47
Specific Aim 3: Determine if concurrent neonatal exposure to genistein and the NOAEL
of BPA has a synergistic affect, thus increasing the perturbations seen on the male
reproductive system.
It has recently been observed that a mixture of estrogens can cause estrogenic
effects in vitro despite the individual components being present below their respective
NOAELs (Silva et al. 2002). The ability of mixtures of estrogens, below their respective
NOAELs, to cause estrogenic effects indicates that they may cause estrogenic effects in
vivo as well. I hypothesized that neonatal exposure of male C57BL/6 mice to
combinations of genistein and BPA at the NOAEL of BPA and below will alter male
reproductive development. The endpoints chosen for study were germ cell proliferation,
efferent duct epithelial cell height, anogenital distance, daily sperm production, testis
weights, and reproductive performance. Three different doses of BPA in combination
with 50 mg/kg genistein were chosen (BPA: 0.5, 5, 50 mg/kg). The doses of BPA were
chosen because 50 mg/kg/day is the NOAEL for reproductive/developmental toxicity, 5
mg/kg is the NOAEL for systemic toxicity, and 0.5 mg/kg is a ten fold lower dose than
the systemic NOAEL (Tyl et al. 2002; Tyl et al. 2008). Since the late 1990s, a large
volume of research has been generated suggesting a possible ‘low’ dose affect for weakly
estrogenic environmental contaminants, such as BPA. The National Toxicology Program
(NTP) defines ‘low’ dose for BPA as ≤ 5 mg/kg bw/day (Melnick et al. 2002). The 0.5
mg/kg dose is less than the defined ‘low’ dose and may give different results from what is
seen at the 50mg/kg and 5 mg/kg dose.
48
2.3: MATERIALS AND METHODS
2.3.A. Mice
Breeder C57BL/6 animals were purchased from Harlan. These animals were bred
to produce litters. Animals were housed in ventilated cages with 1/8 inch corn cob
bedding, with a light: dark cycle of 12h: 12h. Breeder animals were fed Harlan 8604
feed. Dams at day of birth were switched to the phytoestrogen-free 2016 diet from
Harlan. Pups remained on the 2016 diet for the remainder of the experiment.
2.3.B. Chemicals
The vehicle was an emulsion of 24% soy infant formula (Similac Isomil
Advanced soy formula with iron) with 0.01% corn oil. Genistein was a kind gift from
Dr. W. Helferich (Department of Food Sciences and Human Nutrition, University of
Illinois). The genistein stock is a suspension and was delivered in vehicle at 25 mg/L of
emulsion. Soy infant formula was used as a vehicle for BPA treatment to reduce
variability when comparing results from the combined dosing of genistein and BPA.
BPA was purchased from Sigma - Aldrich (St. Louis, MO). The BPA stock is a
suspension and was delivered in vehicle at 25 mg/L of emulsion. The BPA and
genistein stock was made in three separate concentrations. The concentrations were
genistein (25 mg/l) and BPA (25 mg/l), genistein (25 mg/l) and BPA (2.5 mg/l), and
genistein (25 mg/l) and BPA (0.25 mg/l). All stocks were prepared once a week in 5
ml Eppendorf tubes and stored at 4° C. The DES stock was purchased from Sigma Aldrich.
49
2.3.C. Dosing
On the day of birth (postnatal day 1, PND 1), litters were adjusted to eight pups. Pups
were dosed once daily from PND 1 – 5. Each pup was weighed and dosed with vehicle,
50 mg/kg genistein, 50 mg/kg BPA, 50 mg/kg genistein and 0.5 mg/kg BPA, 50 mg/kg
genistein and 5 mg/kg BPA, or 50 mg/kg genistein and 50 mg/kg BPA at 2l per gram of
body weight. Some pups were dosed with 1ng/g DES in olive oil, at 10l per gram of
body weight. Genistein and BPA stock are not soluble and settle quickly; therefore, the
solutions were vortexed 5-7 seconds immediately before each use. Eppendorf tubes with
dosing material were warmed in a water bath in order to enhance the pup’s suckling
reflex. The same 20l pipette was used to measure out the appropriate dose for all
experimental animals. To administer the treatment the tip was placed in the mouth of the
pup and the liquid was slowly dispensed to the pup. Care was taken to ensure the pup
swallowed all of the suspension, and that some was not displaced from the mouth.
2.3.D. Germ Cell Proliferation
The detection of proliferating germ cells was performed through the co-immunolocalization of a nuclear marker of proliferation (Ki67) and a nuclear marker of germ
cells (germ cell nuclear antigen, GCNA). Testis samples were collected at PND 5 and
fixed overnight in neutral buffered formalin. Fixed testis samples were dehydrated in
increasing ethanol concentrations, clarified in xylene and embedded in paraffin wax.
Five-micrometers-thick sections were de-paraffinized in xylene, re-hydrated through
graded ethanol solutions. Antigen retrieval was performed by boiling the slides for 10
50
minutes in 0.01 M citrate buffer (pH 6). Slides were allowed to cool for 20 minutes and
then rinsed in phosphate buffer saline 0.01 M (PBS; 8.5g/L NaCl, 2.17g/L Na2HPO47H2O, 0.26g/L NaH2PO4-H2O, pH=7.4). A permeabilization step was also performed.
Slides were incubated in PBS-Triton X-100 (0.1%) for 20 minutes at room temperature
and then rinsed in PBS. The sections were then incubated for 30 minutes in blocking
buffer (PBS-BSA 3%, tween 0.1%) to block non-specific binding sites for
immunoglobulins. The sections were then incubated overnight at 4 °C in a moist
chamber with monoclonal antibodies to Ki67 (Abcam, Cambridge, MA, USA) diluted
1:1000 in blocking buffer, and GCNA (a gift from Dr. Enders, University of Kansas
Medical Center) diluted 1:5 in blocking buffer. After rinsing for 15 minutes in PBS plus
Tween 0.1%, the slides were incubated under light protection for 2 h with Tritc goat antirat IgM (Southern Biotech, Birmingham, AL) and Alexa Flour 488 donkey anti-rabbit
IgG (Invitrogen, Carlsbad, CA, USA) diluted 1: 100 in blocking buffer. Slides were then
cover slipped. Photographs of slides were taken using an Olympus fluorescent
microscope. Images were analyzed using the NIH Image J software program. To
confirm the specificity of the immunostaining, control-staining procedures were carried
out by omission of the primary antibody.
2.3.E. Efferent Ductule Epithelial Cell Height
Cross-sections of efferent ducts were evaluated using the NIH Image J program.
The height of the efferent duct epithelium was measured using the length tool.
Measurements of the height were taken at right angles from the base of the cell adjacent
to the basement membrane to the luminal surface of the cell. For each animal, at least 25
51
cells were measured, with sampling from a number of different ducts, and calculating the
mean value. Efferent duct epithelial height was assessed at PND 5.
2.3.F. Anogenital Distance
Anogenital distance (AGD) is defined as the distance between the anterior edge of
the anus and the posterior edge of the genital tubercle. AGD of male mice was measured
using a digital micrometer on days 7, 21, 35, and 110. AGDs were recorded by one
person to increase precision and to control for operator variation.
2.3.G. Daily Sperm Production
To determine daily sperm production, testis were removed from adult mice,
weighed, and then homogenized in 10 ml of homogenization solution (0.05% triton X100 in 0.15 M NaCl) for three minutes. Homogenate solution was mixed with 0.4%
trypan blue and observed under the microscope. Sperm numbers were obtained by
counting using a hemocytometer.
2.3.H. Reproductive Performance
Eight-five-day old mice were housed with two adult untreated females for 3 wks
consecutively. Each week, males were placed with two different females. Females from
each breeding pair were observed for 21 days after separation or parturition, whichever
came first. The total number of pregnant females for each male was recorded.
52
2.3.I. Statistical Analysis
Data are reported as means ± SEM. The differences between the means of
vehicle treated group and the means of a treatment group was analyzed using Student’s ttest. Means were considered significantly different if P < 0.05.
2.4: RESULTS
2.4.A. Body weight
Body weights of animals treated with 50 mg/kg genistein were not significantly
different at any time point measured (Figures 4 and 5). Body weights of animals treated
with 50 mg/kg BPA were significantly different at PND 3 and 5, but not at any adult time
points measured (Figures 6 and 7). Body weights of animals treated with 50 mg/kg
genistein and 0.5 mg/kg BPA (Gen/BPA0.5) were significantly different at PND 2, 4, and
5, but not at any adult time points measured (Figures 8 and 9). Body weights of animals
treated with 50 mg/kg genistein and 5 mg/kg BPA (Gen/BPA5) were significantly
different at PND 3, 5, 28, and 84 (Figures 10 and 11). Body weights of animals treated
with 50 mg/kg genistein and 50 mg/kg BPA (Gen/BPA50) were significantly different
from vehicle-treated animals only at PND 28 (Figures 12 and 13). Body weights of
animals treated with 1 ng/g of DES had significant decreases in body weight compared to
vehicle-treated animals at all time points measured except PND 1 (Figure 14 and 15).
53
2.4.B. Germ Cell Proliferation
Germ cell proliferation at PND 5 was also assessed. Treatment with 50 mg/kg
genistein did not significantly alter germ cell proliferation (40% ± 3.1% and 48% ± 5.7%
vehicle - treated and genistein – treated, respectively; Figure 16). Animals treated with
50mg/kg BPA had an 18% increase in germ cell proliferation (40% ± 3.1% and 58% ±
4.3% vehicle - treated and BPA – treated, respectively; p < 0.05; Figure 17). Treatment
with Gen/BPA 0.5 caused a 14% increase in germ cell proliferation (40% ± 3.1% and
54% ± 4.1% vehicle - treated and Gen/BPA0.5 – treated, respectively; p < 0.05; Figure
18). Treatment with Gen/BPA5 caused a 10% increase in germ cell proliferation (40% ±
3.1% and 50% ± 2.2% vehicle - treated and Gen/BPA5 – treated, respectively; p < 0.05;
Figure 19). Gen/BPA50 treatment caused a 10% increase in germ cell proliferation (40%
± 3.1% and 50% ± 2.6% vehicle - treated and Gen/BPA50 – treated, respectively; p <
0.05; Figure 20). Treatment with 1ng/g DES caused a 12% decrease in germ cell
proliferation (40% ± 3.1% and 28% ± 2.7% vehicle - treated and DES – treated,
respectively; p < 0.05; Figure 21).
2.4.C. Efferent Ductule Epithelial Cell Height
Epithelial cell height of the efferent ducts was not significantly changed by
treatment with 50 mg/kg genistein (Figure 22), 50 mg/kg BPA (Figure 23), Gen/BPA5
(Figure 25), Gen/BPA50 (Figure 26), or 1ng/g DES (Figure 27). Treatment with
Gen/BPA0.5 caused a significant increase in effererent duct epithelial cell height
compared to vehicle-treated animals (Figure 24).
54
2.4.D. Anogenital Distance
AGD measurements for male animals were performed on PND 7, 21, 35, and 110.
Males treated with 50 mg/kg genistein had a larger AGD on PND 35 compared to
vehicle-treated males (Figure 28). Animals treated with BPA showed a significant
increase in AGD compared to vehicle-treated males on PND 7, 35, and 110 (Figure 29).
Males treated with Gen/BPA0.5 showed an increase in AGD at PND 7 compared to
vehicle-treated males (Figure 30). Males treated with Gen/BPA5 showed an increase in
AGD at PND 7 and a decrease in AGD on PND 21 compared to vehicle-treated males
(Figure 31). Males treated with Gen/BPA50 showed an increase in AGD compared to
vehicle-treated males on PND 7 and 35 (Figure 32). Males treated with DES showed a
decrease in AGD compared to vehicle-treated males at PND 21 and 110 (Figure 33).
2.4.E. Daily Sperm Production
Daily sperm production was measured at PND 35 and 110. No significant change
in daily sperm production was measured for animals treated with 50 mg/kg genistein
(PND35: 1.06 ± 0.08 x 106 vs. 0.92 ± 0.09 x 106; PND 110: 2.80 ± 0.14 x 106 vs. 3.06 ±
0.12 x 106, vehicle vs. genistein, respectively; Figure 34) or Gen/BPA5 (PND35: 1.06 ±
0.08 x 106 vs. 1.14 ± 0.10 x 106; PND 110: 2.80 ± 0.14 x 106 vs. 2.81 ± 0.19 x 106,
vehicle vs. Gen/BPA5, respectively; Figure 37). Males treated with Gen/BPA0.5 (PND
35: 1.06 ± 0.08 x 106 vs. 1.34 ± 0.15 x 106; PND 110: 2.80 ± 0.14 x 106 vs. 4.10 ± 0.30 x
106; p < 0.05; vehicle vs. Gen/BPA0.5, respectively; Figure 36) had a significant increase
in daily sperm production at PND 35. Males treated with 50 mg/kg bisphenol A (PND35:
1.06 ± 0.08 x 106 vs. 1.49 ± 0.14 x 106; PND 110: 2.80 ± 0.14 x 106 vs. 3.61 ± 0.28 x 106,
55
vehicle vs. bisphenol A, respectively; p < 0.05; Figure 35), or Gen/BPA50 (PND 35: 1.06
± 0.08 x 106 vs. 1.43 ± 0.08 x 106; PND 110: 2.80 ± 0.14 x 106 vs. 3.96 ± 0.26 x 106; p <
0.05; Figure 38) had a significant increase in daily sperm production at both PND 35 and
110. Males treated with DES had a significant decrease in daily sperm production at
PND 35 and 110 (PND35: 1.06 ± 0.08 x 106 vs. 0.74 ± 0.12 x 106; PND 110: 2.80 ± 0.14
x 106 vs. 2.00 ± 0.24 x 106, vehicle vs. DES, respectively; Figure 39).
2.4.F. Testis Weight
Testis weight was not altered from treatment with 50 mg/kg/d genistein (Figure
40), 50 mg/kg/d BPA (Figure 41), Gen/BPA0.5 (Figure 42), Gen/BPA5 (Figure 43), or
Gen/BPA50 (Figure 44) at either PND 35 or 110. A significant decrease in testis weight
was measured for treatment with DES at PND 110 (Figure 45).
2.4.G. Reproductive Performance
Reproductive performance was assessed for males from all treatment groups.
There was no significant difference in reproductive performance between vehicle treated
males and genistein (Figure 46), BPA (Figure 47), Gen/BPA0.5 (Figure 48), Gen/BPA5
(Figure 49), Gen/BPA50 (Figure 50), or DES (Figure 51) treated males. Reproductive
performance was scored as the number of pregnant females out of the total number bred
by each male.
56
Figure 4: Neonatal treatment of mouse pups with 50 mg/kg/d of genistein did not elicit a significant
change in body weight on PND 1 - 5 (n = 15). Data are shown as mean ± SEM.
Figure 5: Neonatal treatment of mouse pups with 50 mg/kg/d of genistein did not elicit a significant
change in body weight on PND 21 - 110 (n = 15). Data are shown as mean ± SEM.
57
Figure 6: Neonatal treatment of mouse pups with 50 mg/kg/d of bisphenol A elicited a significant
change in body weight on PND 3 and 5 when compared to vehicle-treated males indicated by (n =
30, p < 0.05). Data are shown as mean ± SEM.
Figure 7: Neonatal treatment of mouse pups with 50 mg/kg/d of bisphenol A did not elicit a
significant change in body weight on PND 21 - 110 (n = 10). Data are shown as mean ± SEM.
58
Figure 8: Neonatal treatment of mouse pups with 50 mg/kg/d of genistein and 0.5 mg/kg/d bisphenol
A (Gen/BPA0.5) elicited a significant change in body weight on PND 2, 4, and 5 when compared to
vehicle-treated males indicated by (n = 30, p < 0.05). Data are shown as mean ± SEM.
Figure 9: Neonatal treatment of mouse pups with 50 mg/kg/d of genistein and 0.5 mg/kg/d of
bisphenol A (Gen/BPA0.5) did not elicit a significant change in body weight from PND 21 - 110 (n =
10). Data are shown as mean ± SEM.
59
Figure 10: Neonatal treatment of mouse pups with 50 mg/kg/d of genistein and 5 mg/kg/d bisphenol
A (Gen/BPA5) elicited a significant change in body weight on PND 3 and 5 when compared to
vehicle-treated males indicated by (n = 30, p < 0.05). Data are shown as mean ± SEM.
Figure 11: Neonatal treatment of mouse pups with 50 mg/kg/d of genistein and 5 mg/kg/d of
bisphenol A (Gen/BPA5) elicited a significant change in body weight on PND 28 and 84 compared to
vehicle, indicated by (n = 10, p < 0.05). Data are shown as mean ± SEM.
60
Figure 12: Neonatal treatment of mouse pups with 50 mg/kg/d of genistein and 50 mg/kg/d bisphenol
A (Gen/BPA50) did not elicit a significant change in body weight from PND 1 - 5 when compared to
vehicle-treated males (n = 35). Data are shown as mean ± SEM.
Figure 13: Neonatal treatment of mouse pups with 50 mg/kg/d of genistein and 50 mg/kg/d of
bisphenol A (Gen/BPA50) caused a significant change in body weight on PND 28 as compared to
vehicle indicated by (n = 10, p < 0.05). Data are shown as mean ± SEM.
61
Figure 14: Neonatal treatment of mouse pups with 1ng/g/d DES treatment caused a significant
decrease in body weight compared to vehicle at all time points measured except for PND 1 of
treatment indicated by (n = 20, p < 0.05). Data are shown as mean ± SEM.
Figure 15: Neonatal treatment of mouse pups with 1ng/g/d DES treatment caused a significant
decrease in body weight compared to vehicle at all time points measured (n = 10, p < 0.05). Data are
shown as mean ± SEM.
62
Figure 16: Neonatal treatment of mouse pups with 50 mg/kg/d of genistein did not elicit a significant
change in germ cell proliferation at PND 5 (n = 10). Data are shown as mean ± SEM.
Figure 17: Neonatal treatment of mouse pups with 50 mg/kg/d of bisphenol A elicited a significant
increase in germ cell proliferation at PND 5 indicated by
mean ± SEM.
63
(n = 10, p < 0.05). Data are shown as
Figure 18: Neonatal treatment of mouse pups with 50 mg/kg/d of genistein and 0.5 mg/kg of
bisphenol A (Gen/BPA0.5) caused a significant increase in germ cell proliferation at PND 5 indicated
by
(n = 8, p < 0.05). Data are shown as mean ± SEM.
Figure 19: Neonatal treatment of mouse pups with 50 mg/kg/d of genistein and 5 mg/kg/d of
bisphenol A (Gen/BPA5) caused a significant increase in germ cell proliferation at PND 5 indicated
by
(n = 9, p < 0.05). Data are shown as mean ± SEM.
64
Figure 20: Neonatal treatment of mouse pups with 50 mg/kg/d of genistein and 50 mg/kg/d bisphenol
A (Gen/BPA50) elicited a significant increase in germ cell proliferation at PND 5 indicated by
(n = 10, p < 0.05). Data are shown as mean ± SEM.
Figure 21: Neonatal treatment of mouse pups with 1ng/g/d DES elicited a significant decrease in
germ cell proliferation compared to vehicle at PND 5 indicated by
shown as mean ± SEM.
65
(n = 3; p < 0.05). Data are
Figure 22: Neonatal treatment of mouse pups with 50 mg/kg/d of genistein did not elicit a significant
change in efferent ductule epithelial cell height at PND 5 (n = 6). Data are shown as mean ± SEM.
Figure 23: Neonatal treatment of mouse pups with 50 mg/kg/d of bisphenol A did not elicit a
significant change in efferent ductule epithelial cell height at PND 5 (n = 7). Data are shown as mean
± SEM.
66
Figure 24: Neonatal treatment of mouse pups with 50 mg/kg/d of genistein and 0.5 mg/kg/d bisphenol
A (Gen/BPA0.5) elicited a significant increase in efferent ductule epithelial cell height at PND 5
indicated by
(n = 5, p < 0.05). Data are shown as mean ± SEM.
Figure 25: Neonatal treatment of mouse pups with 50 mg/kg/d of genistein and 5 mg/kg/d bisphenol
A (Gen/BPA5) did not elicit a significant change in efferent ductule epithelial cell height at PND 5 (n
= 6). Data are shown as mean ± SEM.
67
Figure 26: Neonatal treatment of mouse pups with 50 mg/kg/d of genistein and 50 mg/kg/d bisphenol
A (Gen/BPA50) did not elicit a significant change in efferent ductule epithelial cell height at PND 5
(n = 8). Data are shown as mean ± SEM.
Figure 27: Neonatal treatment of mouse pups with 1 ng/kg/d DES did not elicit a significant change
in efferent ductule epithelial cell height at PND 5 (n = 3). Data are shown as mean ± SEM.
68
Figure 28: Neonatal treatment of mouse pups with 50 mg/kg/d of genistein caused a significant
increase in anogenital distance on PND 35 compared to vehicle indicated by
Data are shown as mean ± SEM.
(n = 20, p < 0.05).
Figure 29: Neonatal treatment of mouse pups with 50 mg/kg/d of bisphenol A caused a significant
increase in anogenital distance at PND 7, 35, and 110 compared to vehicle indicated by
p < 0.05). Data are shown as mean ± SEM.
69
(n = 20,
Figure 30: Neonatal treatment of mouse pups with 50 mg/kg/d of genistein and 0.5 mg/kg/d of
bisphenol A (Gen/BPA0.5) elicited a significant increase in anogenital distance at PND 7 compared to
vehicle indicated by
(n = 10, p < 0.05). Data are shown as mean ± SEM.
Figure 31: Neonatal treatment of mouse pups with 50 mg/kg/d of genistein and 5 mg/kg/d of
bisphenol A (Gen/BPA5) caused a significant increase in AGD at PND 7 and a significant decrease in
AGD at PND 21 compared to vehicle indicated by
SEM.
70
(n = 10, p < 0.05). Data are shown as mean ±
Figure 32: Neonatal treatment of mouse pups with 50 mg/kg/d of genistein and 50 mg/kg/d of
bisphenol A (Gen/BPA50) elicited a significant increase in anogenital distance at PND 7 and 35
compared to vehicle indicated by
(n = 10, p < 0.05). Data are shown as mean ± SEM.
Figure 33: Neonatal treatment of mouse pups with 1ng/g/d DES treatment caused a significant
decrease in anogenital distance compared to vehicle at PND 21 and PND 110 indicated by
10, p < 0.05). Data are shown as mean ± SEM.
71
(n =
Figure 34: Neonatal treatment of mouse pups with 50 mg/kg/d of genistein did not elicit a significant
change in daily sperm production (n = 10 for PND 35; n = 18 for PND 110). Data are shown as mean
± SEM.
Figure 35: Neonatal treatment of mouse pups with 50 mg/kg/d of bisphenol A caused a significant
increase in daily sperm production at PND 35 and 110 indicated by
for PND 110, p < 0.05). Data are shown as mean ± SEM.
72
(n = 10 for PND 35; n = 12
Figure 36: Neonatal treatment of mouse pups with 50 mg/kg/d of genistein and 0.5 mg/kg/d of
bisphenol A (Gen/BPA0.5) caused a significant increase in daily sperm production at PND 110
indicated by
mean ± SEM.
(n = 10 for PND 35, p = 0.1; n = 10 for PND 110, p < 0.05). Data are shown as
Figure 37: Neonatal treatment of mouse pups with 50 mg/kg/d of genistein and 5 mg/kg/d of
bisphenol A (Gen/BPA5) did not cause a significant increase in daily sperm production at PND 35
and 110 (n = 10). Data are shown as mean ± SEM.
73
Figure 38: Neonatal treatment of mouse pups with 50 mg/kg/d of genistein and 50 mg/kg/d of
bisphenol A (Gen/BPA50) caused a significant increase in daily sperm production at PND 35 and 110
indicated by
(n = 10, p < 0.05). Data are shown as mean ± SEM.
Figure 39: Neonatal treatment of mouse pups with 1ng/g/d DES treatment caused a significant
decrease in daily sperm production compared to vehicle at PND 35 and PND 110 indicated by
(n = 10, p < 0.05). Data are shown as mean ± SEM.
74
Figure 40: Neonatal treatment of mouse pups with 50 mg/kg/d of genistein did not elicit a significant
change in adult testis weight (n = 10 for PND 35; n = 18 for PND 110). Data are shown as mean ±
SEM.
Figure 41: Neonatal treatment of mouse pups with 50 mg/kg/d of bisphenol A did not elicit a
significant change in adult testis weight (n = 10 for PND 35, p = 0.06; n = 12 for PND 110). Data are
shown as mean ± SEM.
75
Figure 42: Neonatal treatment of mouse pups with 50 mg/kg/d of genistein and 0.5 mg/kg/d of
bisphenol A (Gen/BPA0.5) did not elicit a significant change in adult testis weight (n = 10 for PND
35; n = 10 for PND 110, p = 0.06). Data are shown as mean ± SEM.
Figure 43: Neonatal treatment of mouse pups with 50 mg/kg/d of genistein and 5 mg/kg of bisphenol
A (Gen/BPA5) did not elicit a significant change in adult testis weight (n = 10). Data are shown as
mean ± SEM.
76
Figure 44: Neonatal treatment of mouse pups with 50 mg/kg/d of genistein and 50 mg/kg/d of
bisphenol A (Gen/BPA50) did not elicit a significant change in adult testis weight (n = 10). Data are
shown as mean ± SEM.
Figure 45: Neonatal treatment of mouse pups with 1ng/g DES treatment caused a significant
decrease in testis weight compared to vehicle at PND 110 indicated by
are shown as mean ± SEM.
77
(n = 10, p < 0.05). Data
Figure 46: Neonatal treatment of mouse pups with 50 mg/kg/d of genistein did not elicit a significant
change in reproductive performance (n = 11). Reproductive performance was calculated as the
number pregnant out of the total number bred. Data are shown as mean ± SEM.
Figure 47: Neonatal treatment of mouse pups with 50 mg/kg/d of bisphenol A did not elicit a
significant change in reproductive performance (n = 10). Reproductive performance was calculated
as the number pregnant out of the total number bred. Data are shown as mean ± SEM.
78
Figure 48: Neonatal treatment of mouse pups with 50 mg/kg/d of genistein and 0.5 mg/kg/d of
bisphenol A (Gen/BPA0.5) did not elicit a significant change in reproductive performance (n = 11).
Reproductive performance was calculated as the number pregnant out of the total number bred.
Data are shown as mean ± SEM.
Figure 49: Neonatal treatment of mouse pups with 50 mg/kg/d of genistein and 5 mg/kg/d of
bisphenol A (Gen/BPA5) did not elicit a significant change in reproductive performance (n = 12).
Reproductive performance was calculated as the number pregnant out of the total number bred.
Data are shown as mean ± SEM.
79
Figure 50: Neonatal treatment of mouse pups with 50 mg/kg/d of genistein and 50 mg/kg/d of
bisphenol A (Gen/BPA50) did not elicit a significant change in reproductive performance (n = 14).
Reproductive performance was calculated as the number pregnant out of the total number bred.
Data are shown as mean ± SEM.
Figure 51: Neonatal treatment of mouse pups with 1ng/g of DES did not elicit a significant change in
reproductive performance (n = 10). Reproductive performance was calculated as the number
pregnant out of the total number bred. Data are shown as mean ± SEM.
2.5: DISCUSSION
The results from this study illustrate that body weight, germ cell proliferation,
anogenital distance, and daily sperm production were altered with exposure to BPA and
80
combinations of BPA and genistein. However, treatment with genistein alone did not
result in significant differences in the reproductive parameters measured when compared
to vehicle-treated animals except for an increase in anogenital distance at PND 35.
Treatment with 1ng/g DES resulted in a significant decrease in all parameters measured
except for efferent ductule epithelial cell height and reproductive performance. Males
treated with exogenous estrogens did not have a difference in reproductive performance
compared to vehicle treated males.
DES was used as a positive control in these experiments in accordance with the
National Toxicology Program Peer Review Panel that recommends DES as a positive
control in studies using estrogenic chemicals such as genistein and BPA (Program 2001).
DES has previously been shown to have a higher relative binding affinity for ERs than E2
(399% vs. 100%) (Blair et al. 2000). A study by Newbold et al. (2001) determined that
50 mg/kg/day of genistein and 1/ng/g/d DES were capable of increasing uterine weight
by a similar percentage (202% vs. 190%, genistein vs. DES). Therefore, a dose of
1ng/g/day of DES was chosen for use in this study. This DES dose would have greater
estrogenic affects than the genistein administered in this study due to route of
administration. Positive effects of DES would indicate that C57BL/6 mice are
susceptible to exogenous estrogen treatment. Therefore, if results are not seen with any
other estrogen treatment, then potency of the estrogen, or route of administration may be
the cause and the negative affects may not be attributed to sensitivity of animal or the
strain used.
Body weights of animals treated with exogenous estrogens were altered primarily
during the dosing period, except for treatment with DES, which produced lasting
81
changes. Treatment with genistein did not alter body weights at any time point measured,
consistent with previous results (Kang et al. 2002; Fielden et al. 2003; Lewis et al. 2003;
Masutomi et al. 2003; Adachi et al. 2004). Treatment with DES reduced body weights at
all time points measured except the day of birth. Decreased body weights due to DES
treatment has been shown previously (Sharpe et al. 1995; Adachi et al. 2004). The most
likely cause of decreased body weights is due to a decrease in adipocyte size and number.
It has previously been shown that administration of exogenous estrogen is capable of
decreasing adipocyte number (Cooke and Naaz 2004; Cooke and Naaz 2005). A
decrease in body weights during the dosing period of DES-treated animals is most likely
due to the dosing method. Injection with DES is traumatic to the pups and may impair
their suckling ability. Reduced nutrition of DES animals may also be a cause for the
continued low body weights seen in adulthood.
There was an increase in germ cell proliferation with 50 mg/kg/d BPA or the
combination of genistein and BPA in PND 5 testis. At PND 5 the cells present within the
seminiferous epithelium are: 1. Sertoli cells; 2. gonocytes; and 3. primitive type A
spermatogonia (Bellve et al. 1977). The gonocytes, which are the precursors of the germ
cell lineage, proliferate until days 14-16 post coitum, depending on the strain of mice
(Sapsford 1962; Kluin and de Rooij 1981), and then are arrested in the G0/G1 phase of the
cell cycle. After birth gonocytes resume their mitotic activity and eventually give rise to
type A spermatogonia (Sapsford 1962; Kluin and de Rooij 1981). A study by
Vergouwen and colleagues (1991) illustrated that gonocyte proliferation begins at birth
and steadily increases until PND 3. At PND 1 they showed that the [3H]thymidine
labeling index was 10.4 ± 3.1% and increased to 24.1 ± 4.1% on PND 3. They were also
82
able to identify the first primitive type A spermatogonia at PND 3. The results shown by
Vergouwen and colleagues (1991) are in accordance with the results shown by Kluin and
de Rooij (1981) for Cpb-N mice. However, it was 1-2 days earlier than described by
Widmaier (1963) for ‘albino-BLUHM’ mice. A series of studies have identified that the
fetal/neonatal testis as very sensitive to estrogen exposures, raising the possibility that the
precursor cells of spermatogonial stem cells might be preferentially targeted by endocrine
disruptors (Foster 2006; Sharpe 2006). Li et al. (1997), have shown that rat gonocyte
proliferation is stimulated in vitro during the early postnatal period. They showed that E2
was capable of inducing gonocyte proliferation at a level comparable with that of
platelet-derived growth factor (PDGF) and that its effect was blocked by the estrogen
receptor antagonist, ICI 164,384. It has also been shown that exogenous estrogen
exposure is capable of up-regulating the expression of PDGF receptors (PDGFR) in
gonocytes. Immunohistochemical studies of PDGFR and  proteins revealed that both
receptors were present in testis before birth and after birth and that they were up
regulated upon treatment with estrogens in 3-day-old rats, with PDGFR increasing
dramatically in gonocytes (Thuillier et al. 2003). Saunders et al. (1998) has shown that
rat gonocytes express ER the preferential ER isoform of both genistein and bisphenol A.
Therefore, stimulation of gonocyte proliferation with BPA and the combination of
genistein and BPA may be through direct targeting of the ER receptor. Stimulation of
gonocyte proliferation may also be from up regulation of PDGF receptors. Treatment
with genistein alone was incapable of stimulating germ cell proliferation. The results
presented here are constant with other results for genistein treatment. The antiproliferative, cytotoxic and differentiation activities of genistein on some tumors, have
83
been demonstrated both in vivo and in vitro (Kanatani et al. 1993; Wei et al. 1993).
Genistein has also been demonstrated to arrest cell cycle progression at the G2-M stage
(Matsukawa et al. 1993) and to induce apoptosis in thymocytes (McCabe and Orrenius
1993) and leukemia cells (Traganos et al. 1992; Bergamaschi et al. 1993). More recently
genistein has been shown to inhibit the growth and proliferation of testicular cells, TM3
(Leydig, mouse), TM4 (Sertoli, mouse), and GC-1 spg (spermatogonia, mouse) (KumiDiaka et al. 1998). Treatment with DES was capable of decreasing germ cell
proliferation in this study. Previous studies have shown that DES decreases the number
of Sertoli cells (Fielden et al. 2002; Sharpe et al. 2003). With fewer Sertoli cells to
nurture the germ cells there may be a higher percentage of germ cells undergoing
apoptosis and a decrease in germ cell proliferation.
Efferent ductule epithelial cell height was not altered by treatment with genistein
or BPA, except for treatment with Gen/BPA0.5. These results agree with what has
previously been reported by Fisher et al. (1999). Fisher et al. found that following
injection with either 4 mg/kg/day of genistein or 0.5 mg/injection of BPA efferent duct
epithelial cell height was not affected at PND 10. However, efferent duct epithelial cell
height was reduced at PND 18 and/or 25. This reduction in epithelial cell height was
only transient, because changes in efferent duct epithelial cell height were not seen in
animals that were followed through to 35 days and/or adulthood (Fisher et al. 1999).
These researchers and others have not shown an increase in efferent duct epithelial cell
height from exogenous estrogen treatment. It is possible that the increase in efferent
ductule epithelial cell height seen in animals treated with Gen/BPA0.5 is only transient,
and caused by the stimulatory effects of the exogenous estrogen. Therefore, a
84
reevaluation of the efferent duct epithelial cell height of animals in this study should be
reevaluated at multiple different time points. Previous studies have shown that neonatal
treatment of male rats with relatively high doses of DES (10g) causes a range of
reproductive tract abnormalities, including overgrowth/ distension of the rete testis and
reduced epithelial cell height in the efferent ducts, epididymis, vas deferens, seminal
vesicles, and prostate coincident with relative overgrowth of stromal tissue at many of
these sites (Atanassova et al. 1999; Atanassova et al. 2001; McKinnell et al. 2001;
Williams et al. 2001a; Williams et al. 2001b). All of these DES induced changes are
associated with a loss of AR in the effected tissues as well as in Sertoli cells (McKinnell
et al. 2001; Williams et al. 2001). Neonatal treatment with a 100-fold lower dose of DES
(0.1 g) did not induce these changes (McKinnell et al. 2001; Williams et al. 2001;
Williams et al. 2001). The alteration of the androgen: estrogen (low androgen: high
estrogen) balance may be one of the causes of these abnormalities. Our data are
consistent with these results showing that a dose of 1ng DES did not result in a decrease
of efferent duct epithelial cell height.
Anogenital distances of males treated with genistein was increased at PND 35 but
not at any of the other time points measured. Increases in AGD for animals treated with
BPA and the combination of Gen/BPA were also seen. Previous studies have shown that
genistein does not alter AGD (Kang et al. 2002; Fielden et al. 2003; Lewis et al. 2003;
Masutomi et al. 2003). However, these studies have not measured AGD at PND 35. The
increase in AGD seen at 35 days may be due to the onset of spermatogenesis. Anogenital
distance is an androgen sensitive target. Intratesticular levels of testosterone must be
higher than blood levels of testosterone for successful spermatogenesis. So as
85
spermatogenesis begins there may be a rise in intratesticular levels of testosterone which
cause a rise in blood levels of testosterone. The increased testosterone levels may cause a
transient increase in AGD. It has been shown that mice treated with BPA have increased
levels of testosterone (Sharpe et al. 2003). Sharpe et al, hypothesize that the increased
levels of testosterone may be due to the induction of supranormal FSH levels
(Atanassova et al. 2000) leading to increased responsiveness to endogenous LH (Sharpe
1993). These increased levels of testosterone may account for the increase in anogenital
distance seen in animals treated with BPA in the current study. Similarly, the study by
Sharpe et al., ( 2003) showed that treatment with DES caused a decrease in testosterone
concentrations which would indicate a decrease in anogenital distance for these animals.
The decreases in testosterone concentrations in DES treated animals may be due to a
decrease in Leydig cell volume per testis (Sharpe et al. 2003). However, Leydig cell
volume appears to even out so that in adulthood the numbers are comparable between
DES and control animals. Although the Leydig cell volume per testis is comparable DES
treated animals continue to have decreased testosterone levels (Sharpe et al. 2003). DES
treatment in the current study appears to decrease anogenital distance, which may be a
result of decreased testosterone concentrations because of decreased Leydig cell volume
per testis.
Treatment with genistein did not alter daily sperm production, mostlikely due to
genistein’s inability to cause an increase in germ cell proliferation. Previous studies have
also seen no change in sperm numbers following genistein treatment (Nagao et al. 2001;
Shibayama et al. 2001; Kang et al. 2002; Fielden et al. 2003). Atanassova et al. have
shown that BPA causes a stimulatory effect on the first wave of spermatogenesis at
86
puberty (Atanassova et al. 2000). In their study they found that FSH levels mirrored the
effects seen in spermatogenesis, i.e. higher FSH levels were associated with increased
spermatogenesis and lower FSH levels were associated with decreased spermatogenesis.
Results from the current study show an increase of spermatogenesis due to treatment with
BPA and the combination of Gen/BPA0.5 or Gen/BPA50. It is possible that the increase
in spermatogenesis is due to an increase in FSH levels. Another explanation for the
increase in daily sperm production may be due to a direct effect of BPA on Sertoli and/ or
germ cells. Sertoli cells, spermatogonia, and most spermatocytes express ER at all
stages of normal pubertal development (Saunders et al. 1997; Saunders et al. 1998; van
Pelt et al. 1999), and there are several lines of evidence that suggest direct effects of
neonatal estrogen treatment on Sertoli cells (Sharpe et al. 1998; Atanassova et al. 1999).
There are also data in the literature for a number of species that demonstrate stimulatory
effects of estrogens on gonocytes (Li et al. 1997), spermatogonial stem cells (Miura et al.
1999), and spermatogonia (Kula 1988; Slowikowska-Hilczer and Kula 1994) as well as
precocious activation of spermatogenesis in the human testis associated with an estrogensecreting tumor (Kula et al. 1996). Wistuba and colleagues recently demonstrated that
BPA was capable of increasing Sertoli cell numbers (Wistuba et al. 2003). However,
they did not see an increase in spermatogenesis (Wistuba et al. 2003).
Previous research has shown that treatment with DES was capable of reducing
Sertoli cell number (Fielden et al. 2002; Sharpe et al. 2003). The Sertoli cell is
responsible for supporting germ cells throughout spermatogenesis. Therefore, it would
seem likely that if there were fewer Sertoli cells in DES treated animals there would also
be fewer germ cells. The reduction of both Sertoli and germ cell number is expected to
87
cause a decrease in testis weight and daily sperm production. Studies by Majdic et al
(1996; 1997) have demonstrated that in utero exposure to DES alters the expression of
SF-1 and Cyp17 in Leydig cells of fetal rats, suggesting that DES effects on
spermatogenesis are at least in part a result of inhibition of steroidogenesis. Early and
latent alterations in the expression of genes involved in estrogen signaling (ER),
steroidogenesis (SF-1, Star, Cyp17, Cyp11a, and SR-B1), lysosomal function (LGP85
and Psap), and regulation of testicular development (Tr2-11, Inhbc, and Hoxa10) were
observed in prepubertal and adult mice, even long after the cessation of DES exposure
(Fielden et al. 2002). Decreased expression of these genes may contribute, together or in
part, to the decrease in epididymal sperm count and sperm fertilizing ability as a result of
decreased testicular synthesis of C19 steroids or suppression of ER signaling in the
Leydig cells, both of which are essential for fertility (Couse et al. 2001). The findings in
this work for DES treatment are consistent with what has been previously reported
(Sharpe et al. 1995; 2003 Shibayama et al. 2001; Fielden et al. 2002). The most likely
cause for the decrease in daily sperm production of males treated with DES is due to a
decrease in number of Sertoli cells. To identify the true cause of a decrease in daily
sperm production the number of Sertoli cells from animals in this experiment should be
counted. The Sertoli cell is responsible for nurturing germ cells and aiding them in their
progression to spermatozoa. Therefore, any alteration in Sertoli cell numbers/function
would result in changes in spermatogenic output.
Testis weight of animals treated with genistein was also not altered. The majority
of the cells within the testis are germ cells. Therefore a change in germ cell number is
expected to lead to an increase/decrease in testis weight (Atanassova et al. 2000).
88
Therefore, genistein’s inability to cause an increase in germ cell proliferation is
consistent with no increase in testis weight. Previous research has also shown that
genistein does not alter testis weight (Fisher et al. 1999; Atanassova et al. 2000; Nagao et
al. 2001; Shibayama et al. 2001; Kang et al. 2002; Fielden et al. 2003; Masutomi et al.
2003; Adachi et al. 2004). Neonatal treatment with BPA or the combination of genistein
and BPA showed a trend toward an increase in this study. However, no significant
increases in testis weight were seen. Previous reports have shown that treatment with
BPA causes a significant increase in testis weight (Fisher et al. 1999; Atanassova et al.
2000). Animals treated with DES in this study had a significant decrease in testis weight.
Neonatal treatment with DES has previously been shown to decrease testis weight
(Sharpe et al. 1995; 2003; Fisher et al. 1999; Atanassova et al. 2000; Couse et al. 2001;
Shibayama et al. 2001; Fielden et al. 2002; Adachi et al. 2004), which is in accordance
with the results seen in this study.
Treatment with genistein did not alter reproductive performance, defined as the
number of pregnant females out of the total number of females bred. Spermatogenesis in
male mice produces greater numbers of sperm than are needed for maximual fertility.
Therefore, a large increase in daily sperm production would be needed to see an increase
in fertility. Genistein did not alter daily sperm production and therefore had no change in
fertility. The fertility results seen for genistein are consistent with previous research
(Nagao et al. 2001). Similarly treatment with BPA or the combination of BPA and
genistein did not alter reproductive performance. Reproductive performance of males
exposed to DES in utero has previously been shown to decrease fertility (Fielden et al.
2002). Abnormal sperm morphology and motility, lesions in the reproductive tract,
89
abnormal secretions, and inflammation appear to be related to the decreased fertility
(Newbold and McLachlan 1985). The results of this study illustrate that DES treatment
decreases fertility though it was not statistically significant.
Previous studies have shown that treatment with BPA results in a nonmonotonic
dose response curve, indicating that high and low doses have similar effects, whereas
mid-range doses have effects either above or below the high and low doses (Gupta 2000;
Markey et al. 2001; Akingbemi et al. 2004). The results from this study indicate that low
and high doses of BPA in combination with genistein have the greatest affect on daily
sperm production. Males treated with the combination of 50 mg/kg genistein and 5
mg/kg BPA do not have an increase in daily sperm production compared to the vehicletreated males (Figure 37). However, males treated with the combination of 50 mg/kg
genistein and 0.5 mg/kg BPA or 50 mg/kg genistein and 50 mg/kg BPA showed
increased daily sperm production compared to vehicle-treated males (Figures 36 and 38).
These results indicate that BPA has a U-shaped nonmonotonic dose response curve for
daily sperm production (Figure 52). A study conducted by Conolly and Lutz (2004)
sought to identify a mechanistic explanation for nonmonotonic dose response curves.
They found that nonmonotonic dose response curves can be explained by straightforward
biochemical processes or postulated on the basis of reasonable biological hypotheses.
Other mechanisms that they propose are overshooting homeostatic feedback control or
shifts in immune responses (Conolly and Lutz 2004).
The current study utilizes a model for dosing exogenous estrogens that has not
previously been employed. The current model more closely mimics human infant
genistein and BPA exposure, and accounts for first pass metabolism. My data suggest
90
that neonatal exposure to BPA and the combination of genistein and BPA leads to
changes in germ cell proliferation in the neonate which ultimately lead to increased daily
sperm production in the adult. However, the data also demonstrate that exposure to
genistein does not have effects on neonatal germ cell proliferation and thus does not alter
the daily sperm production in adulthood. The data presented also illustrate that C57BL/6
mice are susceptible to exogenous estrogen treatment, indicating that the negative results
seen from genistein treatment are not due to the strain of mouse, but rather the inability of
the treatment to cause estrogenic effects.
Irrespective of whether the increase in germ cell proliferation and daily sperm
production caused by neonatal exposure to BPA or genistein and BPA results from their
estrogenicity, the key question is whether these effects have relevance to humans.
Animal studies relevance to humans is a complex issue which requires detailed doseresponse studies and measurement of the pharmacokinetics of BPA in serum of male
mice. It would be appropriate to consider whether the concentration of exposure of mice
to BPA bears any relationship to the equivalent concentration of human exposure.
Currently the concentration of BPA in serum of bottle-fed human infants is not known.
To more adequately mimic human infant exposure it is necessary to know the serum BPA
concentrations, and to determine a dose of BPA treatment for mice that mimics these
human serum concentrations.
91
Figure 52: Treatment with genistein and BPA at three different doses of BPA reveals a
nonmonotonic dose response curve for daily sperm production. Males treated with 50 mg/kg
genistein and 50 mg/kg bisphenol A (Gen/BPA50) have an increase in daily sperm production when
compared to males treated with 50 mg/kg genistein and 5 mg/kg bisphenol A (Gen/BPA5) at both
PND 35 and 110. Males treated with 50 mg/kg genistein and 0.5 mg/kg bisphenol A (Gen/BPA0.5)
have a significant increase in daily sperm production compared to males treated with Gen/BPA5 at
PND 110. Data are shown as mean ± SEM. Significance indicated by a,b,c. Treatments with the
same letter are not significantly different. Treatments with different letters at a particular age
indicate a significant difference.
92
CHAPTER 3: SUMMARY AND CONCLUSION
In the current study mouse pups were dosed orally with concentrations of
genistein that produced biologically relevant serum concentrations, BPA at the
reproductive NOAEL identified by Tyl et al. (2002; 2008), or with a combination of
genistein and BPA. These compounds were administered by a method that takes
advantage of the natural suckling ability of the pup. The model used in the current study
more adequately mimics human infant exposure. Direct administration of the compound
to the pup allows for first-pass metabolism, which is essential for understanding the
effects of these compounds on the body.
The results from the current study indicate that genistein, at physiological
concentrations, and BPA at the NOAEL or below, do not adversely affect several key
endpoints in male reproductive tract development and function. In fact BPA may have a
stimulatory affect on male spermatogenesis (Table 2). It is likely that exposure to these
two xenoestrogens has not resulted in the decline in sperm numbers that has been seen
over the latter half of the twentieth century. The current study also shows that doses of
BPA below the NOAEL identified by Tyl et al. (2002; 2008) are capable of causing an
effect when administered in combination with physiological concentrations of genistein.
However, in agreement with Tyl et al. (2002; 2008) these effects are not necessarily
adverse. Investigation of BPA and genistein’s combined effects on other body organs
needs to be completed. Results from these studies will paint a complete picture of the
combined effects of these endocrine disruptors. Future studies should aim at using
concentrations of BPA that mimic human infant serum levels. Results from these studies
93
will be better able to answer the questions regarding the safety of using plastic baby
bottles. Animals in this study were only observed till 4 months of age, and it is possible
that testicular tumors/cancer may become evident at 12 months of age or greater.
Therefore, longer term studies on these animals may be necessary to identify the full
effect of these endocrine disruptors. More mechanistic studies should also be conducted
to help identify the cause of the effects seen. Sertoli cell numbers, testosterone, and FSH
concentrations should also be measured. Results from the current study indicate that
consumption of soy infant formula from plastic baby bottles is not detrimental to the
development of the male reproductive tract. It is the conclusion of this author based on
these results, that it is safe for parents to use plastic baby bottles when administering soy
infant formula.
Table 2: Summary table describing the results from treatment with 50 mg/kg/d Genistein, or 50
mg/kg/d BPA, or 50 mg/kg/d genistein and 50 mg/kg/d BPA (Gen/BPA50), or 50 mg/kg/d genistein
and 5 mg/kg/d BPA (Gen/BPA5), or 50 mg/kg/d genistein and 0.5 mg/kg/d BPA (Gen/BPA0.5).
Arrow indicates an up regulation of the measured parameter. Dashes indicate no effect of treatment
on the measured parameter.
94
REFERENCES
Abney, T. O. (1999). "The potential roles of estrogens in regulating Leydig cell
development and function: a review." Steroids 64(9): 610-7.
Aceitero, J., M. Llanero, et al. (1998). "Neonatal exposure of male rats to estradiol
benzoate causes rete testis dilation and backflow impairment of spermatogenesis."
Anat Rec 252(1): 17-33.
Adachi, T., Y. Ono, et al. (2004). "Long-term alteration of gene expression without
morphological change in testis after neonatal exposure to genistein in mice:
toxicogenomic analysis using cDNA microarray." Food Chem Toxicol 42(3):
445-52.
Adlercreutz, C. H., B. R. Goldin, et al. (1995). "Soybean phytoestrogen intake and cancer
risk." J Nutr 125(3 Suppl): 757S-770S.
Adlercreutz, H., H. Markkanen, et al. (1993). "Plasma concentrations of phyto-oestrogens
in Japanese men." Lancet 342(8881): 1209-10.
Akingbemi, B. T. (2005). "Estrogen regulation of testicular function." Reprod Biol
Endocrinol 3: 51.
Akingbemi, B. T., T. D. Braden, et al. (2007). "Exposure to phytoestrogens in the
perinatal period affects androgen secretion by testicular Leydig cells in the adult
rat." Endocrinology 148(9): 4475-88.
Akingbemi, B. T. and M. P. Hardy (2001). "Oestrogenic and antiandrogenic chemicals in
the environment: effects on male reproductive health." Ann Med 33(6): 391-403.
Akingbemi, B. T., C. M. Sottas, et al. (2004). "Inhibition of testicular steroidogenesis by
the xenoestrogen bisphenol A is associated with reduced pituitary luteinizing
hormone secretion and decreased steroidogenic enzyme gene expression in rat
Leydig cells." Endocrinology 145(2): 592-603.
Akiyama, T., J. Ishida, et al. (1987). "Genistein, a specific inhibitor of tyrosine-specific
protein kinases." J Biol Chem 262(12): 5592-5.
Al-Hiyasat, A. S., H. Darmani, et al. (2002). "Effects of bisphenol A on adult male mouse
fertility." Eur J Oral Sci 110(2): 163-7.
Alpert, L. I. (1976). "Veno-occlusive disease of the liver associated with oral
contraceptives: case report and review of literature." Hum Pathol 7(6): 709-18.
Anonymous (2005). "Product Focus: Bishphenol A." Chem. Week October 26: 42.
Anstead, G. M., K. E. Carlson, et al. (1997). "The estradiol pharmacophore: ligand
structure-estrogen receptor binding affinity relationships and a model for the
receptor binding site." Steroids 62(3): 268-303.
Aquila, S., D. Sisci, et al. (2002). "Human ejaculated spermatozoa contain active P450
aromatase." J Clin Endocrinol Metab 87(7): 3385-90.
Arai, Y., T. Mori, et al. (1983). "Long-term effects of perinatal exposure to sex steroids
and diethylstilbestrol on the reproductive system of male mammals." Int Rev
Cytol 84: 235-68.
Aschim, E. L., A. Giwercman, et al. (2005). "The RsaI polymorphism in the estrogen
receptor-beta gene is associated with male infertility." J Clin Endocrinol Metab
90(9): 5343-8.
95
Aschim, E. L., T. Saether, et al. (2004). "Differential distribution of splice variants of
estrogen receptor beta in human testicular cells suggests specific functions in
spermatogenesis." J Steroid Biochem Mol Biol 92(1-2): 97-106.
Ashby, J., H. Tinwell, et al. (1999). "Lack of effects for low dose levels of bisphenol A
and diethylstilbestrol on the prostate gland of CF1 mice exposed in utero." Regul
Toxicol Pharmacol 30(2 Pt 1): 156-66.
Atanassova, N., C. McKinnell, et al. (2000). "Comparative effects of neonatal exposure
of male rats to potent and weak (environmental) estrogens on spermatogenesis at
puberty and the relationship to adult testis size and fertility: evidence for
stimulatory effects of low estrogen levels." Endocrinology 141(10): 3898-907.
Atanassova, N., C. McKinnell, et al. (1999). "Permanent effects of neonatal estrogen
exposure in rats on reproductive hormone levels, Sertoli cell number, and the
efficiency of spermatogenesis in adulthood." Endocrinology 140(11): 5364-73.
Atanassova, N., C. McKinnell, et al. (2001). "Age-, cell- and region-specific
immunoexpression of estrogen receptor alpha (but not estrogen receptor beta)
during postnatal development of the epididymis and vas deferens of the rat and
disruption of this pattern by neonatal treatment with diethylstilbestrol."
Endocrinology 142(2): 874-86.
Auger, J., J. M. Kunstmann, et al. (1995). "Decline in semen quality among fertile men in
Paris during the past 20 years." N Engl J Med 332(5): 281-5.
Axelson, M. and K. D. Setchell (1981). "The excretion of lignans in rats -- evidence for
an intestinal bacterial source for this new group of compounds." FEBS Lett
123(2): 337-42.
Axelson, M., J. Sjovall, et al. (1984). "Soya--a dietary source of the non-steroidal
oestrogen equol in man and animals." J Endocrinol 102(1): 49-56.
Baird, D. D., D. M. Umbach, et al. (1995). "Dietary intervention study to assess
estrogenicity of dietary soy among postmenopausal women." J Clin Endocrinol
Metab 80(5): 1685-90.
Barnes, S., M. Kirk, et al. (1994). Isoflavones and their conjugates in soy foods:
extraction conditions and analysis by HPLC-mass spectrometry. 42: 2466-2474.
Beekman, J. M., G. F. Allan, et al. (1993). "Transcriptional activation by the estrogen
receptor requires a conformational change in the ligand binding domain." Mol
Endocrinol 7(10): 1266-74.
Bellve, A. R., J. C. Cavicchia, et al. (1977). "Spermatogenic cells of the prepuberal
mouse. Isolation and morphological characterization." J Cell Biol 74(1): 68-85.
Bennetts, H. W. and E. J. Underwood (1951). "The oestrogenic effects of subterranean
clover (trifolium subterraneum); uterine maintenance in the ovariectomised ewe
on clover grazing." Aust J Exp Biol Med Sci 29(4): 249-53.
Bennetts, H. W., F. Uuderwood, et al. (1946). "A specific breeding problem of sheep on
subterranean clover pastures in western australia." Australian Veterinary Journal
22(1): 2-12.
Bergamaschi, G., V. Rosti, et al. (1993). "Inhibitors of tyrosine phosphorylation induce
apoptosis in human leukemic cell lines." Leukemia 7(12): 2012-8.
Bern, H. A., R. A. Gorski, et al. (1973). "Long-Term Effects of Perinatal Hormone
Administration." Science 181(4095): 189-190.
96
Bern, H. A., L. A. Jones, et al. (1975). "Exposure of neonatal mice to steroids: longterm
effects on the mammary gland and other reproductive structures." J Steroid
Biochem 6(5): 673-6.
Bibbo, M., W. B. Gill, et al. (1977). "Follow-up study of male and female offspring of
DES-exposed mothers." Obstet Gynecol 49(1): 1-8.
Biles, J. E., T. P. McNeal, et al. (1997). "Determination of Bisphenol A Migrating from
Epoxy Can Coatings to Infant Formula Liquid Concentrates." J. Agric. Food
Chem. 45: 4697-4700.
Biles, J. E., T. P. McNeal, et al. (1997). "Determination of bisphenol-A in reusable
polycarbonate food-contact plastics and migration to food simulating liquids." J.
Agric. Food Chem. 45: 3541-3544.
Bingham, S. A., C. Atkinson, et al. (1998). "Phyto-oestrogens: where are we now?" Br J
Nutr 79(5): 393-406.
Blair, R. M., H. Fang, et al. (2000). "The estrogen receptor relative binding affinities of
188 natural and xenochemicals: structural diversity of ligands." Toxicol Sci 54(1):
138-53.
Bourguiba, S., S. Chater, et al. (2003). "Regulation of aromatase gene expression in
purified germ cells of adult male rats: effects of transforming growth factor beta,
tumor necrosis factor alpha, and cyclic adenosine 3',5'-monosphosphate." Biol
Reprod 69(2): 592-601.
Boutin, J. A. (1994). "Tyrosine protein kinase inhibition and cancer." Int J Biochem
26(10-11): 1203-26.
Braden, A. W. H., N. K. Hart, et al. (1967). Oestrogenic activity and metabolism of
certain isoflavones in sheep. 18: 355-348.
Brotons, J. A., M. F. Olea-Serrano, et al. (1995). "Xenoestrogens released from lacquer
coatings in food cans." Environ Health Perspect 103(6): 608-12.
Brouwers, M. M., W. F. Feitz, et al. (2006). "Hypospadias: a transgenerational effect of
diethylstilbestrol?" Hum Reprod 21(3): 666-9.
Brzozowski, A. M., A. C. Pike, et al. (1997). "Molecular basis of agonism and
antagonism in the oestrogen receptor." Nature 389(6652): 753-8.
Cagen, S. Z., J. M. Waechter, Jr., et al. (1999). "Normal reproductive organ development
in CF-1 mice following prenatal exposure to bisphenol A." Toxicol Sci 50(1): 3644.
Calafat, A. M., X. Ye, et al. (2008). "Exposure of the U.S. population to bisphenol A and
4-tertiary-octylphenol: 2003-2004." Environ Health Perspect 116(1): 39-44.
Carey, F. A. (2003). Organic Chemistry. Boston, McGraw-Hill.
Carreau, S., C. Genissel, et al. (1999). "Sources of oestrogen in the testis and
reproductive tract of the male." Int J Androl 22(4): 211-23.
Champe, P. and R. Harvey (1987). DNA Synthesis. Lippincott's Illustrated Reviews:
Biochemistry. P. Champe and R. Harvey. Philadelphia, Lippincott: 357-368.
Chang, C. J. and R. L. Geahlen (1992). "Protein-tyrosine kinase inhibition: mechanismbased discovery of antitumor agents." J Nat Prod 55(11): 1529-60.
Chang, H. C., M. I. Churchwell, et al. (2000). "Mass spectrometric determination of
Genistein tissue distribution in diet-exposed Sprague-Dawley rats." J Nutr 130(8):
1963-70.
97
Chen, A. and W. J. Rogan (2004). "Isoflavones in soy infant formula: a review of
evidence for endocrine and other activity in infants." Annu Rev Nutr 24: 33-54.
Chitra, K. C., C. Latchoumycandane, et al. (2003). "Induction of oxidative stress by
bisphenol A in the epididymal sperm of rats." Toxicology 185(1-2): 119-27.
Cimafranca, M. A., J. Davila, et al. (2009). A PHYSIOLOGICAL MOUSE MODEL
FOR TESTING NEONATAL EFFECTS OF THE SOY PHYTOESTROGEN
GENISTEIN.
Conolly, R. B. and W. K. Lutz (2004). "Nonmonotonic dose-response relationships:
mechanistic basis, kinetic modeling, and implications for risk assessment."
Toxicol Sci 77(1): 151-7.
Constantinou, A. and E. Huberman (1995). "Genistein as an inducer of tumor cell
differentiation: possible mechanisms of action." Proc Soc Exp Biol Med 208(1):
109-15.
Constantinou, A., K. Kiguchi, et al. (1990). "Induction of differentiation and DNA strand
breakage in human HL-60 and K-562 leukemia cells by genistein." Cancer Res
50(9): 2618-24.
Cook, J. C., L. Johnson, et al. (1998). "Effects of dietary 17 beta-estradiol exposure on
serum hormone concentrations and testicular parameters in male Crl:CD BR rats."
Toxicol Sci 44(2): 155-68.
Cooke, P. S. and A. Naaz (2004). "Role of estrogens in adipocyte development and
function." Exp Biol Med (Maywood) 229(11): 1127-35.
Cooke, P. S. and A. Naaz (2005). "Effects of estrogens and the phytoestrogen genistein
on adipogenesis and lipogenesis in males and females." Birth Defects Res A Clin
Mol Teratol 73(7): 472-3.
Cooke, P. S., V. Selvaraj, et al. (2006). "Genistein, estrogen receptors, and the acquired
immune response." J Nutr 136(3): 704-8.
Cooke, P. S., P. Young, et al. (1991). "Estrogen receptor expression in developing
epididymis, efferent ductules, and other male reproductive organs."
Endocrinology 128(6): 2874-9.
Couse, J. E., D. Mahato, et al. (2001). "Molecular mechanism of estrogen action in the
male: insights from the estrogen receptor null mice." Reprod Fertil Dev 13(4):
211-9.
Couse, J. F., S. C. Hewitt, et al. (1999). "Postnatal sex reversal of the ovaries in mice
lacking estrogen receptors alpha and beta." Science 286(5448): 2328-31.
Couse, J. F. and K. S. Korach (1999). "Estrogen receptor null mice: what have we learned
and where will they lead us?" Endocr Rev 20(3): 358-417.
Coward, L., N. C. Barnes, et al. (1993). "Genistein, daidzein, and their -glycoside
conjugates: antitumor isoflavones in soybean foods from American and Asian
diets." J. Agric. Food Chem. 41(11): 1961-67.
Coward, L., N. C. Barnes, et al. (1993). Genistein, daidzein, and their .beta.-glycoside
conjugates: antitumor isoflavones in soybean foods from American and Asian
diets. 41: 1961-1967.
Coward, L., M. Smith, et al. (1998). Chemical modification of isoflavones in soyfoods
during cooking and processing. 68: 1486S-1491.
Cowley, S. M., S. Hoare, et al. (1997). "Estrogen receptors alpha and beta form
heterodimers on DNA." J Biol Chem 272(32): 19858-62.
98
Crescioli, C., M. Maggi, et al. (2003). "Expression of functional estrogen receptors in
human fetal male external genitalia." J Clin Endocrinol Metab 88(4): 1815-24.
Csaba, G. (2000). "Hormonal imprinting: its role during the evolution and development
of hormones and receptors." Cell Biol Int 24(7): 407-14.
Daston, G. P., J. W. Gooch, et al. (1997). "Environmental estrogens and reproductive
health: a discussion of the human and environmental data." Reprod Toxicol 11(4):
465-81.
Diel, P., T. Schulz, et al. (2000). "Ability of xeno- and phytoestrogens to modulate
expression of estrogen-sensitive genes in rat uterus: estrogenicity profiles and
uterotropic activity." J Steroid Biochem Mol Biol 73(1-2): 1-10.
Dietrich, W., A. Haitel, et al. (2004). "Expression of estrogen receptors in human corpus
cavernosum and male urethra." J Histochem Cytochem 52(3): 355-60.
Dodds, E. C. and W. Lawson (1936). "Synthetic Estrogenic Agents without the
Phenanthrene Nucleus." Nature 137: 996.
Doerge, D. R., N. C. Twaddle, et al. (2002). "Pharmacokinetic analysis in serum of
genistein administered subcutaneously to neonatal mice." Cancer Lett 184(1): 217.
Doerge, D. R., N. C. Twaddle, et al. (2006). "Lactational transfer of the soy isoflavone,
genistein, in Sprague-Dawley rats consuming dietary genistein." Reprod Toxicol
21(3): 307-12.
Dorrington, J. and S. Khan (1993). Steroid production, metabolism and release by Sertoli
cells The Sertoli Cell. L. Russell and M. D. Griswold. Clearwater Cache River
Press: 538-549.
Durkee, T. J., M. Mueller, et al. (1998). "Identification of estrogen receptor protein and
messenger ribonucleic acid in human spermatozoa." Am J Obstet Gynecol 178(6):
1288-97.
Eddy, E. M., T. F. Washburn, et al. (1996). "Targeted disruption of the estrogen receptor
gene in male mice causes alteration of spermatogenesis and infertility."
Endocrinology 137(11): 4796-805.
El-Gehani, F., F. P. Zhang, et al. (1998). "Gonadotropin-independent regulation of
steroidogenesis in the fetal rat testis." Biol Reprod 58(1): 116-23.
Evans, R. M. (1988). "The steroid and thyroid hormone receptor superfamily." Science
240(4854): 889-95.
Faber, K. A. and C. L. Hughes, Jr. (1991). "The effect of neonatal exposure to
diethylstilbestrol, genistein, and zearalenone on pituitary responsiveness and
sexually dimorphic nucleus volume in the castrated adult rat." Biol Reprod 45(4):
649-53.
Faber, K. A. and C. L. Hughes, Jr. (1993). "Dose-response characteristics of neonatal
exposure to genistein on pituitary responsiveness to gonadotropin releasing
hormone and volume of the sexually dimorphic nucleus of the preoptic area
(SDN-POA) in postpubertal castrated female rats." Reprod Toxicol 7(1): 35-9.
Farnsworth, N. R., A. S. Bingel, et al. (1975). "Potential value of plants as sources of new
antifertility agents II." J Pharm Sci 64(5): 717-54.
FDA, U. S. (2008). "Statement of Norris Alderson, PhD., Associate Commissioner or
Science, Food and Drug Administration, Department of Health and Human
Services, before the Subcommittee on Commerce, Trade and Consumer
99
Protection, Committee on Energy and Commerce. U.S. House of Representatives.
June 10, 2008." Retrieved 17 September, 2008, from
http://www.fda.gov/ola/2008/BPA061008.html.
Fielden, M. R., R. G. Halgren, et al. (2002). "Gestational and lactational exposure of male
mice to diethylstilbestrol causes long-term effects on the testis, sperm fertilizing
ability in vitro, and testicular gene expression." Endocrinology 143(8): 3044-59.
Fielden, M. R., S. M. Samy, et al. (2003). "Effect of human dietary exposure levels of
genistein during gestation and lactation on long-term reproductive development
and sperm quality in mice." Food Chem Toxicol 41(4): 447-54.
Fischer, L. J. and J. L. Weissinger (1972). "Development in the newborn rat of the
conjugation and de-conjugation processes involved in the enterohepatic
circulation of diethylstilboestrol." Xenobiotica 2(4): 399-412.
Fisher, J. S. (2004). "Environmental anti-androgens and male reproductive health: focus
on phthalates and testicular dysgenesis syndrome." Reproduction 127(3): 305-15.
Fisher, J. S., M. R. Millar, et al. (1997). "Immunolocalisation of oestrogen receptor-alpha
within the testis and excurrent ducts of the rat and marmoset monkey from
perinatal life to adulthood." J Endocrinol 153(3): 485-95.
Fisher, J. S., K. J. Turner, et al. (1999). "Effect of neonatal exposure to estrogenic
compounds on development of the excurrent ducts of the rat testis through
puberty to adulthood." Environ Health Perspect 107(5): 397-405.
Fisher, J. S., K. J. Turner, et al. (1998). "Immunoexpression of aquaporin-1 in the efferent
ducts of the rat and marmoset monkey during development, its modulation by
estrogens, and its possible role in fluid resorption." Endocrinology 139(9): 393545.
Flouriot, G., C. Griffin, et al. (1998). "Differentially expressed messenger RNA isoforms
of the human estrogen receptor-alpha gene are generated by alternative splicing
and promoter usage." Mol Endocrinol 12(12): 1939-54.
Foster, P. M. (2006). "Disruption of reproductive development in male rat offspring
following in utero exposure to phthalate esters." Int J Androl 29(1): 140-7;
discussion 181-5.
Foster, P. M. and M. W. Harris (2005). "Changes in androgen-mediated reproductive
development in male rat offspring following exposure to a single oral dose of
flutamide at different gestational ages." Toxicol Sci 85(2): 1024-32.
Free, M. J. and R. A. Jaffe (1979). "Collection of rete testis fluid from rats without
previous efferent duct ligation." Biol Reprod 20(2): 269-78.
Gachter, R. and H. Muller, Eds. (1990). Plastics additives handbook: stabilizers,
processing aids, plasticizers, fillers, reinforcements, colorants for thermoplastics.
New York, Hanser Publishers.
Gaido, K. W., S. C. Maness, et al. (2000). "Interaction of methoxychlor and related
compounds with estrogen receptor alpha and beta, and androgen receptor:
structure-activity studies." Mol Pharmacol 58(4): 852-8.
Garreau, B., G. Vallette, et al. (1991). "Phytoestrogens: new ligands for rat and human
alpha-fetoprotein." Biochim Biophys Acta 1094(3): 339-45.
Gaskell, T. L., L. L. Robinson, et al. (2003). "Differential expression of two estrogen
receptor beta isoforms in the human fetal testis during the second trimester of
pregnancy." J Clin Endocrinol Metab 88(1): 424-32.
100
Genissel, C., J. Levallet, et al. (2001). "Regulation of cytochrome P450 aromatase gene
expression in adult rat Leydig cells: comparison with estradiol production." J
Endocrinol 168(1): 95-105.
Gill, W. B., G. F. Schumacher, et al. (1977). "Pathological semen and anatomical
abnormalities of the genital tract in human male subjects exposed to
diethylstilbestrol in utero." J Urol 117(4): 477-80.
Gill, W. B., G. F. Schumacher, et al. (1979). "Association of diethylstilbestrol exposure
in utero with cryptorchidism, testicular hypoplasia and semen abnormalities." J
Urol 122(1): 36-9.
Goodman, M. T., L. R. Wilkens, et al. (1997). "Association of soy and fiber consumption
with the risk of endometrial cancer." Am J Epidemiol 146(4): 294-306.
Gould, J. C., L. S. Leonard, et al. (1998). "Bisphenol A interacts with the estrogen
receptor alpha in a distinct manner from estradiol." Mol Cell Endocrinol 142(1-2):
203-14.
Goyal, H. O., T. D. Braden, et al. (2007). "Estrogen receptor alpha mediates estrogeninducible abnormalities in the developing penis." Reproduction 133(5): 1057-67.
Goyal, H. O., T. D. Braden, et al. (2005). "Estrogen-induced abnormal accumulation of
fat cells in the rat penis and associated loss of fertility depends upon estrogen
exposure during critical period of penile development." Toxicol Sci 87(1): 24254.
Granner, D. (1990). DNA organization and replication. Harper's Biochemistry. R.
Murray, D. Granner, P. Mayes and V. Rodwell. East Norwalk, Appleton &
Lange: 366-382.
Gray, L. E., J. Ostby, et al. (2001). "Effects of environmental antiandrogens on
reproductive development in experimental animals." Hum Reprod Update 7(3):
248-64.
Gupta, C. (2000). "Reproductive malformation of the male offspring following maternal
exposure to estrogenic chemicals." Proc Soc Exp Biol Med 224(2): 61-8.
Gupta, C. (2000). "The role of estrogen receptor, androgen receptor and growth factors in
diethylstilbestrol-induced programming of prostate differentiation." Urol Res
28(4): 223-9.
Hall, J. M. and D. P. McDonnell (1999). "The estrogen receptor beta-isoform (ERbeta) of
the human estrogen receptor modulates ERalpha transcriptional activity and is a
key regulator of the cellular response to estrogens and antiestrogens."
Endocrinology 140(12): 5566-78.
Hammond, G. L. (1995). "Potential functions of plasma steroid-binding proteins." Trends
Endocrinol Metab 6(9-10): 298-304.
Hess, R. A. (2000). "Oestrogen in fluid transport in efferent ducts of the male
reproductive tract." Rev Reprod 5(2): 84-92.
Hess, R. A. (2003). "Estrogen in the adult male reproductive tract: a review." Reprod
Biol Endocrinol 1: 52.
Hess, R. A., D. Bunick, et al. (1997). "A role for oestrogens in the male reproductive
system." Nature 390(6659): 509-12.
Hess, R. A., D. Bunick, et al. (2000). "Morphologic changes in efferent ductules and
epididymis in estrogen receptor-alpha knockout mice." J Androl 21(1): 107-21.
101
Hess, R. A., D. H. Gist, et al. (1997). "Estrogen receptor (alpha and beta) expression in
the excurrent ducts of the adult male rat reproductive tract." J Androl 18(6): 60211.
Hess, R. A., Q. Zhou, et al. (2001). "Estrogens and epididymal function." Reprod Fertil
Dev 13(4): 273-83.
Hirayama, T. (1984). "Epidemiology of stomach cancer in Japan. With special reference
to the strategy for the primary prevention." Jpn J Clin Oncol 14(2): 159-68.
Ho, S. M., W. Y. Tang, et al. (2006). "Developmental exposure to estradiol and bisphenol
A increases susceptibility to prostate carcinogenesis and epigenetically regulates
phosphodiesterase type 4 variant 4." Cancer Res 66(11): 5624-32.
Horwitz, K. B., T. A. Jackson, et al. (1996). "Nuclear receptor coactivators and
corepressors." Mol Endocrinol 10(10): 1167-77.
Howdeshell, K. L., P. H. Peterman, et al. (2003). "Bisphenol A is released from used
polycarbonate animal cages into water at room temperature." Environ Health
Perspect 111(9): 1180-7.
Hunter, T. and J. A. Cooper (1985). "Protein-tyrosine kinases." Annu Rev Biochem 54:
897-930.
Iguchi, T. (1992). "Cellular effects of early exposure to sex hormones and antihormones."
Int Rev Cytol 139: 1-57.
Irvine, C., M. Fitzpatrick, et al. (1995). "The potential adverse effects of soybean
phytoestrogens in infant feeding." N Z Med J 108(1000): 208-9.
Irvine, C. H., M. G. Fitzpatrick, et al. (1998). "Phytoestrogens in soy-based infant foods:
concentrations, daily intake, and possible biological effects." Proc Soc Exp Biol
Med 217(3): 247-53.
Irvine, C. H., N. Shand, et al. (1998). "Daily intake and urinary excretion of genistein and
daidzein by infants fed soy- or dairy-based infant formulas." Am J Clin Nutr 68(6
Suppl): 1462S-1465S.
Janulis, L., J. M. Bahr, et al. (1996). "P450 aromatase messenger ribonucleic acid
expression in male rat germ cells: detection by reverse transcription-polymerase
chain reaction amplification." J Androl 17(6): 651-8.
Janulis, L., J. M. Bahr, et al. (1998). "Rat testicular germ cells and epididymal sperm
contain active P450 aromatase." J Androl 19(1): 65-71.
Janulis, L., R. A. Hess, et al. (1996). "Mouse epididymal sperm contain active P450
aromatase which decreases as sperm traverse the epididymis." J Androl 17(2):
111-6.
Jefferson, W. N., J. F. Couse, et al. (2000). "Expression of estrogen receptor beta is
developmentally regulated in reproductive tissues of male and female mice." Biol
Reprod 62(2): 310-7.
Jefferson, W. N., J. F. Couse, et al. (2002). "Neonatal exposure to genistein induces
estrogen receptor (ER)alpha expression and multioocyte follicles in the maturing
mouse ovary: evidence for ERbeta-mediated and nonestrogenic actions." Biol
Reprod 67(4): 1285-96.
Jesmin, S., C. N. Mowa, et al. (2002). "Evidence for a potential role of estrogen in the
penis: detection of estrogen receptor-alpha and -beta messenger ribonucleic acid
and protein." Endocrinology 143(12): 4764-74.
102
Jesmin, S., C. N. Mowa, et al. (2004). "Aromatase is abundantly expressed by neonatal
rat penis but downregulated in adulthood." J Mol Endocrinol 33(2): 343-59.
Joannou, G. E., G. E. Kelly, et al. (1995). "A urinary profile study of dietary
phytoestrogens. The identification and mode of metabolism of new
isoflavonoids." J Steroid Biochem Mol Biol 54(3-4): 167-84.
Jong, F. H., J. Uilenbroek, et al. (1975). "Oestradiol-17beta, testosterone and
gonadotrophins in oestradiol-17beta-treated intact adult male rats." J Endocrinol
65(2): 281-2.
Kanatani, Y., T. Kasukabe, et al. (1993). "Genistein exhibits preferential cytotoxicity to a
leukemogenic variant but induces differentiation of a non-leukemogenic variant
of the mouse monocytic leukemia Mm cell line." Leuk Res 17(10): 847-53.
Kang, K. S., J. H. Che, et al. (2002). "Lack of adverse effects in the F1 offspring
maternally exposed to genistein at human intake dose level." Food Chem Toxicol
40(1): 43-51.
Karabelyos, C. and G. Csaba (1998). "Effect of fetal digoxin exposure (imprinting) on the
sexual behavior of adult rats." Gen Pharmacol 31(3): 367-9.
Kelly, G. E., C. Nelson, et al. (1993). "Metabolites of dietary (soya) isoflavones in human
urine." Clin Chim Acta 223(1-2): 9-22.
Kiguchi, K., A. I. Constantinou, et al. (1990). "Genistein-induced cell differentiation and
protein-linked DNA strand breakage in human melanoma cells." Cancer Commun
2(8): 271-7.
Kim, K. S., W. Liu, et al. (2002). "Expression of the androgen receptor and 5 alphareductase type 2 in the developing human fetal penis and urethra." Cell Tissue
Res 307(2): 145-53.
Kim, K. S., C. R. Torres, Jr., et al. (2004). "Induction of hypospadias in a murine model
by maternal exposure to synthetic estrogens." Environ Res 94(3): 267-75.
Kimira, M., Y. Arai, et al. (1998). "Japanese intake of flavonoids and isoflavonoids from
foods." J Epidemiol 8(3): 168-75.
Kitamura, S., N. Jinno, et al. (2002). "Thyroid hormonal activity of the flame retardants
tetrabromobisphenol A and tetrachlorobisphenol A." Biochem Biophys Res
Commun 293(1): 554-9.
Kitamura, S., T. Suzuki, et al. (2005). "Comparative study of the endocrine-disrupting
activity of bisphenol A and 19 related compounds." Toxicol Sci 84(2): 249-59.
Klein, K. O. (1998). "Isoflavones, soy-based infant formulas, and relevance to endocrine
function." Nutr Rev 56(7): 193-204.
Klip, H., J. Verloop, et al. (2002). "Hypospadias in sons of women exposed to
diethylstilbestrol in utero: a cohort study." Lancet 359(9312): 1102-7.
Klonisch, T., P. A. Fowler, et al. (2004). "Molecular and genetic regulation of testis
descent and external genitalia development." Dev Biol 270(1): 1-18.
Korach, K. S., J. F. Couse, et al. (1996). "Estrogen receptor gene disruption: molecular
characterization and experimental and clinical phenotypes." Recent Prog Horm
Res 51: 159-86; discussion 186-8.
Kortenkamp, A. and R. Altenburger (1998). "Synergisms with mixtures of
xenoestrogens: a reevaluation using the method of isoboles." Sci Total Environ
221(1): 59-73.
103
Krege, J. H., J. B. Hodgin, et al. (1998). "Generation and reproductive phenotypes of
mice lacking estrogen receptor beta." Proc Natl Acad Sci U S A 95(26): 1567782.
Krishnan, A. V., P. Stathis, et al. (1993). "Bisphenol-A: an estrogenic substance is
released from polycarbonate flasks during autoclaving." Endocrinology 132(6):
2279-86.
Kruger, T., M. Long, et al. (2008). "Plastic components affect the activation of the aryl
hydrocarbon and the androgen receptor." Toxicology 246(2-3): 112-23.
Kuiper, G. G. and J. A. Gustafsson (1997). "The novel estrogen receptor-beta subtype:
potential role in the cell- and promoter-specific actions of estrogens and antiestrogens." FEBS Lett 410(1): 87-90.
Kuiper, G. G., J. G. Lemmen, et al. (1998). "Interaction of estrogenic chemicals and
phytoestrogens with estrogen receptor beta." Endocrinology 139(10): 4252-63.
Kula, K. (1988). "Induction of precocious maturation of spermatogenesis in infant rats by
human menopausal gonadotropin and inhibition by simultaneous administration
of gonadotropins and testosterone." Endocrinology 122(1): 34-9.
Kula, K., J. Slowikowska-Hilczer, et al. (1996). "[Precocious maturation of the testis
associated with excessive secretion of estradiol and testosterone by Leydig cells]."
Pediatr Pol 71(3): 269-73.
Kumi-Diaka, J., R. Rodriguez, et al. (1998). "Influence of genistein (4',5,7trihydroxyisoflavone) on the growth and proliferation of testicular cell lines." Biol
Cell 90(4): 349-54.
Kurzer, M. S. and X. Xu (1997). "Dietary phytoestrogens." Annu Rev Nutr 17: 353-81.
Lang, I. A., T. S. Galloway, et al. (2008). "Association of urinary bisphenol A
concentration with medical disorders and laboratory abnormalities in adults."
Jama 300(11): 1303-10.
Lassurguere, J., G. Livera, et al. (2003). "Time- and dose-related effects of estradiol and
diethylstilbestrol on the morphology and function of the fetal rat testis in culture."
Toxicol Sci 73(1): 160-9.
Lee, B. J., J. K. Kang, et al. (2004). "Exposure to genistein does not adversely affect the
reproductive system in adult male mice adapted to a soy-based commercial diet."
J Vet Sci 5(3): 227-34.
Lee, H. P., L. Gourley, et al. (1991). "Dietary effects on breast-cancer risk in Singapore."
Lancet 337(8751): 1197-200.
Lee, K. H., C. Finnigan-Bunick, et al. (2001). "Estrogen regulation of ion transporter
messenger RNA levels in mouse efferent ductules are mediated differentially
through estrogen receptor (ER) alpha and ER beta." Biol Reprod 65(5): 1534-41.
Leopold, A. S., M. Erwin, et al. (1976). "Phytoestrogens: adverse effects on reproduction
in California quail." Science 191(4222): 98-100.
Lewis, R. W., N. Brooks, et al. (2003). "The effects of the phytoestrogen genistein on the
postnatal development of the rat." Toxicol Sci 71(1): 74-83.
Li, H., V. Papadopoulos, et al. (1997). "Regulation of rat testis gonocyte proliferation by
platelet-derived growth factor and estradiol: identification of signaling
mechanisms involved." Endocrinology 138(3): 1289-98.
104
Lieberman, S. (1996). "Are the differences between estradiol and other estrogens,
naturally occurring or synthetic, merely semantical?" J Clin Endocrinol Metab
81(2): 850-1.
Lieng-Huang, L. (1965). Mechanisms of thermal degradation of phenolic condensation
polymers. III. Cleavage of phenolic segments during the thermal degradation of
uncured epoxy resins. 9: 1981-1989.
Lubahn, D. B., J. S. Moyer, et al. (1993). "Alteration of reproductive function but not
prenatal sexual development after insertional disruption of the mouse estrogen
receptor gene." Proc Natl Acad Sci U S A 90(23): 11162-6.
Lucas, T. F., E. R. Siu, et al. (2008). "17beta-estradiol induces the translocation of the
estrogen receptors ESR1 and ESR2 to the cell membrane, MAPK3/1
phosphorylation and proliferation of cultured immature rat Sertoli cells." Biol
Reprod 78(1): 101-14.
Luconi, M., G. Forti, et al. (2002). "Genomic and nongenomic effects of estrogens:
molecular mechanisms of action and clinical implications for male reproduction."
J Steroid Biochem Mol Biol 80(4-5): 369-81.
Majdic, G., R. M. Sharpe, et al. (1996). "Expression of cytochrome P450 17alphahydroxylase/C17-20 lyase in the fetal rat testis is reduced by maternal exposure to
exogenous estrogens." Endocrinology 137(3): 1063-70.
Majdic, G., R. M. Sharpe, et al. (1997). "Maternal oestrogen/xenoestrogen exposure
alters expression of steroidogenic factor-1 (SF-1/Ad4BP) in the fetal rat testis."
Mol Cell Endocrinol 127(1): 91-8.
Makinen, S., S. Makela, et al. (2001). "Localization of oestrogen receptors alpha and beta
in human testis." Mol Hum Reprod 7(6): 497-503.
Markey, C. M., C. L. Michaelson, et al. (2001). "The mouse uterotrophic assay: a
reevaluation of its validity in assessing the estrogenicity of bisphenol A." Environ
Health Perspect 109(1): 55-60.
Markovits, J., C. Linassier, et al. (1989). "Inhibitory effects of the tyrosine kinase
inhibitor genistein on mammalian DNA topoisomerase II." Cancer Res 49(18):
5111-7.
Marsaud, V., A. Gougelet, et al. (2003). "Various phosphorylation pathways, depending
on agonist and antagonist binding to endogenous estrogen receptor alpha
(ERalpha), differentially affect ERalpha extractability, proteasome-mediated
stability, and transcriptional activity in human breast cancer cells." Mol
Endocrinol 17(10): 2013-27.
Masutomi, N., M. Shibutani, et al. (2003). "Impact of dietary exposure to methoxychlor,
genistein, or diisononyl phthalate during the perinatal period on the development
of the rat endocrine/reproductive systems in later life." Toxicology 192(2-3): 14970.
Matsukawa, Y., N. Marui, et al. (1993). "Genistein arrests cell cycle progression at G2M." Cancer Res 53(6): 1328-31.
Matsumoto, J., H. Yokota, et al. (2002). "Developmental increases in rat hepatic
microsomal UDP-glucuronosyltransferase activities toward xenoestrogens and
decreases during pregnancy." Environ Health Perspect 110(2): 193-6.
105
Mazur, W. and H. Adlercreutz (2000). "Overview of naturally occurring endocrine-active
substances in the human diet in relation to human health." Nutrition 16(7-8): 6548.
McCabe, M. J., Jr. and S. Orrenius (1993). "Genistein induces apoptosis in immature
human thymocytes by inhibiting topoisomerase-II." Biochem Biophys Res
Commun 194(2): 944-50.
McKinnell, C., N. Atanassova, et al. (2001). "Suppression of androgen action and the
induction of gross abnormalities of the reproductive tract in male rats treated
neonatally with diethylstilbestrol." J Androl 22(2): 323-38.
McLachlan, J. A. (2001). "Environmental signaling: what embryos and evolution teach us
about endocrine disrupting chemicals." Endocr Rev 22(3): 319-41.
McLachlan, J. A., R. R. Newbold, et al. (1975). "Reproductive tract lesions in male mice
exposed prenatally to diethylstilbestrol." Science 190(4218): 991-2.
McLachlan, J. A., R. R. Newbold, et al. (2001). "From malformations to molecular
mechanisms in the male: three decades of research on endocrine disrupters."
Apmis 109(4): 263-72.
Medlock, K. L., W. S. Branham, et al. (1992). "Long-term effects of postnatal exposure
to diethylstilbestrol on uterine estrogen receptor and growth." J Steroid Biochem
Mol Biol 42(1): 23-8.
Medlock, K. L., C. R. Lyttle, et al. (1991). "Estradiol down-regulation of the rat uterine
estrogen receptor." Proc Soc Exp Biol Med 196(3): 293-300.
Medlock, K. L., D. M. Sheehan, et al. (1988). "Effects of postnatal DES treatment on
uterine growth, development, and estrogen receptor levels." J Steroid Biochem
29(5): 527-32.
Meegan, M. J. and D. G. Lloyd (2003). "Advances in the science of estrogen receptor
modulation." Curr Med Chem 10(3): 181-210.
Melnick, R., G. Lucier, et al. (2002). "Summary of the National Toxicology Program's
report of the endocrine disruptors low-dose peer review." Environ Health Perspect
110(4): 427-31.
Merritt, R. J. and B. H. Jenks (2004). "Safety of soy-based infant formulas containing
isoflavones: the clinical evidence." J Nutr 134(5): 1220S-1224S.
Miao, S., Z. Gao, et al. (2008). "Influence of bisphenol a on developing rat estrogen
receptors and some cytokines in rats: a two-generational study." J Toxicol
Environ Health A 71(15): 1000-8.
Milligan, S. R., O. Khan, et al. (1998). "Competitive binding of xenobiotic oestrogens to
rat alpha-fetoprotein and to sex steroid binding proteins in human and rainbow
trout (Oncorhynchus mykiss) plasma." Gen Comp Endocrinol 112(1): 89-95.
Miniello, V. L., G. E. Moro, et al. (2003). "Soy-based formulas and phyto-oestrogens: a
safety profile." Acta Paediatr Suppl 91(441): 93-100.
Misao, R., J. Fujimoto, et al. (1997). "Immunohistochemical expressions of estrogen and
progesterone receptors in human epididymis at different ages--a preliminary
study." Int J Fertil Womens Med 42(1): 39-42.
Miura, T., C. Miura, et al. (1999). "Estradiol-17beta stimulates the renewal of
spermatogonial stem cells in males." Biochem Biophys Res Commun 264(1):
230-4.
106
Molteni, A., L. Brizio-Molteni, et al. (1995). "In vitro hormonal effects of soybean
isoflavones." J Nutr 125(3 Suppl): 751S-756S.
Montani, C., M. Penza, et al. (2008). "Genistein is an efficient estrogen in the wholebody throughout mouse development." Toxicol Sci 103(1): 57-67.
Moon, D. G., D. J. Sung, et al. (2001). "Bisphenol A inhibits penile erection via alteration
of histology in the rabbit." Int J Impot Res 13(5): 309-16.
Moriyama, K., T. Tagami, et al. (2002). "Thyroid hormone action is disrupted by
bisphenol A as an antagonist." J Clin Endocrinol Metab 87(11): 5185-90.
Mosconi, G., O. Carnevali, et al. (2002). "Environmental estrogens and reproductive
biology in amphibians." Gen Comp Endocrinol 126(2): 125-9.
Murakami, R. (1986). "Development of the os penis in genital tubercles cultured beneath
the renal capsule of adult rats." J Anat 149: 11-20.
Murakami, R. (1987). "Autoradiographic studies of the localisation of androgen-binding
cells in the genital tubercles of fetal rats." J Anat 151: 209-19.
Murkies, A. L., G. Wilcox, et al. (1998). "Clinical review 92: Phytoestrogens." J Clin
Endocrinol Metab 83(2): 297-303.
Nagao, T., S. Yoshimura, et al. (2001). "Reproductive effects in male and female rats of
neonatal exposure to genistein." Reprod Toxicol 15(4): 399-411.
Nagel, S. C., F. S. vom Saal, et al. (1997). "Relative binding affinity-serum modified
access (RBA-SMA) assay predicts the relative in vivo bioactivity of the
xenoestrogens bisphenol A and octylphenol." Environ Health Perspect 105(1): 706.
Nagel, S. C., F. S. vom Saal, et al. (1999). "Developmental effects of estrogenic
chemicals are predicted by an in vitro assay incorporating modification of cell
uptake by serum." J Steroid Biochem Mol Biol 69(1-6): 343-57.
Nelson, K. G., Y. Sakai, et al. (1994). "Exposure to diethylstilbestrol during a critical
developmental period of the mouse reproductive tract leads to persistent induction
of two estrogen-regulated genes." Cell Growth Differ 5(6): 595-606.
Newbold, R. and J. McLachlan (1985). Diethylstilbestrol associated defects in murine
genial tract development. Estrogens in the Environment. J. McLachlan, Elsevier
Science Publishing Co., Inc.
Newbold, R. R. (2004). "Lessons learned from perinatal exposure to diethylstilbestrol."
Toxicol Appl Pharmacol 199(2): 142-50.
Newbold, R. R., E. P. Banks, et al. (2001). "Uterine adenocarcinoma in mice treated
neonatally with genistein." Cancer Res 61(11): 4325-8.
Newbold, R. R., D. B. Carter, et al. (1984). "Molecular differentiation of the mouse
genital tract: altered protein synthesis following prenatal exposure to
diethylstilbestrol." Biol Reprod 30(2): 459-70.
Newbold, R. R., A. B. Moore, et al. (2002). "Characterization of uterine leiomyomas in
CD-1 mice following developmental exposure to diethylstilbestrol (DES)."
Toxicol Pathol 30(5): 611-6.
Nilsson, S., S. Makela, et al. (2001). "Mechanisms of estrogen action." Physiol Rev
81(4): 1535-65.
Nitta, H., D. Bunick, et al. (1993). "Germ cells of the mouse testis express P450
aromatase." Endocrinology 132(3): 1396-401.
107
North, K. and J. Golding (2000). "A maternal vegetarian diet in pregnancy is associated
with hypospadias. The ALSPAC Study Team. Avon Longitudinal Study of
Pregnancy and Childhood." BJU Int 85(1): 107-13.
O'Donnell, L., K. M. Robertson, et al. (2001). "Estrogen and spermatogenesis." Endocr
Rev 22(3): 289-318.
Okura, A., H. Arakawa, et al. (1988). "Effect of genistein on topoisomerase activity and
on the growth of [Val 12]Ha-ras-transformed NIH 3T3 cells." Biochem Biophys
Res Commun 157(1): 183-9.
Olea, N., R. Pulgar, et al. (1996). "Estrogenicity of resin-based composites and sealants
used in dentistry." Environ Health Perspect 104(3): 298-305.
Papadopoulos, V., S. Carreau, et al. (1986). "Rat testis 17 beta-estradiol: identification by
gas chromatography-mass spectrometry and age related cellular distribution." J
Steroid Biochem 24(6): 1211-6.
Payne, D. W. and J. A. Katzenellenbogen (1979). "Binding specificity of rat alphafetoprotein for a series of estrogen derivatives: studies using equilibrium and
nonequilibrium binding techniques." Endocrinology 105(3): 745-53.
Petersen, D. N., G. T. Tkalcevic, et al. (1998). "Identification of estrogen receptor beta2,
a functional variant of estrogen receptor beta expressed in normal rat tissues."
Endocrinology 139(3): 1082-92.
Pettersson, K., K. Grandien, et al. (1997). "Mouse estrogen receptor beta forms estrogen
response element-binding heterodimers with estrogen receptor alpha." Mol
Endocrinol 11(10): 1486-96.
Ph. M. Kluin, D. G. R. (1981). A comparison between the morphology and cell kinetics
of gonocytes and adult type undifferentiated spermatogonia in the mouse. 4: 475493.
Pike, A. C., A. M. Brzozowski, et al. (1999). "Structure of the ligand-binding domain of
oestrogen receptor beta in the presence of a partial agonist and a full antagonist."
Embo J 18(17): 4608-18.
Pocar, P., B. Fischer, et al. (2005). "Molecular interactions of the aryl hydrocarbon
receptor and its biological and toxicological relevance for reproduction."
Reproduction 129(4): 379-89.
Polack, F. P., N. Khan, et al. (1999). "Changing partners: the dance of infant formula
changes." Clin Pediatr (Phila) 38(12): 703-8.
Powell, C. E., A. M. Soto, et al. (2001). "Identification and characterization of membrane
estrogen receptor from MCF7 estrogen-target cells." J Steroid Biochem Mol Biol
77(2-3): 97-108.
Prins, G. S. and L. Birch (1995). "The developmental pattern of androgen receptor
expression in rat prostate lobes is altered after neonatal exposure to estrogen."
Endocrinology 136(3): 1303-14.
Program, N. T. (2001). National Toxicology Program: Final Report of the Endocrine
Disruptors Low-Dose Peer Review Panel. Endocrine Disruptors Low-Dose Peer
Review. Raleigh, NC, National Institute of Environment and Health Sciences.
Putz, O., C. B. Schwartz, et al. (2001). "Neonatal low- and high-dose exposure to
estradiol benzoate in the male rat: I. Effects on the prostate gland." Biol Reprod
65(5): 1496-505.
108
Rissman, E. F., S. R. Wersinger, et al. (1999). "Sex with knockout models: behavioral
studies of estrogen receptor alpha." Brain Res 835(1): 80-90.
Rivas, A., J. S. Fisher, et al. (2002). "Induction of reproductive tract developmental
abnormalities in the male rat by lowering androgen production or action in
combination with a low dose of diethylstilbestrol: evidence for importance of the
androgen-estrogen balance." Endocrinology 143(12): 4797-808.
Rivas, A., C. McKinnell, et al. (2003). "Neonatal coadministration of testosterone with
diethylstilbestrol prevents diethylstilbestrol induction of most reproductive tract
abnormalities in male rats." J Androl 24(4): 557-67.
Robert J. Gardner, J. R. M. (1979). Humid aging of plastics: Effect of molecular weight
on mechanical properties and fracture morphology of polycarbonate. 24: 12691280.
Robertson, K. M., L. O'Donnell, et al. (1999). "Impairment of spermatogenesis in mice
lacking a functional aromatase (cyp 19) gene." Proc Natl Acad Sci U S A 96(14):
7986-91.
Roy, D., M. Palangat, et al. (1997). "Biochemical and molecular changes at the cellular
level in response to exposure to environmental estrogen-like chemicals." J
Toxicol Environ Health 50(1): 1-29.
Saberwal, G. S., M. K. Sharma, et al. (2002). "Estrogen receptor, calcium mobilization
and rat sperm motility." Mol Cell Biochem 237(1-2): 11-20.
Saceda, M., R. K. Lindsey, et al. (1998). "Estradiol regulates estrogen receptor mRNA
stability." J Steroid Biochem Mol Biol 66(3): 113-20.
Safe, S. H. (1995). "Environmental and dietary estrogens and human health: is there a
problem?" Environ Health Perspect 103(4): 346-51.
Safe, S. H., L. Pallaroni, et al. (2001). "Toxicology of environmental estrogens." Reprod
Fertil Dev 13(4): 307-15.
Sakamoto, H., H. Yokota, et al. (2002). "Excretion of bisphenol A-glucuronide into the
small intestine and deconjugation in the cecum of the rat." Biochim Biophys Acta
1573(2): 171-6.
Sapsford, C. S. (1962). Changes in the cells of the Sex Cords and Seminiferous Tubules
during the development of the Testis of the rat and mouse. 10: 178-192.
Saunders, P. T., J. S. Fisher, et al. (1998). "Expression of oestrogen receptor beta (ER
beta) occurs in multiple cell types, including some germ cells, in the rat testis." J
Endocrinol 156(3): R13-7.
Saunders, P. T., S. M. Maguire, et al. (1997). "Expression of oestrogen receptor beta (ER
beta) in multiple rat tissues visualised by immunohistochemistry." J Endocrinol
154(3): R13-6.
Saunders, P. T., M. R. Millar, et al. (2002). "ERbeta1 and the ERbeta2 splice variant
(ERbetacx/beta2) are expressed in distinct cell populations in the adult human
testis." J Clin Endocrinol Metab 87(6): 2706-15.
Schonfelder, G., B. Flick, et al. (2002). "In utero exposure to low doses of bisphenol A
lead to long-term deleterious effects in the vagina." Neoplasia 4(2): 98-102.
Schonfelder, G., W. Wittfoht, et al. (2002). "Parent bisphenol A accumulation in the
human maternal-fetal-placental unit." Environ Health Perspect 110(11): A703-7.
109
Schultheiss, D., R. Badalyan, et al. (2003). "Androgen and estrogen receptors in the
human corpus cavernosum penis: immunohistochemical and cell culture results."
World J Urol 21(5): 320-4.
Setchell, K. D. (1998). "Phytoestrogens: the biochemistry, physiology, and implications
for human health of soy isoflavones." Am J Clin Nutr 68(6 Suppl): 1333S-1346S.
Setchell, K. D., S. P. Borriello, et al. (1984). "Nonsteroidal estrogens of dietary origin:
possible roles in hormone-dependent disease." Am J Clin Nutr 40(3): 569-78.
Setchell, K. D., N. M. Brown, et al. (2001). "Bioavailability of pure isoflavones in
healthy humans and analysis of commercial soy isoflavone supplements." J Nutr
131(4 Suppl): 1362S-75S.
Setchell, K. D., S. J. Gosselin, et al. (1987). "Dietary estrogens--a probable cause of
infertility and liver disease in captive cheetahs." Gastroenterology 93(2): 225-33.
Setchell, K. D., L. Zimmer-Nechemias, et al. (1997). "Exposure of infants to phytooestrogens from soy-based infant formula." Lancet 350(9070): 23-7.
Setchell, K. D., L. Zimmer-Nechemias, et al. (1998). "Isoflavone content of infant
formulas and the metabolic fate of these phytoestrogens in early life." Am J Clin
Nutr 68(6 Suppl): 1453S-1461S.
Setchell, K. D. R. and H. Adlercreutz (1988). Mammalian lignansand phytoestrogens.
Recent studies on their formation, metabolism and biological role in helath and
disease Role of the Gut Flora in Toxicity and Cancer. I. R. Rowland. London,
Academic: 315-45.
Sfakianos, J., L. Coward, et al. (1997). "Intestinal uptake and biliary excretion of the
isoflavone genistein in rats." J Nutr 127(7): 1260-8.
Sharpe, R. (1993). Experimental evidence for Sertoli-Leydig cell interactions. The Sertoli
Cell. L. D. Russell and M. D. Griswold. Clearwater, FL, Cache River Press: 391419.
Sharpe, R. M. (1993). "Declining sperm counts in men--is there an endocrine cause?" J
Endocrinol 136(3): 357-60.
Sharpe, R. M. (1998). "The roles of oestrogen in the male." Trends Endocrinol Metab
9(9): 371-7.
Sharpe, R. M. (2006). "Pathways of endocrine disruption during male sexual
differentiation and masculinization." Best Pract Res Clin Endocrinol Metab 20(1):
91-110.
Sharpe, R. M., N. Atanassova, et al. (1998). "Abnormalities in functional development of
the Sertoli cells in rats treated neonatally with diethylstilbestrol: a possible role
for estrogens in Sertoli cell development." Biol Reprod 59(5): 1084-94.
Sharpe, R. M., J. S. Fisher, et al. (1995). "Gestational and lactational exposure of rats to
xenoestrogens results in reduced testicular size and sperm production." Environ
Health Perspect 103(12): 1136-43.
Sharpe, R. M., B. Martin, et al. (2002). "Infant feeding with soy formula milk: effects on
the testis and on blood testosterone levels in marmoset monkeys during the period
of neonatal testicular activity." Hum Reprod 17(7): 1692-703.
Sharpe, R. M., A. Rivas, et al. (2003). "Effect of neonatal treatment of rats with potent or
weak (environmental) oestrogens, or with a GnRH antagonist, on Leydig cell
development and function through puberty into adulthood." Int J Androl 26(1):
26-36.
110
Sharpe, R. M. and N. E. Skakkebaek (1993). "Are oestrogens involved in falling sperm
counts and disorders of the male reproductive tract?" Lancet 341(8857): 1392-5.
Shibata, N., J. Matsumoto, et al. (2002). "Male-specific suppression of hepatic
microsomal UDP-glucuronosyl transferase activities toward sex hormones in the
adult male rat administered bisphenol A." Biochem J 368(Pt 3): 783-8.
Shibayama, T., H. Fukata, et al. (2001). "Neonatal exposure to genistein reduces
expression of estrogen receptor alpha and androgen receptor in testes of adult
mice." Endocr J 48(6): 655-63.
Shimizu, M., K. Ohta, et al. (2002). "Sulfation of bisphenol A abolished its estrogenicity
based on proliferation and gene expression in human breast cancer MCF-7 cells."
Toxicol In Vitro 16(5): 549-56.
Shutt, D. A. and A. W. H. Braden (1968). "The significance of equol in relation to the
oestrogenic responses in sheeep ingesting clover with a high formononetin
content." Australian Journal of Agricultural Research 19: 545-53.
Shutt, D. A. and R. I. Cox (1972). "Steroid and phyto-oestrogen binding to sheep uterine
receptors in vitro." J Endocrinol 52(2): 299-310.
Silva, E., N. Rajapakse, et al. (2002). "Something from "nothing"--eight weak estrogenic
chemicals combined at concentrations below NOECs produce significant mixture
effects." Environ Sci Technol 36(8): 1751-6.
Simpson, E. R., M. S. Mahendroo, et al. (1994). "Aromatase cytochrome P450, the
enzyme responsible for estrogen biosynthesis." Endocr Rev 15(3): 342-55.
Slowikowska-Hilczer, J. and K. Kula (1994). "[Comparison between the influence of
estradiol benzoate, testosterone propionate and human chorionic gonadotropin on
initiation of spermatogenesis in the rat]." Ginekol Pol 65(2): 53-7.
Sohoni, P. and J. P. Sumpter (1998). "Several environmental oestrogens are also antiandrogens." J Endocrinol 158(3): 327-39.
Sonnenschein, C. and A. M. Soto (1998). "An updated review of environmental estrogen
and androgen mimics and antagonists." J Steroid Biochem Mol Biol 65(1-6): 14350.
Soto, A. M., M. F. Fernandez, et al. (1997). "Developing a marker of exposure to
xenoestrogen mixtures in human serum." Environ Health Perspect 105 Suppl 3:
647-54.
Srilatha, B. and P. G. Adaikan (2004). "Estrogen and phytoestrogen predispose to erectile
dysfunction: do ER-alpha and ER-beta in the cavernosum play a role?" Urology
63(2): 382-6.
Staples, C. A., P. B. Dorn, et al. (1998). "A review of the environmental fate, effects, and
exposures of bisphenol A." Chemosphere 36(10): 2149-73.
Storgaard, L., J. P. Bonde, et al. (2006). "Male reproductive disorders in humans and
prenatal indicators of estrogen exposure. A review of published epidemiological
studies." Reprod Toxicol 21(1): 4-15.
Strom, A., J. Hartman, et al. (2004). "Estrogen receptor beta inhibits 17beta-estradiolstimulated proliferation of the breast cancer cell line T47D." Proc Natl Acad Sci
U S A 101(6): 1566-71.
Strom, B. L., R. Schinnar, et al. (2001). "Exposure to soy-based formula in infancy and
endocrinological and reproductive outcomes in young adulthood." Jama 286(7):
807-14.
111
Suiko, M., Y. Sakakibara, et al. (2000). "Sulfation of environmental estrogen-like
chemicals by human cytosolic sulfotransferases." Biochem Biophys Res Commun
267(1): 80-4.
Sultan, C., F. Paris, et al. (2001). "Disorders linked to insufficient androgen action in
male children." Hum Reprod Update 7(3): 314-22.
Sun, H., L. C. Xu, et al. (2006). "Effect of bisphenol A, tetrachlorobisphenol A and
pentachlorophenol on the transcriptional activities of androgen receptor-mediated
reporter gene." Food Chem Toxicol 44(11): 1916-21.
Swan, S. H. (2000). "Intrauterine exposure to diethylstilbestrol: long-term effects in
humans." Apmis 108(12): 793-804.
Tajima, K. and S. Tominaga (1985). "Dietary habits and gastro-intestinal cancers: a
comparative case-control study of stomach and large intestinal cancers in Nagoya,
Japan." Jpn J Cancer Res 76(8): 705-16.
Takeuchi, T., O. Tsutsumi, et al. (2006). "Elevated serum bisphenol A levels under
hyperandrogenic conditions may be caused by decreased UDPglucuronosyltransferase activity." Endocr J 53(4): 485-91.
Taylor, J. A., W. V. Welshons, et al. (2008). "No effect of route of exposure (oral;
subcutaneous injection) on plasma bisphenol A throughout 24h after
administration in neonatal female mice." Reprod Toxicol 25(2): 169-76.
Tena-Sempere, M., J. Navarro, et al. (2000). "Neonatal exposure to estrogen
differentially alters estrogen receptor alpha and beta mRNA expression in rat
testis during postnatal development." J Endocrinol 165(2): 345-57.
Thomas, P. and J. Dong (2006). "Binding and activation of the seven-transmembrane
estrogen receptor GPR30 by environmental estrogens: a potential novel
mechanism of endocrine disruption." J Steroid Biochem Mol Biol 102(1-5): 1759.
Thuillier, R., Y. Wang, et al. (2003). "Prenatal exposure to estrogenic compounds alters
the expression pattern of platelet-derived growth factor receptors alpha and beta
in neonatal rat testis: identification of gonocytes as targets of estrogen exposure."
Biol Reprod 68(3): 867-80.
Toppari, J., J. C. Larsen, et al. (1996). "Male reproductive health and environmental
xenoestrogens." Environ Health Perspect 104 Suppl 4: 741-803.
Traganos, F., B. Ardelt, et al. (1992). "Effects of genistein on the growth and cell cycle
progression of normal human lymphocytes and human leukemic MOLT-4 and
HL-60 cells." Cancer Res 52(22): 6200-8.
Tsai-Morris, C. H., D. R. Aquilano, et al. (1985). "Cellular localization of rat testicular
aromatase activity during development." Endocrinology 116(1): 38-46.
Tyl, R. W., C. B. Myers, et al. (2008). "Two-generation reproductive toxicity study of
dietary bisphenol A in CD-1 (Swiss) mice." Toxicol Sci 104(2): 362-84.
Tyl, R. W., C. B. Myers, et al. (2002). "Three-generation reproductive toxicity study of
dietary bisphenol A in CD Sprague-Dawley rats." Toxicol Sci 68(1): 121-46.
Uehar, M., Y. Arai, et al. (2000). "Comparison of plasma and urinary phytoestrogens in
Japanese and Finnish women by time-resolved fluoroimmunoassay." Biofactors
12(1-4): 217-25.
van Lipzig, M. M., A. M. ter Laak, et al. (2004). "Prediction of ligand binding affinity
and orientation of xenoestrogens to the estrogen receptor by molecular dynamics
112
simulations and the linear interaction energy method." J Med Chem 47(4): 101830.
van Pelt, A. M., D. G. de Rooij, et al. (1999). "Ontogeny of estrogen receptor-beta
expression in rat testis." Endocrinology 140(1): 478-83.
Vandenberg, L. N., R. Hauser, et al. (2007). "Human exposure to bisphenol A (BPA)."
Reprod Toxicol 24(2): 139-77.
Vandenberg, L. N., M. V. Maffini, et al. (2009). "Bisphenol-A and the great divide: a
review of controversies in the field of endocrine disruption." Endocr Rev 30(1):
75-95.
Vandenberg, L. N., P. R. Wadia, et al. (2006). "The mammary gland response to
estradiol: monotonic at the cellular level, non-monotonic at the tissue-level of
organization?" J Steroid Biochem Mol Biol 101(4-5): 263-74.
Varayoud, J., J. G. Ramos, et al. (2005). "The estrogen receptor alpha sigma3 mRNA
splicing variant is differentially regulated by estrogen and progesterone in the rat
uterus." J Endocrinol 186(1): 51-60.
Verdeal, K., R. R. Brown, et al. (1980). "Affinity of phytoestrogens for estradiol-binding
proteins and effect of coumestrol on growth of 7,12-dimethylbenz[a]anthraceneinduced rat mammary tumors." J Natl Cancer Inst 64(2): 285-90.
Vergouwen, R. P., S. G. Jacobs, et al. (1991). "Proliferative activity of gonocytes, Sertoli
cells and interstitial cells during testicular development in mice." J Reprod Fertil
93(1): 233-43.
Vidaeff, A. C. and L. E. Sever (2005). "In utero exposure to environmental estrogens and
male reproductive health: a systematic review of biological and epidemiologic
evidence." Reprod Toxicol 20(1): 5-20.
Visser, J. A., A. McLuskey, et al. (1998). "Effect of prenatal exposure to
diethylstilbestrol on Mullerian duct development in fetal male mice."
Endocrinology 139(10): 4244-51.
Volkel, W., T. Colnot, et al. (2002). "Metabolism and kinetics of bisphenol a in humans
at low doses following oral administration." Chem Res Toxicol 15(10): 1281-7.
vom Saal, F. S. (2006). "Bisphenol A eliminates brain and behavior sex dimorphisms in
mice: how low can you go?" Endocrinology 147(8): 3679-80.
vom Saal, F. S., B. T. Akingbemi, et al. (2007). "Chapel Hill bisphenol A expert panel
consensus statement: integration of mechanisms, effects in animals and potential
to impact human health at current levels of exposure." Reprod Toxicol 24(2): 1318.
vom Saal, F. S., P. S. Cooke, et al. (1998). "A physiologically based approach to the
study of bisphenol A and other estrogenic chemicals on the size of reproductive
organs, daily sperm production, and behavior." Toxicol Ind Health 14(1-2): 23960.
vom Saal, F. S. and C. Hughes (2005). "An extensive new literature concerning low-dose
effects of bisphenol A shows the need for a new risk assessment." Environ Health
Perspect 113(8): 926-33.
Vom Saal, F. S., C. A. Richter, et al. (2005). "The importance of appropriate controls,
animal feed, and animal models in interpreting results from low-dose studies of
bisphenol A." Birth Defects Res A Clin Mol Teratol 73(3): 140-5.
113
vom Saal, F. S., B. G. Timms, et al. (1997). "Prostate enlargement in mice due to fetal
exposure to low doses of estradiol or diethylstilbestrol and opposite effects at high
doses." Proc Natl Acad Sci U S A 94(5): 2056-61.
Wang, H. and P. A. Murphy (1994). Isoflavone Content in Commercial Soybean Foods.
42: 1666-1673.
Wang, H. J. and P. A. Murphy (1994). "Isoflavone content in commercial soybean
foods." J. Agric. Food Chem. 42(8): 1666-73.
Ward, I. L. and J. Weisz (1984). "Differential effects of maternal stress on circulating
levels of corticosterone, progesterone, and testosterone in male and female rat
fetuses and their mothers." Endocrinology 114(5): 1635-44.
Watson, C. S., N. N. Bulayeva, et al. (2007). "Xenoestrogens are potent activators of
nongenomic estrogenic responses." Steroids 72(2): 124-34.
Watson, C. S., N. N. Bulayeva, et al. (2005). "Signaling from the membrane via
membrane estrogen receptor-alpha: estrogens, xenoestrogens, and
phytoestrogens." Steroids 70(5-7): 364-71.
Wei, H., L. Wei, et al. (1993). "Inhibition of tumor promoter-induced hydrogen peroxide
formation in vitro and in vivo by genistein." Nutr Cancer 20(1): 1-12.
Weihua, Z., S. Makela, et al. (2001). "A role for estrogen receptor beta in the regulation
of growth of the ventral prostate." Proc Natl Acad Sci U S A 98(11): 6330-5.
Wellejus, A., H. E. Jensen, et al. (2008). "Expression of aquaporin 9 in rat liver and
efferent ducts of the male reproductive system after neonatal diethylstilbestrol
exposure." J Histochem Cytochem 56(5): 425-32.
Welshons, W. V., S. C. Nagel, et al. (2006). "Large effects from small exposures. III.
Endocrine mechanisms mediating effects of bisphenol A at levels of human
exposure." Endocrinology 147(6 Suppl): S56-69.
Welshons, W. V., K. A. Thayer, et al. (2003). "Large effects from small exposures. I.
Mechanisms for endocrine-disrupting chemicals with estrogenic activity."
Environ Health Perspect 111(8): 994-1006.
Weniger, J. P., J. Chouraqui, et al. (1993). "Production of estradiol by the fetal rat testis."
Reprod Nutr Dev 33(2): 121-7.
Wetherill, Y. B., B. T. Akingbemi, et al. (2007). "In vitro molecular mechanisms of
bisphenol A action." Reprod Toxicol 24(2): 178-98.
Whitten, P. L., C. Lewis, et al. (1995). "Potential adverse effects of phytoestrogens." J
Nutr 125(3 Suppl): 771S-776S.
Whitten, P. L. and F. Naftolin (1998). "Reproductive actions of phytoestrogens."
Baillieres Clin Endocrinol Metab 12(4): 667-90.
Widmaier, R. (1963). "[on Postnatal Testis Development and Germ Cell Maturation in
Mice.]." Z Mikrosk Anat Forsch 70: 215-41.
Wijayaratne, A. L. and D. P. McDonnell (2001). "The human estrogen receptor-alpha is a
ubiquitinated protein whose stability is affected differentially by agonists,
antagonists, and selective estrogen receptor modulators." J Biol Chem 276(38):
35684-92.
Willhite, C. C., G. L. Ball, et al. (2008). "Derivation of a bisphenol A oral reference dose
(RfD) and drinking-water equivalent concentration." J Toxicol Environ Health B
Crit Rev 11(2): 69-146.
114
Williams-Ashman, H. G. and A. H. Reddi (1991). "Differentiation of mesenchymal
tissues during phallic morphogenesis with emphasis on the os penis: roles of
androgens and other regulatory agents." J Steroid Biochem Mol Biol 39(6): 87381.
Williams, K., J. S. Fisher, et al. (2001a). "Relationship between expression of sex steroid
receptors and structure of the seminal vesicles after neonatal treatment of rats with
potent or weak estrogens." Environ Health Perspect 109(12): 1227-35.
Williams, K., C. McKinnell, et al. (2001b). "Neonatal exposure to potent and
environmental oestrogens and abnormalities of the male reproductive system in
the rat: evidence for importance of the androgen-oestrogen balance and
assessment of the relevance to man." Hum Reprod Update 7(3): 236-47.
Wilson, N. K., J. C. Chuang, et al. (2004). "Design and sampling methodology for a large
study of preschool children's aggregate exposures to persistent organic pollutants
in their everyday environments." J Expo Anal Environ Epidemiol 14(3): 260-74.
Wisniewski, A. B., S. L. Klein, et al. (2003). "Exposure to genistein during gestation and
lactation demasculinizes the reproductive system in rats." J Urol 169(4): 1582-6.
Wistuba, J., M. H. Brinkworth, et al. (2003). "Intrauterine bisphenol A exposure leads to
stimulatory effects on Sertoli cell number in rats." Environ Res 91(2): 95-103.
Woodham, C., L. Birch, et al. (2003). "Neonatal estrogen down-regulates prostatic
androgen receptor through a proteosome-mediated protein degradation pathway."
Endocrinology 144(11): 4841-50.
Wozniak, A. L., N. N. Bulayeva, et al. (2005). "Xenoestrogens at picomolar to nanomolar
concentrations trigger membrane estrogen receptor-alpha-mediated Ca2+ fluxes
and prolactin release in GH3/B6 pituitary tumor cells." Environ Health Perspect
113(4): 431-9.
Xu, X., K. S. Harris, et al. (1995). "Bioavailability of soybean isoflavones depends upon
gut microflora in women." J. Nutr. 125(9): 2307-15.
Ye, X., Z. Kuklenyik, et al. (2005). "Quantification of urinary conjugates of bisphenol A,
2,5-dichlorophenol, and 2-hydroxy-4-methoxybenzophenone in humans by online
solid phase extraction-high performance liquid chromatography-tandem mass
spectrometry." Anal Bioanal Chem 383(4): 638-44.
Yokota, H., H. Iwano, et al. (1999). "Glucuronidation of the environmental oestrogen
bisphenol A by an isoform of UDP-glucuronosyltransferase, UGT2B1, in the rat
liver." Biochem J 340 ( Pt 2): 405-9.
Yoo, S. D., B. S. Shin, et al. (2000). "Pharmacokinetic disposition and tissue distribution
of bisphenol A in rats after intravenous administration." J Toxicol Environ Health
A 61(2): 131-9.
Yoo, S. D., B. S. Shin, et al. (2001). "Bioavailability and mammary excretion of
bisphenol a in Sprague-Dawley rats." J Toxicol Environ Health A 64(5): 417-26.
Zadina, J. E., J. L. Dunlap, et al. (1979). "Modifications induced by neonatal steroids in
reproductive organs and behavior of male rats." J Comp Physiol Psychol 93(2):
314-22.
Zalko, D., A. M. Soto, et al. (2003). "Biotransformations of bisphenol A in a mammalian
model: answers and new questions raised by low-dose metabolic fate studies in
pregnant CD1 mice." Environ Health Perspect 111(3): 309-19.
115
Zava, D. T. and G. Duwe (1997). "Estrogenic and antiproliferative properties of genistein
and other flavonoids in human breast cancer cells in vitro." Nutr Cancer 27(1):
31-40.
Zhou, Q., L. Clarke, et al. (2001). "Estrogen action and male fertility: roles of the
sodium/hydrogen exchanger-3 and fluid reabsorption in reproductive tract
function." Proc Natl Acad Sci U S A 98(24): 14132-7.
Zhou, W., Z. Liu, et al. (2006). "Identification and characterization of two novel splicing
isoforms of human estrogen-related receptor beta." J Clin Endocrinol Metab
91(2): 569-79.
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CURRICULUM VITAE
Heather N. Schlesser
187 Paddock Dr. East
Savoy, Il. 61874
(217) 714-1837
[email protected]
Education:
PhD in Veterinary Biosciences, University of Illinois expected August 2009
Title: Effect of Endocrine Disruptors on the Male Mouse Reproductive System
Teaching Certificate: Agricultural Education and Biology December 2008
Masters in Agricultural Education, University of Illinois May 2008
Masters in Animal Sciences, University of Illinois December 2004
Title: Graphical Approach to Evaluate Genetic Estimates of Calf Survival
Bachelors in Animal Sciences, University of Illinois December 2002
Minor in Chemistry, University of Illinois December 2002
Certificate in Business Administration, University of Illinois 2001
Fellowships:
Agriculture Genome Sciences and Public Policy Training Program 2004-2006
Professional Experience:
Minooka High School:
Student teaching internship (January 2008 – May 2008): Lectures on agriculture,
animal science, pre-veterinary medicine, horticulture and greenhouse
management.
University of Illinois Teaching Assistant:
Introduction to Genetics (2002-2003): Basic understanding of plant and animal
genetics
Genetics and Animal Breeding (2003): Knowledge of animal selection and
genetic inheritance
University of Illinois Guest Lecturer:
Quantitative Genetics (2002-2003): Lectures on probability and statistics
Publications:
Schlesser HN, Shanks RD, Berger, PJ, Healey MH. Graphical approach to evaluate
genetic estimates of calf survival. J Dairy Sci 2009; 92: 2166-2173.
Schlesser HN, Simon L, Hofmann MC, Murphy KM, Murphy T, Hess RA, Cooke PS.
Effects of ETV5 (ets variant gene 5) on testis and body growth, time course of
spermatogonial stem cell loss, and fertility in mice. Biol Reprod 2008; 78: 483-489.
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Meeting Abstracts and Presentations:
Schlesser, HN., Cimafranca, MA., Hess, RA., Cooke, P.S. 2008. Effect of neonatal
genistein exposure on the male mouse reproductive system. Abstract-Poster Session,
29th Annual Minisymposium on Reproductive Biology, Evenston IL.
Schlesser, H.N., Hofmann, M-C., Carnes, K., Murphy, K.M., Hess, R.A., Cooke, P.S.
2007. Time course of spermatogonial stem cell loss in ERM knockout mice: Critical
neonatal effects of ERM. Abstract-Poster Session, Testis Workshop, Tampa, FL.
Schlesser, H.N., Hofmann, M-C., Carnes, K., Murphy, K.M., Hess, R.A., Cooke, P.S.
2006. Time course of spermatogonial stem cell loss in ERM knockout mice: Critical
neonatal effects of ERM. Abstract-Oral Session, 27th Annual Minisymposium on
Reproductive Biology, Evenston IL.
Bora, A., Schlesser, H.N., Southey, B.R., Ko, Y., Stearns, T., Le Conte, Y., Brillet, C.,
Whitfield, C.W., Robinson, G.E., Rodriguez-Zas, S.L. 2005. Statistical Approaches to
Brain Gene ExpressionTrajectories across Honey Bees Fostered in Different Social
Environments. Abstract-Poster Session, PAG XIII Annual Meeting, San Diego, CA.
Ko, Y., Southey, B.R., Bora, A., Schlesser, H.N., Stearns, T. M., Le Conte, Y., Brillet,
C., Whitfield, C.W., Robinson, G.E., Rodriguez-Zas, S.L. 2005. Statistical
characterization of gene expression trajectories across developmental stages in honey
bees. Abstract-Poster Session, PAG XIII Annual Meeting, San Diego, CA.
Stearns, T. M., Southey, B.R., Ko, Y., Bora, A., Schlesser, H., Le Conte, Y., Brillet, C.,
Whitfield, C.W., Robinson, G.E., Rodriguez-Zas, S.L. 2005. Delineation Of
Differential Gene Gxpression Patterns Between Maturation Ages In Apis mellifera
Mellifera Bees Using Different Statistical Approaches. Abstract-Poster Session, PAG
XIII Annual Meeting, San Diego, CA.
Schlesser, H., R.D. Shanks, P.J. Berger, M.H. Healey. 2004. Allele effect for calf survival
estimated for US Holstein population. Abstract-Poster Session, Dairy Science Society
Annual Meeting, St. Louis, MO
Schlesser, H., R.D. Shanks, P.J. Berger, M.H. Healey. 2003. Graphical approach to
evaluate genetic estimates of calf survival. J. Dairy Sci. 86(Suppl. 1): 196 (Abstr).
Berger, P.J., J. Koltes, M.H. Healey, M.S. Ashwell, R.D. Shanks, H. Schlesser, and H.A.
Lewin. 2003. Putative quantitative trait loci affecting perinatal survival in eleven
Holstein families. J. Dairy Sci. 86(Suppl. 1): 196 (Abstr).
Kolter, J., P.J.Berger, J.C.M. Dekkers, M.H. Healey, M.S. Ashwell, R.D. Shanks, H.
Schlesser, and H.A. Lewin. 2003. Evidence for major quantitative trait loci affecting
perinatal survival in two elite Holstein sire families. Proc. The John M. Airy Beef
Cattle Symposium. Visions for genetics and breeding. p131. Ed. J. M. Reecy.
Other Credentials:
PADI Master Instructor for Scuba Diving 2005 – Current
Tae Kwon Do Instructor 1997-1999
SAS Programming
PhD Duties:
Mouse colony maintenance and care, Animal breeding, Genotyping, Gel electrophoresis,
Dosing of mouse pups, Immunohistochemistry, Western blot analysis, Cell Culture
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(Primary and cell line), Chromatin Immunoprecipitation Assay (ChIP), In vitro
fertilization, Artificial Insemination, Sperm capacitation, Sperm acrosome reaction,
Assessment of daily sperm production, Seminar presentations, Tissue
collection/embedding/sectioning
Offices Held:
Treasurer Amateur Radio Emergency Services (ARES) 2004 - 2008
Assistant Emergency Coordinator of Public Service ARES 2005 - 2009
Professional Memberships:
Endocrinology Society 2008 – present
American Society of Andrology 2005 – present
Society for the Study of Reproduction 2005 – 2006
American Dairy Science Association 2002 - 2004
Honors and Awards:
Gamma Sigma Delta – The Honor Society of Agriculture 2004
Edmund J. James Scholar for Academic Year 2000-2001, 2001-2002
Deans List: Fall 1999, Spring 2000, Fall 2000, Spring 2002, Fall 2002
Phi Eta Sigma – National Honor Society for Freshman 2000
Alpha Lambda Delta – National Academic Honor Society 2000
The National Society of Collegiate Scholars 2000
Graduated undergraduate with High Honors
References:
Dr. Paul Cooke: PhD advisor, [email protected], (217) 333-6825
Dr. Roger Shanks: Masters advisor, [email protected], (540) 553-1630
Dr. James Anderson: Professor in Human and Community Development,
[email protected], (217) 244-0285
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