Environmental Toxicology and Pharmacology 51 (2017) 56–70

Contents lists available at ScienceDirect

Environmental Toxicology and Pharmacology

journal homepage: www.elsevier.com/locate/etap

Human exposure to endocrine disrupting chemicals: effects on the

male and female reproductive systems
Stavros Sifakis a , Vasilis P. Androutsopoulos b,∗ , Aristeidis M. Tsatsakis c ,
Demetrios A. Spandidos b
a
Department of Obstetrics and Gynecology, University of Crete, Medical School, Heraklion, GR 71003, Greece
b
Department of Clinical Virology, University of Crete, Medical School, Heraklion, GR 71003, Greece
c
Department of Toxicology, University of Crete, Medical School, Heraklion, GR 71003, Greece

a r t i c l e i n f o a b s t r a c t

Article history: Endocrine disrupting chemicals (EDCs) comprise a group of chemical compounds that have been exam-
Received 20 December 2016 ined extensively due to the potential harmful effects in the health of human populations. During the past
Received in revised form 22 February 2017 decades, particular focus has been given to the harmful effects of EDCs to the reproductive system. The
Accepted 26 February 2017
estimation of human exposure to EDCs can be broadly categorized into occupational and environmental
Available online 6 March 2017
exposure, and has been a major challenge due to the structural diversity of the chemicals that are derived
by many different sources at doses below the limit of detection used by conventional methodologies. Ani-
Keywords:
mal and in vitro studies have supported the conclusion that endocrine disrupting chemicals affect the
Endocrine disrupting chemicals
Reproductive system
hormone dependent pathways responsible for male and female gonadal development, either through
Exposure direct interaction with hormone receptors or via epigenetic and cell–cycle regulatory modes of action. In
Fertility human populations, the majority of the studies point towards an association between exposure to EDCs
Cancer and male and/or female reproduction system disorders, such as infertility, endometriosis, breast cancer,
Mechanism testicular cancer, poor sperm quality and/or function. Despite promising discoveries, a causal relation-
ship between the reproductive disorders and exposure to specific toxicants is yet to be established, due
to the complexity of the clinical protocols used, the degree of occupational or environmental exposure,
the determination of the variables measured and the sample size of the subjects examined. Future stud-
ies should focus on a uniform system of examining human populations with regard to the exposure to
specific EDCs and the direct effect on the reproductive system.
© 2017 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
2. EDCs and pesticide occupational and environmental exposure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
3. Endocrine disrupting mechanism of action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
3.1. Enzyme and receptor-mediated mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
3.2. Epigenetic mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
3.3. Alternative mechanisms of action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
3.4. Metabolism of EDCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
4. EDCs and female reproductive system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
5. EDCs and male reproductive system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
5.1. Semen quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
5.2. Testicular cancer and cryptorchidism. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .64
5.3. EDCs modes of action on the male reproductive system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
5.4. EDCs, obesity, and male fertility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

∗ Corresponding author.
E-mail address: androuts@uoc.gr (V.P. Androutsopoulos).

http://dx.doi.org/10.1016/j.etap.2017.02.024
1382-6689/© 2017 Elsevier B.V. All rights reserved.
 S. Sifakis et al. / Environmental Toxicology and Pharmacology 51 (2017) 56–70 57

6. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

1. Introduction

Endocrine disrupting chemicals (EDCs) are exogenous chem-

ical entities or mixtures of compounds that interfere with any
aspect of hormone action responsible for the maintenance of
homeostasis and the regulation of developmental processes. The
research conducted in the field of EDCs has increased consider-
ably over the last two decades, due to the potentially harmful
effects to the human body and the increasing knowledge in the
areas of developmental biology and environmental toxicology. It
is well known that compounds with endocrine disrupting mech-
anism of action can seriously affect human reproduction and
several studies have demonstrated a considerable decrease in fer-
tility biomarkers, notably sperm counts, in human populations
that have been exposed to EDCs (Safe, 2013; Slutsky et al., 1999;
Perry et al., 2007; Jouannet et al., 2001). The toxic effects of EDCs
have resulted in the restriction of their use in countries where
evidence of extensive exposure is ample (Knez, 2013). In some
westernized countries, the use of certain EDCs, such as polychlo-
rinated biphenyls or polybrominated diphenyl ethers has been
banned. However, in some cases the human exposure to EDCs is Fig. 1. Chemical structures of the main endocrine disrupting agents investigated in
inevitable, when such chemicals are used in the occupation or this study.
are widely dispersed across the environment. A specific example
includes the chemical class of pesticides. A pesticide is “any sub-
stance or a combination of substances used to prevent or eradicate 2. EDCs and pesticide occupational and environmental
unwanted insects, including vectors of diseases in human-beings exposure
and animals, weeds or fungi, in order to enhance food produc-
tion and aid production processing, storage, transport or marketing Organophosphate (OPs) pesticides are one of the most widely
of the food and agricultural commodities” (Sifakis et al., 2011; used class of pesticides for agricultural purposes (Koureas et al.,
Mehrpour et al., 2014; Abdollahi et al., 2004). Pesticides can act 2014). They are metabolized by xenobiotic metabolizing enzymes,
as EDCs, depending on their structure and their precise mecha- notably the cytochrome P450 (CYP) and the Paraoxonase (PON)
nism of action. Examples of pesticides with endocrine disrupting families of enzymes and are therefore not persistent in the envi-
properties include the organochlorines and the organophospho- ronment (Androutsopoulos et al., 2013). The exposure to OPs
rus pesticides. The exposure of human populations to pesticides is is assessed by the detection of their corresponding secondary
a matter of major concern, irrespective of their structural diver- metabolites notably the dialkyl phosphates in biological matrices
sity. It has been estimated that approximately 750,000 individuals such as urine (Androutsopoulos et al., 2013; Sokoloff et al., 2016;
are exposed annually to new forms of pesticides in the USA (Perry, Omoike et al., 2015). The exposure of humans to OPs is either
2008). The fate of pesticide chemicals depends on their metabolism through the occupation or the environment. Occupational expo-
by xenobiotic detoxification enzymes and their degradation to sure depends on a variety of factors, such as personal protective
smaller molecular weight compounds that may persist in the equipment (gloves, boots, and glasses), precaution practice (safety
environment. clothes and routine washing of hands), the type of job that deter-
The input of pesticides and EDCs on the reproductive system mines the frequency and extent of exposure (farmers, loaders, and
has been well defined. The adverse effects of the latter compounds applicators), specific training in the safe use and handling of pes-
as regards the health of the reproductive system include infertility, ticides and legislation (directives and laws to protect employees
cancer, cryptorchidism, decreased semen quality and hypospadias. from occupational toxins, periodic health surveillance) (Mehrpour
The major determinants of the toxic effects caused by pesticides et al., 2014). Dermal exposure is considered to be the main route for
and EDCs on the human reproductive system include the dose, occupational exposure and is defined as the transfer of the pesticide
the frequency of exposure, the route of exposure and the geno- from the surface of the foliage to the skin of the worker (Kapka-
typic characteristics of the exposed subjects (Hernández et al., Skrzypczak et al., 2011). The general quantitative levels for the
2013). In this review, a comprehensive synopsis of EDCs on the dermal exposure are difficult to estimate, due to the wide variabil-
human reproductive system is presented, with particular focus on ity in exposure parameters, such as the surface area exposed and
the class of pesticide chemicals and the commonly encountered the frequency of contact, although it has been shown that expo-
endocrine disrupting molecule Bisphenol A (BPA). The aspects of sure can occur in the presence of protective clothing, as the latter
endocrine disruption, with regard to reproductive disorders are could be the source of additional exposure (Jurewicz et al., 2009;
thoroughly addressed by evidence derived from in vitro, in vivo and Poet and McDougal, 2002). The activity of plasma cholinesterase
epidemiological studies. Information regarding EDCs and/or pes- has been used as a marker of chronic exposure to OP components
ticide exposure is also presented. The chemical structures of the (De Silva et al., 2006).
most common EDCs that are investigated in this study are outlined The second route of exposure to pesticides and EDCs involves
in Fig. 1. the environment. The factors that influence the extent of exposure
58 S. Sifakis et al. / Environmental Toxicology and Pharmacology 51 (2017) 56–70
are namely, the type of chemical, the route of exposure, the timing
and duration, the age, the gender and the genetic predisposition
(Mehrpour et al., 2014). For example, the US environmental pro-
tection agency (EPA) reference dose of the EDC BPA that is used as
a synthetic of plastic and epoxy resins is 50 ␮g/kg/day, although
the actual daily human exposure may be much higher (Taylor
et al., 2011; Tzatzarakis et al., 2016). As regards organochlorine-
type compounds this class includes environmental pollutants with
EDC-type of activity and organochlorine pesticides that are either
in use or have been banned for several years and are present in the
environment due to the lack of metabolism and biodegradation.
Organochlorine compounds such as DDT and dioxins are not metab-
olized by the human body and accumulate for a long period of time.
In addition, such compounds appear to be a lot more persistent
in the environment compared to organophosphorus compounds.
Risk assessment studies have shown that risk quotient estimates
for heptachlor epoxide, endrin, dieldrin, DDT, and endosulfan pos-
sessed potential risk for aquatic organisms based on the observed
mean concentrations in the water sediments Clarias gariepinus
and Tilapia zilli from the Owan river (Ogbeide et al., 2015). The
estimated average daily intake for the aforementioned organochlo-
rine pesticides was above their respective acceptable average daily
intake (Ogbeide et al., 2015). The organochlorine exposure of DDT
and sumDDT-compounds and hexachlorobenzene was found to be
elevated in fish samples on St. Lawrence Island that was previously
used as a defense site, indicating that contamination of pesticides
in the food chain is possible and that it can have a major impact
of exposure in human populations (Byrne et al., 2015). In contrast
to the aforementioned observations, the contamination of soil by
DDT, DDD and DDE in the Xiangfen County in China was not an
issue of concern as regards the long-term health impact for adults
and children (Ma et al., 2016). This is possibly due to the low con-
centration of the organochlorine compounds that was estimated to
427.81 ng/g and is unlikely to affect significantly the health of the
exposed population (Ma et al., 2016). Thus, the exact contamina-
tion of the environment and the consequent exposure of the human
populations to DDT-related compounds depend on the regional and
geographical burden and require extensive risk-assessment and
monitoring studies regarding their bioaccumulation and environ-
mental degradation.
The detection of pesticide exposure in several biological matri-
ces has been employed as a method to identify potential cases
of reproductive system disorders. The research work conducted
by our group has been pioneering in the determination of expo-
sure assessment of pesticides to human populations. The detection
of Dialkyl phosphates (DAPs) and pyrethroid pesticides has been
documented in meconium, maternal hair, infant hair, cord blood,
amniotic fluid and reproductive tissue samples of pregnant women
and normal volunteers by HPLC/MS methodologies (Tsatsakis
et al., 2009; Tsatsakis, 2008; Tsatsakis, 2006; Relakis et al., 1999;
Koutroulakis et al., 2014; Koureas et al., 2012). In addition BPA
was detected in the hair samples of 69 Greek volunteers using the
aforementioned assays (Tzatzarakis et al., 2015).
3. Endocrine disrupting mechanism of action
3.1. Enzyme and receptor-mediated mechanisms
EDCs exert their toxicity by interfering with the normal
hormonal homeostatic mechanisms that promote growth and
development of tissues. The classical action with respect to the
reproductive system involves interference of EDCs with hormone
binding to the corresponding receptor, notably the androgen recep-
tor (AR) or the estrogen receptor (ER). Following binding to a
receptor the EDC can trigger two types of responses: a hormonal
response that is termed an agonistic effect, and/or a lack of hor-
monal response that is termed an antagonistic action. Agonistic
effects of MTX, an organochlorine pesticide used as an insecti-
cide that was indented to replace DDT, have been reported for
the estrogen receptor subtypes ER␣ and ER␤, whereas an oppo-
site response was noted for the androgen receptor (Gaido et al.,
2000; Waters et al., 2001; Mrema et al., 2013). A similar anti-
androgenic effect has been noted for the environmental polluting
chemical tetrachlorodibenzo-p-dioxin (TCDD) that has been shown
to be an inhibitor or antagonist of hormone synthesis (Gore et al.,
2015). An additional example is the insecticide endosulfan that
has demonstrated estrogenic activity and has been shown to cause
ovarian regression in in vitro and in vivo models (Senthilkumaran,
2015). Endosulfan competes with estradiol (E2) for binding to
the estrogen receptor, yet with lower affinity (Lemaire et al.,
2006). This in turn affects gene expression of sex specific genes.
In contrast to the ovarian-mediated biological effects, endosulfan
caused the decrease of several male reproductive system associated
genes such as testis-related transcription factors (Sox9a and wt1)
(Rajakumar et al., 2012), whereas BPA has been classified as a pro-
totypical non-steroidal estrogen receptor that possesses binding
affinity for the ER␣ and ER␤ receptors (Welshons et al., 2003).
In addition to the hormone-related receptors, EDCs act on
enzymes involved in steroidogenesis and the metabolism of hor-
mones. For example phthalates, which constitute a particular class
of pesticides, exert anti-androgenic effects by inhibiting the syn-
thesis of testosterone in Leydig cells, as a result of direct CYP17
inhibition (Foster, 2005). Furthermore, EDCs have been shown to
inhibit the activity of 5-␣ reductase, which is one of the most impor-
tant enzymes involved in the production of dihydrotestosterone
from testosterone and thus in the regulation of the masculin-
ization of the external gentalia and the prostate (Fisher, 2004).
Thiophosphates, a class of organophosphorous pesticides, inhibit
P450 enzymes namely, CYP3A4 and CYP1A2 that are involved in
the metabolism of estrone and testosterone in the liver (Usmani
et al., 2003, Usmani et al., 2006).
In addition, EDCs have been shown to affect hormone receptor
expression in animal models. Bisphenol A (BPA) caused dysregula-
tion of steroid receptors and co-regulators in the rat testes, whereas
in utero exposure of Sprague–Dawley rats to diethylhexyl phthalate
(DEHP) resulted in increased mineralocorticoid receptor mRNA and
protein expression in adult interstitial Leydig cells (Skinner et al.,
2011; Martinez–Arguelles et al., 2009).
It is important to highlight that EDCs exhibit multiple hormone-
binding activities irrespective of binding to hormonal receptors. For
example, dichlorodiphenyltrichloroethane (DDT) is an agonist for
the oestrogen receptor, whereas one of the metabolites of DDT is an
anti-androgen (Kelce et al., 1995). BPA is a thyroid hormone antago-
nist in addition to its estrogenic and androgenic activity (Moriyama
et al., 2002; Wetherill et al., 2005).
As regards the effects of EDCs on the thyroid function, sev-
eral studies have indicated that EDCs interfere with TH and TSH
signaling via a majority of pathways that result in alteration of
deiodinase activity, inhibition of TH excretion and/or metabolism,
blockage of iodine uptake by thyroid cells, competitive inhibi-
tion of the thyroid transport protein TTR and antagonism of
complexes that originate from the thyroid hormone responsive
elements (TREs) (Noyes et al., 2013; Viluksela et al., 2004; Butt
and Stapleton, 2013; Nishimura et al., 2002; Meerts et al., 2002;
Hamers et al., 2011). The structural similarity of TH with spe-
cific TH-EDCs namely, brominated flame retardants, hydroxylated
polychlorinated biphenyls (PCBs) metabolites, and dioxin (PCDD)
results in binding with the high affinity TH transport protein TTR
and consequently inhibition of T4-TTR binding (Grimm et al., 2013).
The effects of EDCs are extended beyond the disruption of nor-
mal TH levels to the alteration of the lipid profile and glucose
 S. Sifakis et al. / Environmental Toxicology and Pharmacology 51 (2017) 56–70
homeostasis of the exposed individuals, since TH regulates lipid and
glucose metabolism. For example, BPA has been shown to cause
hyperlipidemia and increase abdominal adipose tissue in mice
(Miyawaki et al., 2007). Finally, certain TH-EDCs further interfere
with the actions of hormones that act via nuclear receptors such as
sex hormones (estrogen, progesterone, and androgen) and/or inter-
act with their corresponding nuclear receptors (ER, PR, and AR).
At the molecular level, EDCs can affect the expression of
steroid and sex hormone related enzymes by inducing their corre-
sponding transcription, via binding to nuclear receptors. Notably,
organochlorine pesticides and dioxins have been documented to
bind with considerable potency to the aryl hydrocarbon recep-
tor (AhR) that induces the expression of CYP1 genes that in turn
metabolizes estradiol (E2) to hydroxylated derivatives (Park et al.,
2017). TCDD can further interfere with ovarian steroidogenesis and
previous studies have shown that it can decrease progesterone,
androstenedione, testosterone and E2 levels in vitro and in vivo
(Karman et al., 2012a,b; Chen et al., 2009). The organochlorine pes-
ticide MTX can inhibit ovarian steroidogenesis and consequently
the production of E2, testosterone and androstenedione by the
downregulation of the expression of CYP19␣1, CYP17␣1, CYP11␣1
and 17␤-hydroxysteroid dehydrogenase enzymes (Basavarajappa
et al., 2011). A similar anti-estrogenic effect has been noted for the
MTX metabolite 2,2-bis(p-hydroxyphenyl)-1,1, 1-trichloroethane
(HPTE) that can reduce the FSH-stimulated synthesis of proges-
terone and estrogen by decreasing the expression of CYP11␣1 and
CYP19␣1 (Zachow and Uzumcu, 2006).
The induction of endogenous-compound-metabolizing and/or
xenobiotic-metabolizing enzymes has been reported to occur by
several EDCs and affects their metabolic fate in the human body. For
example, the induction of the AhR and the E2 metabolizing enzyme
CYP1B1 has been documented to occur for the EDC class of phtha-
lates (Zachow and Uzumcu, 2006). As in the cases of MTX and TCDD
phthalates decrease the levels of E2 and DEHP in vivo as a result of
an additional downregulation of CYP19a1 expression (Gupta et al.,
2010; Hannon et al., 2015).
3.2. Epigenetic mechanisms
The epigenetic effects of EDCs refer to heritable changes in gene
function in the absence of DNA sequence alterations. Notably, epi-
genetic effects are mediated by transcription factors that repress
or enhance the transcription of specific genes. The main epige-
netic mechanisms include DNA methylation, post-translational
modifications of histone proteins (histone acetylation and histone
deacetylation) and non-coding RNA (Jirtle and Skinner, 2007). DNA
methylation usually leads to a reduction of gene expression, since
it affects the binding of transcription factors to the DNA. Post-
translational modifications of the histone proteins at specific amino
acid residues, such as lysine may alter the structure and function
of chromatin (Turner, 2009).
Generally it is accepted that acetylation of histones results
in the activation of transcription as a result of the relaxation of
chromatin, whereas deacetylation results in the silencing of genes
and transcriptional repression. Non-coding RNAs are transcripts of
sequences that do not code for proteins but regulate the expression
of genes in a cis and trans manner (Chang et al., 2006). They are
involved in specific functions such as X-chromosome inactivation,
genomic imprinting and developmental patterning and differenti-
ation.
Studies in laboratory animals have demonstrated that EDCs such
as diethylstilbestrol (DES) can activate the expression of immedi-
ately early genes in neonatal development, such as c-fos, c-jun,
c-myc and lactoferrin that are upregulated in childhood (Nelson
et al., 1994). This effect was accompanied by hypomethylation of
the promoter region of the lactoferrin gene in the adult uterus (Li
59
et al., 1997), whereas when the animals were exposed during adult-
hood to the same interval, no such pattern of methylation was
observed. The exact etiology was attributed to hypomethylation
of the exon-4 of the fos gene that was discovered in subsequent
studies (Li et al., 2003). DES is further associated with hypermeth-
ylation in the promoter and intron regions of the Hoxa-10 gene
that in turn induces anterior transformations of the reproductive
tract (Block et al., 2000). Importantly, the interference of EDCs to
the epigenetic regulation of several genes has been associated with
additional cell signaling pathways that act upstream, including the
ER and the PI3 K/Akt pathways (Bredfeldt et al., 2010; Falck and
Forsberg, 1996).
The epigenetic changes in the ovary have been documented
for the organochlorine pesticide methoxychlor (MTX). The use of
bisulfite-sequencing PCR and methylation-specific PCR has demon-
strated that MTX caused hypermethylation in multiple CpGs in
the ER␤ promoter sequences in a study conducted by Zama and
Uzumcu in 2009 (Zama and Uzumcu, 2009). The extent of DNA
methylation in the promoter regions appears to be age-dependent,
as the aforementioned genes reveal a different methylation pattern
in neonatal models of ovaries following exposure to EDCs (MTX,
DES) (Zama and Uzumcu, 2009; Tang et al., 2008). With regard
to the gene targets that are methylated by MTX, genome-wide
methylation analyses have indicated that the majority of candi-
date genomic regions include transcription factors and ribosomal
proteins. Specifically targets such as PTEN and IGF have been iden-
tified as critical signaling pathways that are hypermethylated by
MTX (Zama and Uzumcu, 2009).
3.3. Alternative mechanisms of action
Several other modes of action of EDCs as regards the disruption
of the normal reproductive system development have been pro-
posed that are not directly related to the regulation of hormonal
function. For example, mono-(2ethyl-hexyl-phthalate) (MEHP) has
been shown to affect ovarian follicle health by inducing the gen-
eration of reactive oxygen species and by increasing the level of
oxidative stress, while concomitantly disrupting the expression of
the enzymes superoxide dismutase 1 (SOD1) and glutathione per-
oxidase (GPX) (Wang et al., 2012). The disruption of the normal cell
cycle control has also been documented for the phthalate dibutyl
phthalate (DBP) that inhibits the expression of cyclin E1, cyclin A2
and cyclin B1 and results in cell cycle arrest and subsequent inhibi-
tion of antral follicle growth (Craig et al., 2013). In addition, BPA was
shown to induce apoptosis, via a caspase 3-mediated mechanism
in female rat follicles and to regress the luteum development that
is considered as the main cause of infertility (Lee et al., 2013). Fig. 2
represents a summary of the modes of EDCs action with regard to
the adverse effects caused in the reproductive system.
3.4. Metabolism of EDCs
The in vitro metabolism of EDCs is considered a major factor
that contributes to their corresponding adverse effects. Gener-
ally organochlorine compounds such as dioxin and DDT are not
metabolized readily due to the extensive number of chlorine
atoms in their structure that remain unconjugated. Consequently,
such compounds exhibit a considerably long half-life in the
human body that contributes to the continuous stimulation of
the adverse effects (Androutsopoulos et al., 2013). Organochlo-
rine compounds including environmental pollutants and pesticides
can induce the expression of phase I and II metabolizing enzymes
notably CYP1A1, CYP1B1, UGT1A6 and NQQ1 via the Aryl hydro-
carbon receptor (AhR) (Androutsopoulos et al., 2009). This in
turn facilitates metabolism of polycyclic aromatic hydrocarbons,
nitrosamines and various environmental chemicals to biologically
60 S. Sifakis et al. / Environmental Toxicology and Pharmacology 51 (2017) 56–70

Fig. 2. The molecular mechanism of action of EDCs with regard to the disorders of the reproductive system. (A) Enzyme inhibition and enzyme up/down–regulation. Pesticides,
such as methoxychlor can down–regulate the expression of steroid metabolizing enzymes via antagonism with the estrogen receptor (ER). Environmental pollutants, such as
TCDD can induce the expression of steroid and carcinogen–metabolizing enzymes via the Aryl hydrocarbon receptor (AhR). (B) The epigenetic effects of EDCs. Methoxychlor
has been shown to hypermethylate the promoter of the ER␤ receptor and IGF and PTEN sequences. (C) The effects of EDCs on the antral follicle cell cycle. BPA has been reported
to down–regulate the expression of cyclin E1, cyclin A2 and cyclin B1 and results in cell cycle arrest and consequently apoptosis via a caspase–3 mediated mechanism.

active metabolites (Androutsopoulos et al., 2009). With regard
to the EDC methoxychlor, it has been shown that the active
metabolite hydroxyphenyltrichloroethane exerts approximately
100-fold greater estrogenic activity and potency in the target
organs of animals exposed to methoxychlor compared with the
parent compound (Aoyama and Chapin, 2014). Methoxychlor
mainly undergoes demethylation reactions and hydroxylation by
cytochrome P450, such as CYP3A4, CYP3A5 and CYP2B6. In contrast
to MTX, BPA has been shown to be notably metabolized via sulfa-
tion and glucuronidation reactions to the metabolites Bisphenol
A-4-ulfate and Bisphenol A-4D-glucuronide, respectively that do
not possess activity on endocrine receptors (Gramec Skledar et al.,
2016). Hydroxylation reactions have further been documented for
BPA by phase I enzymes. The metabolite 5-hydroxybisphenol A
is considered to be less potent than the parent compound (Elsby
et al., 2001). The metabolism of phthalates follows a similar path-
way to that of BPA with glucuronidation reactions demonstrating
a key process in the detoxification of the compound activity,
whereas organophosphorus pesticides are notably metabolized to
the common metabolite dialkyl phosphate (DAP) via the enzyme
Paraoxonase 1 (PON1). An initial desulfuration reaction occurs by
cytochrome P450 enzymes namely, CYP2B6, CYP2C19 and CYP3A4
to the corresponding oxon metabolite that is in turn hydrolyzed by
PON1 to DAP (Androutsopoulos et al., 2013). The metabolites of OPs
retain a part of the activity of the parent compound, although the
majority of the studies in human populations have focused notably
on the detection of OPs metabolites rather than the evaluation of
their corresponding biological activity.
4. EDCs and female reproductive system
The main biological adverse effects of EDCs with regard to the
development of the reproductive system are attributed to folliculo-
genesis. The primordial follicles evolve to primary, pre-antral and
antral follicles. EDCs, such as BPA, MTX, TCDD, and phthalates can
interfere with the development of the aforementioned types of fol-
licles (Fig. 3A). Toxicity caused to the antral follicles by EDCs can
lead to infertility. BPA has been associated with female fertility
problems, polycystic ovary syndrome and endometriosis, whereas
in women undergoing fertility treatments BPA levels have been
associated with decreased antral follicle counts and a reduction
in the number of oocytes (Kandaraki et al., 2011; Caserta et al.,
2014; Souter et al., 2013). The results reported in human popu-
lations have been verified by experimental animal studies, where
 S. Sifakis et al. / Environmental Toxicology and Pharmacology 51 (2017) 56–70 61

Fig. 3. The pathophysiological and biochemical mechanism of action of EDCs with regard to the disorders of the reproductive system. (A) The effects of EDCs on folliculogenesis.
The growth of primordial follicles to antral follicles is affected by exposure to EDCs. The antral follicle is responsible for the ovulation and the production of sex steroid
hormones. The exposure to EDCs can lead to infertility and impaired production of steroid hormones. (B) The life stages of the individuals exposed to EDCs are a major
factor that determines the extent of the adverse effects. Notably severe health disorders are initiated at the early stages of life (infancy, childhood) that manifest following
a considerably period of time (adulthood) to specific symptoms and diseases. (C) Biochemical modes of EDCs action with regard to infertility and adverse effects on the
female reproductive system. Inhibition of aromatase by EDCs leads to reduced plasma E2 levels, impaired oocyte development and consequently infertility. (D) Inhibition
of 5␣–reductase by EDCs interferes with the metabolism of testosterone to dihydrotestosterone (DHT) in the male reproductive system and in turn with the binding and
activation of the androgen receptor(AR)-DHT complex, resulting in impairment in the transcription of the corresponding AR target genes.

the latter compound was shown to reduce the primordial follicle
pool and lead to premature ovarian failure (Wang et al., 2014).
Similarly, MTX inhibits folliculogenesis in vivo by increasing the
expression of the anti-Müllerian hormone in the pre-antral and
early antral follicles (Uzumcu et al., 2006), whereas TCDD pos-
sesses anti-proliferative effects on the rat ovary and can interfere
with ovarian steroidogenesis (Heimler et al., 1998; Karman et al.,
2012a,b). An increase in the atretic follicles and a decrease in the
primary and secondary follicle numbers have been noted in rats
following 600 mg/kg of exposure to the phthalate DHEP for 60 con-
secutive days (Xu et al., 2010).
Although the toxic effects of EDCs in female reproduction in
experimental animals are apparent, in human populations this
association is not directly related to causality, as some studies have
indicated positive associations and others no associations of EDC
exposure and female reproductive system disorders (Rochester,
2013; Ehrlich et al., 2012a,b; Yang et al., 2009; Philippat et al.,
2012). This apparent contradiction occurs mainly due to the sever-
ity of exposure that determines the actual dose of daily intake. It
is a lot more difficult to determine the environmental exposure
to EDCs and the dose of human exposure, compared to laboratory
investigations where the conditions and notably the dose treat-
ment are adequately controlled. Consequently, the differences that
are noted among studies with regard to the biological effects of
EDCs are notably attributed to the different experimental condi-
tions and study protocols that define the dose of exposure. Although
the mechanism of EDCs action can be adequately demonstrated
in animal models, the dose of exposure used is frequently super-
ficial compared with the actual exposure that occurs in human
populations. For example, mineralocorticoid receptor dysfunction
was demonstrated in rats, following exposure to DEHP at a dose
range of 100 to 950 mg from gestational day 14 to gestational
day 19 (Martinez–Arguelles et al., 2009). This dose is consider-
ably greater than the actual exposure of humans to DEHP, whereas
the life stage of animal exposure corresponds to early childhood
in humans, which will in theory deteriorate the adverse effects
caused compared with the later stages of life. Similarly, Uzumcu and
coworkers used a very high concentration range of 1–500 mg/kg of
methoxychlor (MTX) in postnatal female rats in order to demon-
strate reduction in ovarian weight by 50, 100, and 500 mg/kg of
MTX treatment (Uzumcu et al., 2006). In contrast to the aforemen-
tioned studies, Wang et al., reported doses of exposure of 0.5, 20,
and 50 ␮g/kg of BPA in newborn FVB mice in order to demonstrated
fertility reduction as an adverse effect of BPA (Wang et al., 2014).
Although the doses used in the latter study are similar with the
exposure to BPA noted in human populations (2.78 ng/ml in urine,
1.6 ␮g/L in serum), the majority of the studies conducted in humans
comprised adult and not newborn subjects. Taken together, the
findings derived by the aforementioned studies suggested that the
experimental dose of exposure and the selection of the target group
under investigation were major determinants of the “adversity” of
EDCs.
In addition, research in humans and EDCs is less in quantity,
compared to animal and in vitro studies (Rochester, 2013). For
example BPA has been studied in humans with regard to the
adverse health outcomes on the reproductive system by various
research groups during the period of 2002 and 2012 and it was
found that higher BPA exposure, as demonstrated by higher BPA
concentration in plasma or urine, was associated with poorer ovar-
ian response, reduced number of mature oocytes, reduced number
of fertilized oocytes, lower peak E2 in response to hyperstimulation
with human chorionic gonadotrophin (hCG), decreased probability
of fertilization, higher implantation failure and infertility (Ehrlich
et al., 2012a,b; Mok-Lin et al., 2010; Bloom et al., 2011; Fujimoto
et al., 2011; Ehrlich et al., 2012b; Caserta et al., 2013) (Table 1).
As regards the concentration of the sex hormone prolactin, it was
found that it was significantly higher in BPA exposed women vs.
non-exposed women in an occupational case control study of 155
subjects (Hao et al., 2011;), whereas BPA exposure has been further
linked to polycystic ovary syndrome as documented by a cross-
sectional study in 171 women (Kandaraki et al., 2011).
In terms of breast cancer development and BPA, non-significant
associations have been reported by the studies of Yang et al. and
Aschengrau et al. (Yang et al., 2009; Aschengrau et al., 1998), while
62 S. Sifakis et al. / Environmental Toxicology and Pharmacology 51 (2017) 56–70

Table 1
A summary of the studies that have assessed the associations between the exposure of human populations to pesticides and EDC agents and the incidence of disorders
affecting the female reproductive system. (CS: cross sectional study; CC: case-control study; CHS: Cohort study).

Authors, Year Chemical N Results Variables Study Type

Kandaraki et al., BPA 171 (100 PCOS, 71 Normal) Higher BPA levels in PCOS, Hormonal parameters, BMI CS
2011 association of BPA with
androgens
Caserta et al., 2013 BPA, phthalates 61 women Higher BPA and receptor Urinary BPA, EDC levels in CS
levels in infertile group serum, oestrogen and
nuclear receptors
Souter et al., 2013 BPA 209 women on fertility Higher BPA levels Urinary BPA, Antral Follicle CHS
treatment associated with lower AFC count (AFC), FSH
Ehrlich et al., BPA 147 women with IVF cycles Negative association Urinary BPA, oocyte CHS
2012a,b between urinary BPA and maturation, fertilization,
serum peak E2 and oocyte embryo quality
yield
Ehrlich et al., BPA 137 women Positive association Urinary BPA, ␤-hCG, CHS
2012a,b between urinary BPA and implantation failure
implantation failure
Mok-Lin et al., 2010 BPA 84 women Urinary BPA is inversely Urinary BPA, E2 levels, CHS
correlatedwith serum peak number of oocytes
E2
Bloom et al., 2011 BPA 44 women with IVF Serum BPA is associated Serum BPA, E2 levels, CHS
with reduced E2 levels number of oocytes
Fujimoto et al., BPA 58 infertile females, 37 Inverse association Serum BPA, fertilization CHS
2011 male partners between BPA and
fertilization
Yang et al., 2009 BPA 167 women with breast No association with BPA Blood BPA, incidence of CC
cancer, 152 controls and breast cancer breast cancer
Aschengrau et al., BPA, BHA, benzyl 261 women with breast Association with PCBs, Levels of EDCs and CC
1998 phthalate, nonylphenol cancer, 753 controls 4-octylphenol and breast pesticides and incidence of
cancer breast cancer
Philippat et al., BPA, phenols, 288 mother-newborn pairs Positive/Negative Levels of EDC and CC
2012 phthalates (72 cases, 216 controls) association of pesticides, birth weight,
benzophenone-3/2,5 DCP head circumference, birth
with male birth weight, length
respectively
Sugiura–Ogasawara BPA 77 women (45 cases with Serum BPA is associated Serum BPA, ANA, NK cells, CC
et al., 2005 miscarriages, 32 controls) with recurrent miscarriage prolactin, progesterone,
TSH, fT4
Buck Louis et al., BPA, phthalates 626 women mECPP, mEHHP, mEOHP, BPA, 14 phthalate CHS
2013 mEHP, mBP and mCMHP metabolites,
were associated with endometriosis, age, BMI,
endometriosis creatinine
Raanan et al., 2015 DAP, DE, DM 359 mothers and children Early life exposure (1–7 Urinary DAP, DE, DM and CHS
years) to DAP, DE, DM is metabolites, respiratory
associated with respiratory symptoms in childhood
symptoms
Mahalingajah et al., HCB, DDT, DDE 720 women with IVF Serum HCB is associated Serum HCB, DDT, DDE and CHS
2012 with failed implantation metabolites, fertilization

the latter chemical has been linked with developmental and birth
weight abnormalities of infants, premature delivery and miscar-
riages (Sugiura–Ogasawara et al., 2005; Philippat et al., 2012). The
results of the aforementioned studies are indicative of the severe
adversity of BPA on the female reproductive system. In contrast to
the observations by Sugiura-Ogasawa et al., Cantorinine et al., and
Phillipant and coworkers, the major disadvantage encountered by
such studies was the small number of subjects that were analyzed.
With the exception of the study conducted by Aschengrau et al., the
aforementioned studies comprised a sample size of less than 200
participants. This indicates that more work is required to confirm
these findings. As regards infertility, the studies were not directly
related to the general population, since infertile couples and/or cou-
ples undergoing IVF appeared more sensitive to BPA based on the
endpoints: beta-hCG, embryo quality and decreased probability of
fertilization (Bloom et al., 2011; Chen et al., 2013; Ehrlich et al.,
2012a,b). However, approximately 1/3 of these women had infer-
tility diagnoses due to the female factor, 1/3 due to the male factor
and 1/3 had unexplained infertility (Bloom et al., 2011; Chen et al.,
2013; Ehrlich et al., 2012a,b; Ehrlich et al., 2012b; Mok-Lin et al.,
2010; Fujimoto et al., 2011). This suggests that neither male, nor
female infertility can alone explain the sensitivity to BPA in the
populations examined. A case control study that was published by
Caserta and colleagues revealed that serum BPA was detected in a
significantly higher number of women, compared with controls in
a sample size of 61 participants, whereas the presence of specific
phthalate metabolites and not BPA in urine were associated with
the diagnosis of endometriosis in a study that comprised 14 clini-
cal centers in the US. (Caserta et al., 2013; Buck Louis et al., 2013).
Overall, the data in the literature support that there is some evi-
dence that BPA may contribute to infertility in humans (Rochester,
2013).
Similar effects with those noted in the case of BPA in humans
have been reported for a variety of other EDCs. For exam-
ple epidemiological evidence has supported the conclusion that
there is a strong association between developmental exposure to
organochlorine pesticides and subsequent female fertility prob-
lems (Zama and Uzumcu, 2009; Zama and Uzumcu, 2010). Although
phthalates have been extensively studied for their effects in male
reproduction, there is accumulating evidence that they exert addi-
tional adverse effects in the female reproductive system. The
most commonly studied phthalates are di-(2-ethyl-hexyl) phtha-
late and the metabolite mono-(2-ethyl-hexyl) phthalate (MEHP)
(Craig et al., 2011). The levels of phthalates have been associated
 S. Sifakis et al. / Environmental Toxicology and Pharmacology 51 (2017) 56–70
with significantly higher risk of implantation failure in women
undergoing infertility treatments (Hauser et al., 2016). As regards
DDT and dioxin-type compounds, the evidence in humans is lesser,
since these molecules have been banned for more than 30 years
worldwide. The levels of HCB, DDT and DDE were examined in
the serum of 720 women undergoing in vitro fertilization (IVF) and
the HCB concentrations were associated with failed implantation
compared with DDE and DDT, where no significant difference was
noted (Mahalingajah et al., 2012). The results of the clinical studies
conducted with regard to the association of EDCs with the female
reproductive system are summarized in Table 1.
Accumulating evidence suggests a potential association with
OPs and the disruption of normal female reproductive functions.
Raanan and coworkers reported higher prenatal DAP concen-
trations with the occurrence of respiratory symptoms in early
childhood during the period of 0.5–5 years of age (Raanan et al.,
2015). A similar study in China that comprised 249 pregnant
women indicated that higher levels of DAPs and their metabolites
in the urine of the women was the predominant risk factor for
neonatal neurobehavioral development (Zhang et al., 2014). With
regard to the association of the female reproductive dysfunction
and infertility with the exposure to OPs, the majority of the data in
the literature has been derived by in vivo and experimental studies.
Evidence regarding the impairment of fetal growth and develop-
ment caused by prenatal exposure to OPs in humans has been
associated with the activity of the paraoxonase 1 enzyme (PON1)
in the fetus and during early childhood. This process increases the
vulnerability of the fetus and child to OP poisoning and suggests
that the exposure to OPs has a greater impact on the fetal and
infant growth compared with the respective exposure of the adults
to the same concentration of pesticides (Fig. 3B) (Peiris–John and
Wickremasinghe, 2008).
Overall, the data reported in the literature in human popula-
tions are suggestive of an association between EDCs and disorders
in the female reproductive system. Several discrepancies between
studies are attributed to the complexity of the protocols, the degree
of occupational or environmental exposure and the sample size of
the subjects examined. The timing of exposure is of great impor-
tance since the same dosage exerts diverse effects in individuals of
different age. Furthermore, the exposure during critical develop-
mental phases (intrauterine life, neonatal period, and childhood)
is considered to affect the reproductive system greater compared
with the adulthood (Fig. 3B). The effects noted may be the result of
synergism and/or antagonism, since exposure is determined by a
mixture of toxicants. Furthermore, the results may appear imme-
diately, and/or they may manifest later in life and in the following
generations, with different susceptibility, depending on the genetic
polymorphisms of the exposed individuals. With regard to the
mechanism involved, the contribution of aromatase inhibition by
EDCs to the development of impaired oocytes is one of the main
factors responsible for infertility (Fig. 3C).
5. EDCs and male reproductive system
The adverse effects caused by EDCs in the male reproductive sys-
tem have been a matter of increasing alert during the last decades.
Although several studies from the early 1940s have proposed the
decline in semen quality as the main adverse effect caused by EDCs
on the male reproductive system, the diverging results from the
different study designs were not able to suggest a definitive conclu-
sion regarding the dose, the magnitude of exposure and the type of
chemical that contributed to the occurrence of the adverse effects.
Similar trends of adverse effects on the male reproductive system
have been reported, such as the incidence of cryptorchidism and
testicular cancer that may be related to EDC exposure (Nordkap
63
et al., 2012). The latter adverse effects along with the decrease in
the quality of semen share similar pathophysiological patterns and
are important risk factors for the development of the male fertility
disorders.
5.1. Semen quality
The quality of semen has been investigated as an important vari-
able for the determination of the adverse effects caused by EDCs.
However, initial studies that were conducted in early 1980s were
observational and did not aim at investigating the contribution of
EDC exposure on the developmental defects, with regard to the
male reproductive system. The initial hypothesis was to explore
the cause of a deteriorating semen quality in specific human popu-
lations. A meta-analysis conducted by Carlsen (Carlsen et al., 1992)
demonstrated a significant decline in mean sperm concentrations
from 113 to 66 million/ml. During that period, additional confound-
ing factors, such as the period of abstinence, the methodological
aspects of semen analysis, the age and the fertility were of con-
sideration regarding the decrease in semen quality, although they
were not proven sufficient for a definitive conclusion (Jouannet
et al., 2001). In addition, this trend could have been the outcome
of geographical variation. The follow-up study that was carried out
by Swan and coworkers confirmed that the afore mentioned find-
ings were not the result of bias, as the confounding factors were
accordingly adjusted and an overall decline in the sperm concen-
tration was noted annually at 1.5 and 3.1% of the population in the
USA and Europe, respectively (Swan et al., 1997). The meta-analysis
that was conducted by Swan did however confirm that sperm qual-
ity trends could be affected by the variation in the geographical
regions (Swan et al., 2000). Furthermore, studies that were con-
ducted in North Europe and particularly Denmark have provided
supportive evidence that semen quality can be largely affected by
the location of the human population that is examined (Jørgensen
et al., 2001; Jørgensen et al., 2002). Danish men were reported
to have the lowest semen quality, followed by Norwegian, Scot-
tish and French, whereas the Finnish population had the greatest
(Jørgensen et al., 2001; Jørgensen et al., 2002). In contrast to the
early observations by Jorgensen and colleagues in 2001, subsequent
studies that were conducted in Finland revealed a discouraging
temporal decrease in semen quality in men from the general pop-
ulation in the period between 1998 and 2006 (Jørgensen et al.,
2011; Jørgensen et al., 2012). The data derived by the studies of
Jorgensen and similar researchers suggest that a large proportion
of young men from the general population (approximately 43%)
may exhibit suboptimal sperm counts (less than 40 million/ml)
(Andersson et al., 2008; Jørgensen et al., 2012; Jørgensen et al.,
2011). The aforementioned epidemiological studies suggest that
the different geographical locations of the human population res-
idency, cannot be the sole contributing factor for the decrease in
the sperm count. Thus, alternative environmental factors have to
be considered for the increased incidence of poor semen quality in
specific human populations of North Europe.
Recent studies that aimed at investigating the association of
the exposure to pesticides and the sperm quality have corrobo-
rated the findings by Jørgensen and colleagues. The occupational
exposure to pesticides increases the risk of morphological abnor-
malities in the sperm of farm workers that includes a decline in
sperm count and a decreased percentage of viable sperms. OP pes-
ticides such as parathion and methyl parathion can decrease the
concentration of the sperm by damaging the seminiferous epithe-
lium, while it has been suggested that pesticide exposure affects sex
accessory glands that may also reduce the seminal volume (Perry
et al., 2011; Yucra et al., 2006). The exposure to pesticides reduces
the seminal volume, increases the seminal pH and increases the
abnormal sperm head morphology (Yucra et al., 2006). In addition,
64 S. Sifakis et al. / Environmental Toxicology and Pharmacology 51 (2017) 56–70
Lifeng et al. demonstrated that sperm motility could be affected
by a limited number of pyrethroid pesticides, such as fenvalerate
(Lifeng et al., 2006). Specific adverse effects on the male repro-
ductive system have been reported to occur by organochlorine
pesticides, such as endosulfan and DDT due to the disruption of the
hypothalamic–pituitary testes axis and the direct interaction with
the sex steroid receptors in the target tissue (Mehrpour et al., 2014).
As regards organophosphate pesticides, the exposure of male farm-
ers from three different communities in Malaysia to malathion
and/or paraquat resulted in significantly lower values of sperm
concentration, pH, mean volumes motility of sperm cells, com-
pared with the non-exposed group (Hossain et al., 2010). The results
have been supported by experimental animal studies where both
malathion and parathion were shown to lower the body weights,
the reproductive organ weights and the sperm counts of adult rats
(Narayana et al., 2006; Geng et al., 2015). The mechanism of action
was attributed to the induction of spermatogenic apoptosis via the
modulation of the expression of the proteins Bax and Bcl-2 and
the concomitant reduction in the levels of the reproductive hor-
mones LH, FSH and testosterone (Geng et al., 2015). The indirect
modes of pesticide action involve the interference with the neu-
roendocrine control at the testicular level that results in the altered
production and release of testosterone, the excess production of
free radicals and the induction of oxidative stress (Talamanca et al.,
2001; Abdollahi et al., 2004). A direct interaction with regard to
the modulation of the testosterone/oestradiol hormonal concen-
tration has been noted for the class of polychlorinated biphenyls in
266 fertile men, indicating that structurally similar compounds to
the class of organochlorine pesticides that possess endocrine dis-
rupting activity can affect the male reproductive system (Petersen
et al., 2015).
The adverse effects of phthalates on sperm quality were con-
firmed by ex vivo studies, where spermatozoa were incubated in
vitro and exposed with high concentrations of phthalates (Pant
et al., 2011). It was reported that the sperm motility was decreased
and that cytotoxicity was caused at long-term exposures (> 3 days)
of human semen samples to the metabolite DEHP (Pant et al., 2011).
In parallel DHEP has been shown to inhibit testosterone production,
when cultured in vitro with explants derived from human testes
(Desdoits–Lethimonier et al., 2012).
Studies that were conducted in human populations corrobo-
rated the in vitro findings and suggested that exposure to phthalate
metabolites is correlated with lower motility of spermatozoa in
men from subfertile couples (Duty et al., 2003). The DNA damage
induced in spermatozoa, the sperm motility and the morphology
of the spermatozoa were weakly associated with the exposure to
phthalate (Hauser et al., 2007; Duty et al., 2004; Hauser et al.,
2006; Liu et al., 2012), whereas with regard to the disruption of the
hormonal function, an inverse association between MEHP metabo-
lite increase and testosterone and oestradiol levels was reported
(Meeker et al., 2009).
In addition to the phthalates, the human exposure to the EDC
BPA affects the concentration of testosterone in plasma in men
and women, whereas the hormones acting upstream such as FSH
are downregulated, possibly due to the severity of the endocrine
disrupting action. Mendiola and colleagues investigated the expo-
sure of 360 fertile men to BPA and highlighted that the levels of
the compound were inversely correlated with the concentration of
indices of androgens, such as testosterone (Mendiola et al., 2010). In
contrast to the findings by Mendiola et al., non-significant trends
between urinary BPA levels and oestradiol/testosterone index as
well as lower sperm counts and DNA damage, were reported by
Meeker and co-workers, suggesting the lack of sufficient infor-
mation in the literature to support conclusive evidence regarding
strong associations between the aforementioned 2 parameters
(Meeker et al., 2010).
The urinary levels of BPA have been associated with lower sperm
quality parameters (Li et al., 2011) in men that were occupationally
exposed to this chemical. Of note is that the median urine con-
centration estimated was approximately 70 times lower than the
accepted daily intake according to the USEPA, whereas the control
group exhibited exposure doses of approximately 2,000 times less
than the accepted daily intake. This finding highlighted that BPA can
cause harmful effects at doses much lower than the USEPA accepted
daily intake (Li et al., 2011). BPA was further shown to be associ-
ated with lower sperm count; sperm morphology, sperm motion
and DNA damage (Meeker et al., 2010). As regards hormonal levels,
BPA was associated with higher FSH, lower inhibin B and a lower
oestradiol: testosterone ratio (Meeker et al., 2010). BPA was further
associated with a decreased free androgen-index and higher testos-
terone in men in a study that comprised 375 fertile men (Mendiola
et al., 2010).
Similar data have been published for PCBs, which is a struc-
turally related class of environmental pollutants. In the study
conducted by Petersen and colleagues, 266 fertile men were exam-
ined with regard to the exposure to PCBs, hormone concentration
and semen quality (Petersen et al., 2015). The ratio of testos-
terone/oestradiol and the levels of FSH and sex hormone binding
globulin (SHBG) were positively correlated with serum PCB lev-
els, whereas no association was noted between the latter and
the semen quality variables, such as semen volume, sperm con-
centration, total sperm count, sperm morphology and period of
abstinence (Petersen et al., 2015). The results of the clinical stud-
ies conducted with regard to the association of EDCs with the male
reproductive system are summarized in Table 2.
5.2. Testicular cancer and cryptorchidism
The incidence of testicular cancer has notably been investigated
in cohorts located in Denmark and Norway, where it was shown
that approximately 1% of men will develop testicular cancer during
their life (Skakkebaek et al., 2007). It is important to note that during
the period between 1998 and 2002 the incidence of testicular can-
cer in men in Norway was approximately 5-fold higher compared
with the corresponding incidence in Spain (Chia et al., 2010; Llanes
et al., 2008). This finding suggests that the incidence may be higher
in developed rather than developing countries. Such differences
were attributed initially to the heterogeneity of the populations
examined, with regard to the ethnic origin and the immigration
frequency from countries with a high morbidity rate of this can-
cer (Nordkap et al., 2012). The prevalence of cryptorchidism has
received a considerable increase in Denmark during the period
of 1997–2001 (Boisen et al., 2004), whereas a lower incidence
was observed in England and Finland, respectively (Boisen et al.,
2004; Boisen et al., 2005). Such geographic trends were followed
by specialized studies where it was shown that maternal-lifestyle
factors, such as the increased intake of pain killers, notably para-
cetamol and ibuprofen might be associated with cryptorchidism in
the offsprings (Jensen et al., 2007; Thorup et al., 2006; Stoorgaard
et al., 2003). It has been suggested that the exposure to environmen-
tal chemicals, such as phthalates that results in poor semen quality
may coincide with the development of testicular cancer, cryptor-
chidism and hypospadias (Nordkap et al., 2012). The “so called”
testicular dysgenesis syndrome hypothesis is based on the combi-
nation of clinical observations with the aforementioned 3 disorders
of the male reproductive system. Particularly the link between tes-
ticular cancer and impaired semen quality has been proposed as
an explanation for the aberrant differentiation of the gonads and
the subsequent formation of the Leydig cells that produces the
hormone testosterone and results in the initiation of the masculin-
ization of the male fetus (Sharpe, 2006; Sharpe et al., 2003). Despite
such promising observations, the exact contribution of pesticide
 S. Sifakis et al. / Environmental Toxicology and Pharmacology 51 (2017) 56–70 65

Table 2
A summary of the studies that have assessed the associations between the exposure of human populations to pesticides and EDC agents and the incidence of disorders
affecting the male reproductive system. (CS: cross sectional study; CC: case-control study; CHS: Cohort study; L: longitudinal study; PS: prospective study).

Authors, Year Chemical N Results Variables Study Type

Carlsen et al., 1992 N/A 14,947 men A genuine decline in semen Mean sperm density and L
quality is observed over the mean seminal volume
last 50 years in USA, Europe
and Australia
Swan et al., 1997 N/A 14,947 men Decline in sperm density in Mean sperm density L
USA and Europe
Swan et al., 2000 N/A 20,833 men Decline in sperm density Mean sperm density L
1.5%/year and 3%/year in USA
and Europe/Australia
respectively
Jørgensen et al., N/A 1082 fertile men Danish men had the lowest Sperm concentration and CS
2001 sperm concentrations, morphology
followed by French and
Scottish men. Finnish men had
the highest sperm counts
Jørgensen et al., N/A 968 men Finish and Estonian Sperm concentration CS
2002 populations had higher sperm
concentrations than
Norwegian and Dannish
Jørgensen et al., N/A 858 men (sperm Decline in sperm Sperm concentration, PS–CS
2011 parameters), 5974 men concentrations/counts in Finish sperm counts and
(testicular cancer registry) men over the years 1978–1987 incidence of testicular
with a simultaneous increase cancer
in testicular cancer 1954–2008
Jørgensen et al., N/A 4867 men Increase in sperm Sperm concentration, PS–CS
2012 concentration and/or counts in sperm counts, semen
Danish men over the years volume, sperm
1996–2010 morphology
Perry et al., 2011 OPs 94 cases and 95 controls Odds of being a case were Sperm concentration, CC
greater in men with higher sperm motility, urine DMP
urinary concentrations of and DAP concentrations
dimethylphosphate (DMP)
Yucra et al., 2006 OPs 31 sprayers and 80 controls Pesticide sprayers had Sperm concentration, CC
significantly reduced seminal sperm motility, semen
volume, percentage of motility, volume, sperm
percentage of sperm with morphology, LH, T levels
normal morphology, serum LH
and T levels, increased time of
liquefaction, seminal pH,
percentage of immature sperm
morphology,
Lifeng et al., 2006 Fenvalerate, 32 cases, 46 internal Sperm motion parameters; fenvalerate, toluene, CC
toluene, xylene controls, 22 external sperm progression and beat xylene concentrations in
controls cross frequency in the work place, dermal
exposure group were contamination of
decreased significantly fenvalerate, semen
compared with the internal qualities
and the external control
groups. Opposite effect for
abnormality rate of viscidity
and coagulation
Hossain et al., 2010 Paraquat, 152 farmers (62 exposed) The mean values of volume, Volume, pH, sperm CS
malathion pH, sperm concentration, concentration, motility,
motility, and WBC count were morphology and WBC
significantly less in the count were examined and
exposed group. Greater risk of recorded
abnormal semen parameters
was noted in the exposed
group
Petersen et al., PCBs 266 fertile men positive association between PCB exposure, semen CS
2015 serum–PCB and quality and reproductive
testosterone/estradiol ratio, hormones
elevated PCB exposure was
associated with increased
serum concentrations of SHBG,
no association between serum
PCB concentration and semen
quality variables
Pant et al., 2011 Phthalates 180 men, 65 correlation between phthalate Semen analysis, phthalate CC
asthenospermic, 65 ester diethylhexyl phthalate, ester concentration, in vitro
oligoasthenospermic, 50 di-n-butyl phthalate (DEHP cell viability
fertile and DBP) and sperm motility
both in vitro and in vivo
conditions
66 S. Sifakis et al. / Environmental Toxicology and Pharmacology 51 (2017) 56–70

Table 2 (Continued)

Duty et al., 2003 Phthalates 168 men from subfertile Dose–response relations Urinary phthalate CC
couples between tertiles of mono-butyl metabolites, semen
phthalates and tertiles of analysis, sperm
mono-benzyl phthalate and concentration, sperm
sperm motility and sperm motility and morphology
concentration, respectively
Liu et al., 2012 Phthalates 150 men Borderline–significant Urinary levels of MMP, CHS
dose–response relationship MEP, MBP, MBzP, MEHP,
between MBP and sperm MEOHP, semen volume,
concentration, significant sperm concentration,
positive correlation between motility and sperm motion
MEP and straight–line velocity parameters
of sperm motion
Hauser et al., 2007 Phthalates 379 men from an infertility Monoethyl phthalate (MEP), Urinary concentrations of CS
clinic Mono-(2-ethylhexyl) phthalate phthalate metabolites,
(MEHP) were associated with sperm DNA damage
increased DNA damage measurements (comet
assay)
Hauser et al., 2006 Phthalates 463 male partners of Dose–response relationships of Urinary concentrations of CHS
subfertile couples MBP with low sperm phthalates, semen
concentration and motility parameters
Meeker et al., 2009 Phthalates 425 men from an infertility MEHP was inversely associated Urinary concentrations of CS
clinic with testosterone, estradiol, MEHP and other phthalate
and free androgen index, the monoester metabolites,
ratio of testosterone to serum levels of
estradiol was positively testosterone, estradiol,
associated with MEHP SHBG, FSH, LH, inhibin B,
prolactin
Mendiola et al., BPA 375 fertile men no significant associations FSH, LH, testosterone, CS
2010 between semen parameter and inhibin B, estradiol, SHBG,
urinary BPA concentration, androgen index
significant inverse association
between urinary BPA
concentration and FAI levels,
SHBG and the FAI/LH ratio
Meeker et al., 2010 BPA 190 men from an infertility Urinary BPA concentration was urinary BPA CS
clinic associated with declines in concentrations, semen
sperm concentration, motility, quality, and sperm DNA
and morphology of along with damage
a 10% increase in sperm DNA
damage
Li et al., 2011 BPA 218 men increasing urine BPA level was Semen parameters CHS
statistically significantly
associated with decreased
sperm concentration,
decreased total sperm count,
decreased sperm vitality
decreased sperm motility, no
association of urine BPA level
with semen volume or
abnormal sperm morphology
Boisen et al., 2004 1068 Danish boys, 1494 Prevalence of cryptorchidism Testicular position, L
Finnish boys, 5798 Finnish at birth was 9.0% in Denmark demographics,
newborns and 2.4% in Finland. The rate in confounding factors
Denmark was significantly
higher than that reported 40
years ago.

and EDC exposure to the development of testicular cancer has not
been fully elucidated to date, although a combination of genetic
background and environmental factors have been demonstrated to
account for the reduced sperm counts and the formation of the dis-
ease. In addition, the latter effects may not be recognized until 2–3
decades following fetal exposure to the corresponding chemical
compounds. This highlights the limitations of the clinical stud-
ies and the importance of the human population epidemiological
assessment studies (Publicover et al., 2007; Nordkap et al., 2012).
5.3. EDCs modes of action on the male reproductive system
The effects of EDCs on the male reproductive system are notably
attributed to the interactions of these chemicals with the normal
production of steroid hormones that are responsible for the ini-
tiation of prostate development and the masculinization of the
Wolffian ducts in order to form the epididymis, seminal vesi-
cles, and vas deferens (Sweeney et al., 2015). The inhibition of
the enzymes 5␣-reductase and aromatase by EDCs is one of the
main mechanisms responsible for the adverse effects noted, as
5␣-reductase is required for the conversion of the androgens
to testosterone, whereas aromatase catalyzes the metabolism of
androgens to estrogen (Fig. 3C, D) (Sweeney et al., 2015). The modu-
lation of the expression levels of the two estrogen receptor isoforms
namely, ER␣ and ER␤ is an additional mode of action of certain
EDCs. The expression of ER␤ is maintained during morphogene-
sis and cellular differentiation in humans and is decreased in early
puberty. The expression of the receptor is increased in response to
elevated levels of androgens in plasma that may be associated with
benign prostate hyperplasia and/or prostate cancer. It has been
shown that early life exposure of neonatal rats to BPA enhances the
prostate susceptibility to estrogen-induced hyperplasia (Ho et al.,
 S. Sifakis et al. / Environmental Toxicology and Pharmacology 51 (2017) 56–70
2006). This effect is possibly due to the androgenic action of BPA,
although epigenetic etiologies with regard to genes involved in
prostate cellular proliferation have been proposed (Sweeney et al.,
2015; Ho et al., 2006).
5.4. EDCs, obesity, and male fertility
The interaction of EDCs with male fertility is further associated
with the obesity status of the individuals exposed. It has been sug-
gested that the biological effects of certain EDCs are prolonged in
obese individuals, due to the greater retention of the substances in
the subcutaneous fat mass (Cardoso et al., 2017). Fat soluble tox-
icants, such as PCBs and dioxin-type compounds can be stored in
the subcutaneous adipose tissue, resulting in an exacerbation of
the endocrine disrupting effects. The bioaccumulation of obeso-
gens is greater in obese individuals compared with lean subjects,
due to a larger fat mass that reduces the clearance rate of the tox-
icants over time (Jung et al., 1997). Mechanistically, EDCs such as
BPA and phthalates may promote insulin resistance accounting for
reduced gonadotrophin secretion at the hypothalamic–pituitary
level (Cardoso et al., 2017). Furthermore, permanent exposure to
EDCs stored in adipose tissue (obesogens), may increase aromatase
activity that in turn increases E2 levels. The increased produc-
tion of E2 can compromise downstream signaling events of the
Hypothalamic–pituitary axis and consequently alter the spermato-
genic process (Cardoso et al., 2017). Animal studies have shown that
exposure to DDT induced transgenerational actions that promoted
obesity. The next generations of the gestating female rats that were
exposed to 50 mg/kg/day of DDT, at gestational days of 8–14, devel-
oped obesity and contained sperm epimutations (Skinner et al.,
2013). With regard to human populations there are few epidemi-
ological studies that have focused on the effects of obesogens in
sperm quality and consequently it is not possible to draw definite
conclusions on the exact interaction of the aforementioned com-
pounds with the molecular mechanisms involved in the production
and function of human sperm (Cardoso et al., 2017).
6. Conclusion
Endocrine disrupting chemicals comprise a class of compounds
that can potentially cause harmful effects to the male and female
reproductive systems. There has been significant progress in the
identification of the biological action of EDCs by in vivo and in vitro
experimental studies. In addition to the classical action of EDCs
that includes the agonism and/or antagonism with hormone and
nuclear receptors, the significant scientific advances in the field
of molecular biology have demonstrated that endocrine disrup-
tion by several compounds can interfere with the cell cycle, the
apoptotic machinery and the epigenetic regulation of the target
cells. In contrast to the experimental evidence, data derived from
clinical and epidemiological studies have been somewhat contro-
versial as to what extent EDCs can affect the male and female
reproductive systems. This discrepancy among clinical studies is
attributed notably to the complexity of the clinical protocols used,
the degree of occupational or environmental exposure, the selec-
tion of the target group under investigation, the determination of
the variables measured and the sample size of the subjects exam-
ined. The findings can be broadly categorized into three different
classes: 1) studies that have shown strong evidence of associa-
tion between the adverse effects caused by EDCs and the extent
of exposure, 2) studies that have shown evidence of some associ-
ation between the adverse effects caused by EDCs and the extent
of exposure and 3) studies that have shown no evidence of associ-
ation between the adverse effects caused by EDCs and the extent
of exposure. The explanations for such contradictory conclusions
67
are not clear, although several factors have been proposed to affect
the overall outcome of the study results as mentioned previously.
With regard to the male reproductive system, the contribution of
geographical and seasonal variation of the semen parameters must
be considered. The majority of the epidemiological studies that
have examined chemical exposure and the associated semen qual-
ity deterioration are cross-sectional. The measurement of exposure
and the possible health impairment are conducted simultaneously
in the cross-sectional design that may result in skewed findings.
Consequently, a longitudinal design is required in studies of semen
quality. Despite contradictory observations the overall conclu-
sion points toward a positive association between exposure to
EDCs and reproductive system disorders such as female infertility,
miscarriage, endometriosis, cancer, semen quality, and testicular
dysgenesis, although the most critical parameter that is subject
to consideration is the frequency and dose of exposure. Future
studies should focus on a uniform system of the investigation of
human populations with regard to the exposure to specific EDCs
and the direct effect on the reproductive system. In addition, the
use of advanced molecular biology techniques that are employed to
evaluate DNA damage in spermatozoa namely, single cell gel elec-
trophoresis assay (COMET), terminal transferase dUTP Nick End
Labelling (TUNEL), sperm chromatin structure assay (SCSA) and
in situ nick translation (ISNT) as well as and sperm aneuploidy
must be included. Finally, scientists should adopt a methodologi-
cal strategy for the implementation of stricter rules and guidelines
on the safety of the use of chemicals and their effects on human
reproduction.
Acknowledgments
This work did not receive funding from a grant application.
References
Abdollahi, M., Ranjbar, A., Shadnia, S., Nikfar, S., Rezaie, A., 2004. Pesticides and
oxidative stress: a review. Med. Sci. Monit. 10, RA141–RA147.
Andersson, A.M., Jørgensen, N., Main, K.M., Toppari, J., Rajpert–De Meyts, E., Leffers,
H., Juul, A., Jensen, T.K., Shakkebaek, N.E., 2008. Adverse trends in male
reproductive health: we may have reached a crucial “tipping point”. Int. J.
Androl. 31, 74–80.
Androutsopoulos, V.P., Tsatsakis, A.M., Spandidos, D.A., 2009. Cytochrome P450
CYP1A1: wider roles in cancer progression or prevention. BMC Cancer 9, 187.
Androutsopoulos, V.P., Hernandez, A.F., Liesivuori, J., Tsatsakis, A.M., 2013. A
mechanistic overview of health associated effects of low levels of
organochlorine and organophosphorous pesticides. Toxicology 307, 89–94.
Aoyama, H., Chapin, R.E., 2014. Reproductive toxicities of methoxychlor based on
estrogenic properties of the compound and its estrogenic metabolite,
hydroxyphenyltrichloroethane. Vitam. Horm. 94, 193–210.
Aschengrau, A., Coogan, P.F., Quinn, M., Cashins, L.J., 1998. Occupational exposure
to estrogenic chemicals and the occurrence of breast cancer: an exploratory
analysis. Am. J. Ind. Med. 34, 6–14.
Basavarajappa, M.S., Craig, Z.R., Hernandez-Ochoa, I., Paulose, T., Leslie, T.C., Flaws,
J.A., 2011. Methoxychlor reduces estradiol levels by altering steroidogenesis
and metabolism in mouse antral follicles in vitro. Toxicol. Appl. Pharmacol.
253, 161–169.
Block, K., Kardana, A., Igarashi, P., Taylor, H.S., 2000. In utero diethylstilbestrol
(DES) exposure alters Hox gene expression in the developing mullerian
system. FASEB J. 14, 1101–1108.
Bloom, M.S., Kim, D., Vom Saal, F.S., Taylor, J.A., Cheng, G., Lamb, J.D., Fujimoto, V.Y.,
2011. Bisphenol A exposure reduces the estradiol response to gonadotropin
stimulation during in vitro fertilization. Fertil. Steril. 96, 672–677.
Boisen, K.A., Chellakooty, M., Schmidt, I.M., Kai, C.M., Damgaard, I.N., Suomi, A.M.,
Toppari, J., Skakkebaek, N.E., Main, K.M., 2005. Hypospadias in a cohort of 1072
Danish newborn boys: prevalence and relationship to placental weight,
anthropometrical measurements at birth, and reproductive hormone levels at
three months of age. J. Clin. Endocrinol. Metab. 90, 4041–4046.
Boisen, K.A., Kaleva, M., Main, K.M., Virtanen, H.E., Haavisto, A.M., Schmidt, I.M.,
Chellakooty, M., Damgaard, I.N., Mau, C., Reunanen, M., Skakkebaek, N.E.,
Toppari, J., 2004. Difference in prevalence of congenital cryptorchidism in
infants between two Nordic countries. Lancet 363, 1264–1269.
Bredfeldt, T.G., Greathouse, K.L., Safe, S.H., Hung, M.C., Bedford, M.T., Walker, C.L.,
2010. Xenoetsrogen-induced regulation of EZH2 and histone methylation via
estrogen receptor signalling to PI3 K/AKT. Mol. Endocrinol. 24, 993–1006.
68 S. Sifakis et al. / Environmental Toxicology and Pharmacology 51 (2017) 56–70
Buck Louis, G.M., Peterson, C.M., Chen, Z., Croughan, M., Sundaram, R., Stanford, J.,
Varner, M.W., Kennedy, A., Giudice, L., Fujimoto, V.Y., Sun, L., Wang, L., Guo, Y.,
Kannan, K., 2013. Bisphenol A and phthalates and endometriosis: the
endometriosis: Natural history, diagnosis and outcomes study. Fertil. Steril.
100, 162–169.
Butt, C.M., Stapleton, H.M., 2013. Inhibition of thyroid hormone sulfotransferase
activity by brominated flame retardants and halogenated phenolics. Chem.
Res. Toxicol. 26, 1692–1702.
Byrne, S., Miller, P., Waghiyi, V., Buck, C.L., von Hippel, F.A., Carpenter, D.O., 2015.
Persistent organochlorine pesticide exposure related to a formerly used
defence site on St. Lawrence Island, Alaska: data from sentinel fish and human
sera. J. Toxicol. Environ. Health A. 78, 976–992.
Cardoso, A.M., Alves, M.G., Mathur, P.P., Oliveira, P.F., Cavaco, J.E., Rato, L., 2017.
Obesogens and male fertility. Obes. Rev. 18, 109–125.
Carlsen, E., Giwercman, A., Keiding, N., Skakkebaek, N.E., 1992. Evidence for
decreasing quality of semen during past 50 years. BMJ 305, 609–613.
Caserta, D., Bordi, G., Ciardo, F., Marci, R., La Rocca, C., Tait, S., Bergamasco, B.,
Stecca, L., Mantovani, A., Guerranti, C., Fanello, E.L., Perra, G., Borghini, F.,
Focardi, S.E., Moscarini, M., 2013. The influence of endocrine disruptors in a
selected population of infertile women. Gynecol. Endocrinol. 29, 444–447.
Caserta, D., Di Segni, N., Mallozi, M., Giovanale, V., Mantovani, A., Marci, R.,
Moscarini, M., 2014. Bisphenol A and the female reproductive tract: an
overview of recent laboratory evidence and epidemiological studies. Reprod.
Biol. Endocrinol. 12, 37.
Chang, S.C., Tucker, T., Thorogood, N.P., Brown, C.J., 2006. Mechanisms of
X-chromosome inactivation. Front. Biosci. 11, 852–866.
Chen, M., Tang, R., Fu, G., Xu, B., Zhu, P., Qiao, S., Chen, X., Xu, B., Qin, Y., Lu, C., Hang,
B., Xia, Y., Wang, X., 2013. Association of exposure to phenols and idiopathic
male fertility. J. Hazard. Material. 250-251, 115–121.
Chen, X., Ma, X.M., Ma, S.W., Goenraads, P.J., Zhang, C.M., Liu, J., Zhao, L.J., Sun, M.,
Tang, N.J., 2009. Proteomic analysis of the rat ovary following chronic low-dose
exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). J. Toxicol. Environ.
Health A 72, 717–726.
Chia, V.M., Quraishi, S.M., Devesa, S.S., Purdue, M.P., Cook, M.B., McGlynn, K.A.,
2010. Interventional trends in the incidence of testicular cancer, 1973–2002.
Cancer Epidemiol. Biomarkers Prev. 19, 1151–1159.
Craig, Z.R., Hannon, P.R., Wang, W., Ziv-Gal, A., Flaws, J.A., 2013. Di-n-butyl
phthalate disrupts the expression of genes involved in cell cycle and apoptotic
pathways in mouse ovarian antral follicles. Biol. Reprod. 88, 23.
Craig, Z.R., Wang, W., Flaws, J.A., 2011. Endocrine-disrupting chemicals in ovarian
function: effects on steroidogenesis, metabolism and nuclear receptor
signaling. Reproduction 142, 633–646.
Desdoits–Lethimonier, C., Albert, O., Le Bizec, B., Perdu, E., Zalko, D., Courant, F.,
Lesné, L., Guillé, F., Dejucq-Rainsford, N., Jégou, B., 2012. Human testis
steroidogenesis is inhibited by phthalates. Hum. Reprod. 27, 1451–1459.
De Silva, H.J., Samarawickrema, N.A., Wickremasinghe, A.R., 2006. Toxicity due to
organophosphorous compounds: what about chronic exposure? Trans. R. Soc.
Trop. Med. Hyg. 100, 803–806.
Duty, S.M., Silva, M.J., Barr, D.B., Brock, J.W., Ryan, L., Chen, Z., Herrick, R.F.,
Christiani, D.C., Hauser, R., 2003. Phthalate exposure and human semen
parameters. Epidemiology 14, 269–277.
Duty, S.M., Calafat, A.M., Silva, M.J., Brock, J.W., Ryan, L., Chen, Z., Overstreet, J.,
Hauser, R., 2004. The relationship between environmental exposure to
phthalates and computer-aided sperm analysis motion parameters. J. Androl.
25, 293–302.
Ehrlich, S., Williams, P.L., Missmer, S.A., Flaws, J.A., Berry, K.F., Calafat, A.M., Ye, X.,
Petrozza, J.C., Wright, D., Hauser, R., 2012a. Urinary bisphenol A concentrations
and implantation failure among women undergoing in vitro fertilization.
Environ. Health Perspect. 120, 978–983.
Ehrlich, S., Williams, P.L., Missmer, S.A., Flaws, J.A., Ye, X., Calafat, A.M., Petrozza,
J.C., Wright, D., Hauser, R., 2012b. Urinary bisphenol A concentrations and early
reproductive health outcomes among women undergoing IVF. Hum. Reprod.
27, 3583–3592.
Elsby, R., Maggs, J.L., Ashby, J., Park, B.K., 2001. Comparison of the modulatory
effects of human and rat liver microsomal metabolism on the estrogenicity of
bisphenol A: implications for extrapolation to humans. J. Pharmacol. Exp. Ther.
297, 103–113.
Falck, L., Forsberg, J.G., 1996. Immunohistochemical studies on the expression and
estrogen dependency of EGF and its receptor and C-fos proto-oncogene in the
uterus and vagina of normal and neonatally estrogen-treated mice. Anat. Rec.
245, 459–471.
Figà–Talamanca, I., Traina, M.E., Urbani, E., 2001. Occupational exposures to
metals, solvents and pesticides: recent evidence on male reproductive effects
and biological markers. Occup. Med. (Lond.) 51, 174–188.
Fisher, J.S., 2004. Environmental anti-androgens and male reproductive health:
focus on phthalates and testicular dysgenesis syndrome. Reproduction 127,
305–315.
Foster, P.M., 2005. Mode of action: impaired fetal leydig cell function—effects on
male reproductive development produced by certain phthalate esters. Crit.
Rev. Toxicol. 35, 713–719.
Fujimoto, V.Y., Kim, D., vom Saal, F.S., Lamb, J.D., Taylor, J.A., Bloom, M.S., 2011.
Serum unconjugated bisphenol A concentrations in women may adversely
influence oocyte quality during in vitro fertilization. Fertil. Steril. 95,
1816–1819.
Gaido, K.W., Maness, S.C., McDonnell, D.P., Dehal, S.S., Kupfer, D., Safe, S., 2000.
Interaction of methoxychlor and related compounds with estrogen receptor
alpha and beta, and androgen receptor: structure-activity studies. Mol.
Pharmacol. 58, 852–858.
Geng, X., Shao, H., Zhang, Z., Ng, J.C., Peng, C., 2015. Malathion–induced testicular
toxicity is associated with spermatogenic apoptosis and alterations in
testicular enzymes and hormone levels in male Wistar rats. Environ. Toxicol.
Pharmacol. 39, 659–667.
Gore, A.C., Chappell, V.A., Fenton, S.E., Flaws, J.A., Nadal, A., Prins, G.S., Toppari, J.,
Zoeller, R.T., 2015. Executive summary to EDC-2: The endocrine society’s
second scientific statement on endocrine-disrupting chemicals. Endocr. Rev.
36, 593–602.
Gramec Skledar, D., Peterlin, Mašič, L., 2016. Bisphenol A and its analogs: do their
metabolites have endocrine activity? Environ. Toxicol. Pharmacol. 47, 182–199.
Grimm, F.A., Lehmler, H.-J., He, X., Robertson, L.W., Duffel, M.W., 2013. Sulfated
metabolites of polychlorinated biphenyls are high-affinity ligands for the
thyroid hormone transport protein transthyretin. Environ. Health Perspect.
121, 657–662.
Gupta, R.K., Singh, J.M., Leslie, T.C., Meachum, S., Flaws, J.A., Yao, H.H., 2010.
Di-(2-ethylhexyl) phthalate and mono-(2-ethylhexyl) phthalate inhibit growth
and reduce estradiol levels of antral follicles in vitro. Toxicol. Appl. Pharmacol.
242, 224–230.
Hamers, T., Kamstra, J.H., Cenijn, P.H., Pencikova, K., Palkova, L., Simeckova, P.,
et al., 2011. In vitro toxicity profiling of ultrapure non-dioxin-like
polychlorinated biphenyl congeners and their relative toxic contribution to
PCB mixtures in humans. Toxicol. Sci. 121, 88–100.
Hannon, P.R., Brannick, K.E., Wang, W., Gupta, R.K., Flaws, J.A., 2015.
Di(2-ethylhexyl) phthalate inhibits antral follicle growth, induces atresia, and
inhibits steroid hormone production in cultured mouse antral follicles. Toxicol.
Appl. Pharmacol. 284, 42–53.
Hao, J., Wang, J., Zhao, W., Ding, L., Gao, E., Yuan, W., 2011. Effect of bisphenol A
exposure on sex hormone level in occupational women. Wei Sheng Yan Jiu 40,
312–314.
Hauser, R., Gaskins, A.J., Souter, I., Smith, K.W., Dodge, L.E., Ehrlich, S., Meeker, J.D.,
Calafat, A.M., Williams, P.L., EARTH Study Team, 2016. Urinary phthalate
metabolite concentrations and reproductive outcomes among women
undergoing in vitro fertilization: results from the EARTH study. Environ.
Health Perspect. 124, 831–839.
Hauser, R., Meeker, J.D., Duty, S., Silva, M.J., Calafat, A.M., 2006. Altered semen
quality in relation to urinary concentrations of phthalate monoester and
oxidative metabolites. Epidemiology 17, 682–691.
Hauser, R., Meeker, J.D., Singh, N.P., Silva, M.J., Ryan, L., Duty, S., Calafat, A.M., 2007.
DNA damage in human sperm is related to urinary levels of phthalate
monoester and oxidative metabolites. Hum. Reprod. 22, 688–695.
Heimler, I., Trewin, A.L., Chaffin, C.L., Rawlins, R.G., Hutz, R.J., 1998. Modulation of
ovarian follicle maturation and effects on apoptotic cell death in Holtzman rats
exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in utero and
lactationally. Reprod. Toxicol. 12, 69–73.
Hernández, A.F., Parrón, T., Tsatsakis, A.M., Requena, M., Alarcón, R.,
López–Guarnido, O., 2013. Toxic effects of pesticide mixtures at a molecular
level: their relevance to human health. Toxicology 307, 136–145.
Hossain, F., Ali, O., D’Souza, U.J., Naing, D.K., 2010. Effects of pesticide use on semen
quality among farmers in rural areas of Sabah, Malaysia. J. Occup. Health 52,
353–360.
Ho, S.M., Tang, W.Y., Belmonte de Frausto, J., Prins, G.S., 2006. Developmental
exposure to estradiol and bisphenol A increases susceptibility to prostate
carcinogenesis and epigenetically regulates phosphodiesterase type 4 variant.
Cancer Res. 66, 5624–5632.
Jensen, M.S., Toft, G., Thulstrup, A.M., Bonde, J.P., Olsen, J., 2007. Cryptorchidism
according to maternal gestational smoking. Epidemiology 18, 220–225.
Jirtle, R.L., Skinner, M.K., 2007. Environmental epigenomics and disease
susceptibility. Nat. Rev. Genet. 8, 253–262.
Jørgensen, N., Andersen, A.G., Eustache, F., Irvine, D.S., Suominen, J., Petersen, J.H.,
Andersen, A.N., Auger, J., Cawood, E.H., Horte, A., Jensen, T.K., Jouannet, P.,
Keiding, P., Vierula, M., Toppari, J., Skakkebaek, N.E., 2001. Regional differences
in semen quality in Europe. Hum. Reprod. 16, 1012–1019.
Jørgensen, N., Carlsen, E., Nermoen, I., Punab, M., Suominen, J., Andersen, A.G.,
Andersson, A.M., Haugen, T.B., Horte, A., Jensen, T.K., Magnus, Ø., Petersen, J.H.,
Vierula, M., Toppari, J., Skakkebaek, N.E., 2002. East–west gradient in semen
quality in the Nordic–Baltic area: a study of men from the general population
in Denmark, Norway, Estonia and Finland. Hum. Reprod. 17, 2199–
2208.
Jørgensen, N., Joensen, U.N., Jensen, T.K., Jensen, M.B., Almstrup, K., Olesen, I.A.,
Juul, A., Andersson, A.M., Carlsen, E., Petersen, J.H., Toppari, J., Skakkebaek, N.E.,
2012. Human semen quality in the new millennium: a prospective
cross–sectional population based study of 4867 men. BMJ Open 2 (pii),
e000990.
Jørgensen, N., Vierula, M., Jacobsen, R., Pukkala, E., Perheentupa, A., Virtanen, H.E.,
Skakkebaek, N.E., Toppari, J., 2011. Recent adverse trends in semen quality and
testis cancer incidence among Finnish men. Int. J. Androl. 34, e37–e48.
Jouannet, P., Wang, C., Eustache, F., Kold–Jensen, T., Auger, J., 2001. Semen quality
and male reproductive health: the controversy about human sperm
concentration decline. APMIS 109, 333–344.
Jung, D., Becher, H., Edler, L., Flesch–Janys, D., Gurn, P., Konietzko, J., et al., 1997.
Elimination of beta–hexachlorocyclohexane in occupationally exposed
persons. J. Toxicol. Environ. Health 51, 23–34.
Jurewicz, J., Hanke, W., Sobala, W., Ligocka, D., 2009. Assesment of the dermal
exposure to azoxystrobin among women tending cucumbers in selected polish
 S. Sifakis et al. / Environmental Toxicology and Pharmacology 51 (2017) 56–70
greenhouses after restricted entry intervals expired—the role of the protective
gloves. Int. J. Occup. Med. Environ. Health 22, 261–267.
Kandaraki, E., Chatzigeorgiou, A., Livadas, S., Palioura, E., Economou, F., Koutsilieris,
M., Palimeri, S., Panidis, D., Diamanti-Kandarakis, E., 2011. Endocrine
disruptors and polycystic ovary syndrome (PCOS): elevated serum levels of
bisphenol A in women with PCOS. J. Clin. Endocrinol. Metab. 96, E480–E484.
Kapka-Skrzypczak, L., Cyranka, M., Skrzypczak, M., Kruszewski, M., 2011.
Biomonitoring and biomarkers of organophosphate pesticides exposure-state
of the art. Ann. Agricult. Environ. Med. 18, 294–303.
Karman, B.N., Basavarajappa, M.S., Craig, Z.R., Flaws, J.A., 2012a.
2,3,7,8-Tetrachlorodibenzo-p-dioxin activates the aryl hydrocarbon receptor
and alters sex steroid hormone secretion without affecting growth of mouse
antral follicles in vitro. Toxicol. Appl. Pharmacol. 261, 88–96.
Karman, B.N., Basavarajappa, M.S., Hannon, P., Flaws, J.A., 2012b. Dioxin exposure
reduces the steroidogenic capacity of mouse antral follicles mainly at the level
of HSD17B1 without altering atresia. Toxicol. Appl. Pharmacol. 264, 1–12.
Kelce, W.R., Stone, C.R., Laws, S.C., Gray, L.E., Kemppainen, J.A., Wilson, E.M., 1995.
Persistent DDT metabolite p,p –DDE is a potent androgen receptor antagonist.
Nature 375, 581–585.
Knez, 2013. Endocrine-disrupting chemicals and male reproductive health.
Reprod. Biomed. Online 26, 440–448.
Koureas, M., Tsakalof, A., Tsatsakis, A., Hadjichristodoulou, C., 2012. Systematic
review of biomonitoring studies to determine the association between
exposure to organophosphorus and pyrethroid insecticides and human health
outcomes. Toxicol. Lett. 210, 155–168.
Koureas, M., Tsakalof, A., Tzatzarakis, M., Vakonaki, E., Tsatsakis, A.,
Hadjichristodoulou, C., 2014. Biomonitoring of organophosphate exposure of
pesticide sprayers and comparison of exposure levels with other population
groups in Thessaly (Greece). Occup. Environ. Med. 71, 126–133.
Koutroulakis, D., Sifakis, S., Tzatzarakis, M.N., Alegakis, A.K., Theodoropoulou, E.,
Kavvalakis, M.P., Kappou, D., Tsatsakis, A.M., 2014. Dialkyl phosphates in
amniotic fluid as a biomarker of fetal exposure to organophosphates in Crete,
Greece; association with fetal growth. Reprod. Toxicol. 46, 98–105.
Lee, S.G., Kim, J.Y., Chung, J.Y., Kim, Y.J., Park, J.E., Oh, S., Yoon, Y.D., Yoo, K.S., Yoo,
Y.H., Kim, J.M., 2013. Bisphenol A exposure during adulthood causes
augmentation of follicular atresia and luteal regression by decreasing
17␤-estradiol synthesis via downregulation of aromatase in rat ovary. Environ.
Health Perspect. 121, 663–669.
Lemaire, G., Mnif, W., Mauvais, P., Balaguer, P., Rahmani, R., 2006. Activation of
alpha- and beta-estrogen receptors by persistent pesticides in reporter cell
lines. Life Sci. 79, 1160–1169.
Li, D.K., Zhou, Z., Miao, M., He, Y., Wang, J., Ferber, J., Herrinton, L.J., Gao, E., Yuan,
W., 2011. Urine bisphenol–A (BPA) level in relation to semen quality. Fertil.
Steril. 95, 625–630.
Li, S., Hansman, R., Newbold, R., Davis, B., McLachlan, J.A., Barrett, J.C., 2003.
Neonatal diethylstilbestrol exposure induces persistent elevation of c-fos
expression and hypomehtylation in its exon-4 in mouse uterus. Mol. Carcinog.
38, 78–84.
Li, S., Washburn, K.A., Moore, R., Uno, T., Teng, C., Newbold, R.R., McLachlan, J.A.,
Negishi, M., 1997. Developmental exposure to diethylstilbestrol elicits
demethylation of estrogen-responsive lactoferrin gene in mouse uterus.
Cancer Res. 57, 4356–4359.
Lifeng, T., Shoulin, W., Junmin, J., Xuezhao, S., Yannan, L., Qianli, W., Longsheng, C.,
2006. Effects of fenvalerate exposure on semen quality among occupational
workers. Contraception 73, 92–96.
Liu, L., Bao, H., Liu, F., Zhang, J., Shen, H., 2012. Phthalates exposure of Chinese
reproductive age couples and its effect on male semen quality, a primary
study. Environ. Int. 42, 78–83.
Llanes Gonzalez, L., Lujan Galan, M., Rodriguez Garcia, N., Garcia Tello, A.,
Berenguer Sanchez, A., 2008. Trends in the incidence of testicular germ cell
cancer in 300.000 inhabitants Spanish population (1991–2005). Actas Urol.
Esp. 32, 691–695.
Ma, J., Pan, L.B., Yang, X.Y., Liu, X.L., Tao, S.Y., Zhao, L., Qin, X.P., Sun, Z.J., Hou, H.,
Zhou, Y.Z., 2016. DDT, DDD, and DDE in soil of Xiangfen County, China:
residues, sources, spatial distribution, and health risks. Chemosphere 163,
578–583.
Mahalingajah, S., Missmer, S.A., Maity, A., Williams, P.L., Meeker, J.D., Berry, K.,
Ehrlich, S., Perry, M.J., Cramer, D.W., Hauser, R., 2012. Association of
hexachlorobenzene (HCB), dichlorodiphenyltrichloroethane (DDT), and
dichlorodiphenyltrichloroethylene (DDE) with in vitro fertilization outcomes.
Environ. Health Perspect. 120, 316–320.
Martinez–Arguelles, D.B., Culty, M., Zirkin, B.R., Papadopoulos, V., 2009. In utero
exposure to di–(2–ethylhexyl) phthalate decreases mineralocorticoid receptor
expression in the adult testis. Endocrinology 150, 5575–5585.
Meeker, J.D., Calafat, A.M., Hauser, R., 2009. Urinary metabolites of
di(2–ethylhexyl) phthalate are associated with decreased steroid hormone
levels in adult men. J. Androl. 30, 287–297.
Meeker, J.D., Ehrlich, S., Toth, T.L., Wright, D.L., Calafat, A.M., Trisini, A.T., Ye, X.,
Hauser, R., 2010. Semen quality and sperm DNA damage in relation to urinary
bisphenol A among men from an infertility clinic. Reprod. Toxicol. 30,
532–539.
Meerts, I.A., Assink, Y., Cenijn, P.H., Van Den Berg, J.H., Weijers, B.M., Bergman, A.,
et al., 2002. Placental transfer of a hydroxylated polychlorinated biphenyl and
effects on fetal and maternal thyroid hormone homeostasis in the rat. Toxicol.
Sci. 68, 361–371.
69
Mehrpour, O., Karrari, P., Zamani, N., Tsatsakis, A.M., Abdollahi, M., 2014.
Occupational exposure to pesticides and consequences on male semen and
fertility: a review. Toxicol. Lett. 230, 146–156.
Mendiola, J., Jørgensen, N., Andersson, A.M., Calafat, A.M., Ye, X., Redmon, J.B.,
Drobnis, E.Z., Wang, C., Sparks, A., Thurston, S.W., Liu, F., Swan, S.H., 2010. Are
environmental levels of bisphenol A associated with reproductive function in
fertile men? Environ. Health Perspect. 118, 1286–1291.
Miyawaki, J., Sakayama, K., Kato, H., Yamamoto, H., Masuno, H., 2007. Perinatal and
postnatal exposure to bisphenol A increases adipose tissue mass and serum
cholesterol level in mice. J. Atheroscler. Thromb. 14, 245–252.
Mok-Lin, E., Ehrlich, S., Williams, P.L., Petrozza, J., Wright, D.L., Calafat, A.M., Ye, X.,
Hauser, R., 2010. Urinary bisphenol A concentrations and ovarian response
among women undergoing IVF. Int. J. Androl. 33, 385–393.
Moriyama, K., Tagami, T., Akamizu, T., Usui, T., Saijo, M., Kanamoto, N., et al., 2002.
Thyroid hormone action is disrupted by bisphenol A as an antagonist. J. Clin.
Endocrinol. Metab. 87, 5185–5190.
Mrema, E.J., Rubino, F.M., Brambilla, G., Moretto, A., Tsatsakis, A.M., Colosio, C.,
2013. Persistent organochlorinated pesticides and mechanisms of their
toxicity. Toxicology 307, 74–88.
Narayana, K., Prashanthi, N., Nayanatara, A., Kumar, H.H., Abhilash, K., Bairy, K.L.,
2006. Neonatal methyl parathion exposure affects the growth and functions of
the male reproductive system in the adult rat. Folia Morphol. (Warsz) 65,
26–33.
Nelson, K.G., Sakai, Y., Eitzman, B., Steed, T., McLachlan, J., 1994. Exposure to
diethylstilbesterol during a critical developmental period of the mouse
reproductive tract leads to persistent induction of two estrogen-regulated
genes. Cell Growth Differ. 5, 595–606.
Nishimura, N., Miyabara, Y., Sato, M., Yonemoto, J., Tohyama, C., 2002.
Immunohistochemical localization of thyroid stimulating hormone induced by
a low oral dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin in female
Sprague-Dawley rats. Toxicology 171, 73–82.
Nordkap, L., Joensen, U.N., Blomberg Jensen, M., Jørgensen, N., 2012. Regional
differences and temporal trends in male reproductive health disorders: semen
quality may be a sensitive marker of environmental exposures. Mol. Cell
Endocrinol. 355, 221–230.
Noyes, P.D., Lema, S.C., Macaulay, L.J., Douglas, N.K., Stapleton, H.M., 2013. Low
level exposure to the flame retardant BDE-209 reduces thyroid hormone levels
and disrupts thyroid signaling in fathead minnows. Environ. Sci. Technol. 47,
10012–10021.
Ogbeide, O., Tongo, I., Ezemonye, L., 2015. Risk assesment of agricultural pesticides
in water, sediment, and fish from Owan river, Edo State, Nigeria. Environ.
Monit. Assess. 187, 654.
Omoike, O.E., Lewis, R.C., Meeker, J.D., 2015. Associations between urinary
biomarkers of exposure to organophosphate insecticides and serum
reproductive hormones in men from NHANES 1999–2002. Reprod. Toxicol. 53,
99–104.
Pant, N., Pant, A., Shukla, M., Mathur, N., Gupta, Y., Saxena, D., 2011. Environmental
and experimental exposure of phthalate esters: the toxicological consequence
on human sperm. Hum. Exp. Toxicol. 30, 507–514.
Park, S.A., Lee, M.H., Na, H.K., Surh, Y.J., 2017. 4-hydroxyestradiol induces
mammary epithelial cell transformation through Nrf2-mediated heme
oxygenase-1 overexpression. Oncotarget 8, 164–178.
Peiris–John, R.J., Wickremasinghe, R., 2008. Impact of low-level exposure to
organophosphates on human reproduction and survival. Trans. R. Soc. Trop.
Med. Hyg. 102, 239–245.
Perry, M.J., Venners, S.A., Barr, D.B., Xu, X., 2007. Environmental pyrethroid and
organophosphorous insecticide exposures and sperm concentration. Reprod.
Toxicol. 23, 113–118.
Perry, M.J., Venners, S.A., Chen, X., Liu, X., Tang, G., Xing, H., Barr, D.B., Xu, X., 2011.
Organophosphorous pesticide exposures and sperm quality. Reprod. Toxicol.
31, 75–79.
Perry, M.J., 2008. Effects of environmental and occupational pesticide exposure on
human sperm: a systematic review. Hum. Reprod. Update 14, 233–242.
Petersen, M.S., Halling, J., Weihe, P., Jensen, T.K., Grandjean, P., Nielsen, F.,
Jørgensen, N., 2015. Spermatogenic capacity in fertile men with elevated
exposure to polychlorinated biphenyls. Environ. Res. 138, 345–351.
Philippat, C., Mortamais, M., Chevrier, C., Petit, C., Calafat, A.M., Ye, X., Silva, M.J.,
Brambilla, C., Pin, I., Charles, M.A., Cordier, S., Slama, R., 2012. Exposure to
phthalates and phenols during pregnancy and offspring size at birth. Environ.
Health Perspect. 120, 464–470.
Poet, T.S., McDougal, J.N., 2002. Skin absorption and human risk assessment. Chem.
Biol. Interact. 140, 217–227.
Publicover, S., Harper, C.V., Barratt, C., 2007. [Ca2+]i signalling in sperm–making
the most of what you ‘ve got. Nat. Cell Biol. 9, 235–242.
Raanan, R., Harley, K.G., Balmes, J.R., Bradman, A., Lipsett, M., Eskenazi, B., 2015.
Early-life exposure to organophosphate pesticides and pediatric respiratory
symptoms in the CHAMACOS cohort. Environ. Health Perspect. 123, 179–185.
Rajakumar, A., Singh, R., Chakrabarty, S., Murugananthkumar, R., Laldinsangi, C.,
Prathibha, Y., Sudhakumari, C.C., Dutta-Gupta, A., Senthilkumaran, B., 2012.
Endosulfan and flutamide impair testicular development in the juvenile Asian
catfish Clarias batrachus. Aquat. Toxicol. 110-111, 123–132.
Relakis, K., Sifakis, S., Froudarakis, G., Papadopoulou, E., Michalodimitrakis, E.N.,
Koumantakis, E., Tsatsakis, A.M., 1999. Disposition of pesticides and toxicants
in the human reproductive system in cases of acute poisoning. Clin. Exp.
Obstet. Gynecol. 26, 207–210.
70 S. Sifakis et al. / Environmental Toxicology and Pharmacology 51 (2017) 56–70
Rochester, J.R., 2013. Bisphenol A and human health: a review of the literature.
Reprod. Toxicol. 42, 132–155.
Safe, S., 2013. Endocrine disruptors and falling sperm counts: lessons learned or
not! Asian J. Androl. 15, 191–194.
Senthilkumaran, B., 2015. Pesticide- and sex steroid analogue-induced endocrine
disruption differentially targets hypothalamo-hypophyseal-gonadal system
during gametogenesis in teleosts- a review. Gen. Comp. Endocrinol. 219,
136–142.
Sharpe, R.M., McKinnell, C., Kivlin, C., Fisher, J.S., 2003. Proliferation and functional
maturation of Sertoli cells, and their relevance to disorders of testis function in
adulthood. Reproduction 125, 769–784.
Sharpe, R.M., 2006. Pathways of endocrine disruption during male sexual
differentiation and masculinization. Best Pract. Res. Clin. Endocrinol. Metab.
20, 91–110.
Sifakis, S., Tsatsakis, A., Mparmpas, M., Soldin, O.P., 2011. Pesticide exposure and
health related issues in male and female reproductive system. INTECH.
Skakkebaek, N.E., Rajpert–De Meyts, E., Jørgensen, N., Main, K.M., Leffers, H.,
Andersson, A.M., Juul, A., Jensen, T.K., Toppari, J., 2007. Testicular cancer trends
as ‘whistle blowers’ of testicular developmental problems in populations. Int. J.
Androl. 30, 198–204.
Slutsky, M., Levin, J.L., Levy, B.S., 1999. Azoospermia and oligospermia among a
large cohort of DBCP applicators in 12 countries. Int. J. Occup. Environ. Health
5, 116–122.
Sokoloff, K., Fraser, W., Arbuckle, T.E., Fisher, M., Gaudreau, E., LeBlanc, A., Morisset,
A.S., Bouchard, M.F., 2016. Determinants of urinary concentrations of dialkyl
phosphates among pregnant women in Canada–resutls from the MIREC study.
Environ. Int. 94, 133–140.
Souter, I., Smith, K.W., Dimitriadis, I., Ehrlich, S., Williams, P.L., Calafat, A.M.,
Hauser, R., 2013. The association of bisphenol-A urinary concentrations with
antral follicle counts and other measures of ovarian reserve in women
undergoing infertility treatments. Reprod. Toxicol. 42, 224–231.
Stoorgaard, L., Bonde, J.P., Ernst, E., Spano, M., Andersen, C.Y., Frydenberg, M.,
Olsen, J., 2003. Does smoking during pregnancy affect sons’ sperm counts?
Epidemiology 14, 278–286.
Skinner, M.K., Manikham, M., Guerrero–Bosagna, C., 2011. Epigenetic
transgenerational actions of endocrine disruptors. Reprod. Toxicol. 31,
337–343.
Skinner, M.K., Manikkam, M., Tracey, R., Guerrero–Bosagna, C., Haque, M., Nilsson,
E.E., 2013. Ancestral dichlorodiphenyltrichloroethane (DDT) exposure
promotes epigenetic transgenerational inheritance of obesity. BMC Med. 11,
228.
Sugiura–Ogasawara, M., Ozaki, Y., Sonta, S., Makino, T., Suzumori, K., 2005.
Exposure to bisphenol A is associated with recurrent miscarriage. Hum.
Reprod. 20, 2325–2329.
Swan, S.H., Elkin, E.P., Fenster, L., 1997. Have sperm densities declined? a
reanalysis of global trend data. Environ. Health Perspect. 105, 1228–1232.
Swan, S.H., Elkin, E.P., Fenster, L., 2000. The question of declining sperm density
revisited: an analysis of 101 studies published 1934–1996. Environ. Health
Perspect. 108, 961–966.
Sweeney, M.F., Hasan, N., Soto, A.M., Sonnenschein, C., 2015. Environmental
endocrine disruptors: effects on the human male reproductive system. Rev.
Endocr. Metab. Disord. 16, 341–357.
Tang, W.Y., Newbold, R., Mardilovich, K., Jefferson, W., Cheng, R.Y., Medvedovic, M.,
Ho, S.M., 2008. Persistent hypomethylation in the promoter of nucleosomal
binding protein 1 (Nsbp1) correlates with overexpression of Nsbp1 in mouse
uteri neonatally exposed to diethylstilbestrol or genistein. Endocrinology 149,
5922–5931.
Taylor, J.A., Richter, C.A., Ruhlen, R.L., vom Saal, F.S., 2011. Estrogenic
environmental chemicals and drugs: mechanisms for effects on the developing
male urogenital system. J. Steroid Biochem. Mol. Biol. 127, 83–95.
Thorup, J., Cortes, D., Petersen, B.L., 2006. The incidence of bilateral cryptorchidism
is increased and the fertility potential is reduced in sons born to mothers who
have smoked during pregnancy. J. Urol. 176, 734–737.
Tsatsakis, A.M., Tzatzarakis, M.N., Koutroulakis, D., Toutoudaki, M., Sifakis, S., 2009.
Dialkyl phosphates in meconium as a biomarker of prenatal exposure to
organophosphate pesticides: a study on pregnant women of rural areas in
Crete, Greece. Xenobiotica 39, 364–373.
Tsatsakis, A.M., 2008. Commentary on the levels of pesticide exposure in general
and rural population based on the article titled “A comparison of infant hair,
cord blood and meconium analysis to detect fetal exposure to environmental
pesticides” by Ostrea et al. Environ. Res. 106, 277–283, Environ. Res. 109
(2009) 352–353.
Tsatsakis, A.M., 2006. ‘Maternal hair–an appropriate matrix for detecting maternal
exposure to pesticides during pregnancy’. Environ. Res. 102, 365.
Turner, B.M., 2009. Epigenetic responses to environmental change and their
evolutionary implications. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 364,
3403–3418.
Tzatzarakis, M.N., Karzi, V., Vakonaki, E., Goumenou, M., Kavvalakis, M., Stivaktakis,
P., et al., 2016. Bisphenol A in soft drinks and canned foods and data evaluation.
Food Addit. Contam. Part B Surveill. 11, 1–6.
Tzatzarakis, M.N., Vakonaki, E., Kavvalakis, M.P., Barmpas, M., Kokkinakis, E.N.,
Xenos, K., Tsatsakis, A.M., 2015. Biomonitoring of bisphenol A in hair of Greek
population. Chemosphere 118, 336–341.
Usmani, K.A., Rose, R.L., Hodgson, E., 2003. Inhibition and activation of the human
liver and human cytochrome P450 3A4 metabolism of testosterone by
deployment–related chemicals. Drug Metab. Dispos. 31, 384–391.
Usmani, K.A., Cho, T.M., Rose, R.L., Hodgson, E., 2006. Inhibition of the human liver
microsomal and human cytochrome P450 1A2 and 3A4 metabolism of
estradiol by deployement–related and other chemicals. Drug Metab. Dispos.
34, 1606–1614.
Uzumcu, M., Kuhn, P.E., Marano, J.E., Armenti, A.E., Passantino, L., 2006. Early
postnatal methoxychlor exposure inhibits folliculogenesis and stimulates
anti-Mullerian hormone production in the rat ovary. J. Endocrinol. 191,
549–558.
Viluksela, M., Raasmaja, A., Lebofsky, M., Stahl, B.U., Rozman, K.K., 2004.
Tissue-specific effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the
activity of 5 -deiodinases I and II in rats. Toxicol. Lett. 147, 133–142.
Wang, W., Craig, Z.R., Basavarajappa, M.S., Hafner, K.S., Flaws, J.A., 2012.
Mono-(2-ethylhexyl) phthalate induces oxidative stress and inhibits growth of
mouse ovarian antral follicles. Biol. Reprod. 87, 152.
Wang, W., Hafner, K.S., Flaws, J.A., 2014. In utero bisphenol A exposure disrupts
germ cell nest breakdown and reduces fertility with age in the mouse. Toxicol.
Appl. Pharmacol. 276, 157–164.
Waters, K.M., Safe, S., Gaido, K.W., 2001. Differential gene expression in response
to methoxychlor and estradiol through ERalpha, ERbeta and AR in
reproductive tissues of female mice. Toxicol. Sci. 63, 47–56.
Welshons, W.V., Thayer, K.A., Judy, B.M., Taylor, J.A., Curran, E.M., vom Saal, F.S.,
2003. Large effects from small exposures I. Mechanisms for
endocrine-disrupting chemicals with estrogenic activity. Environ. Health
Perspect. 111, 994–1006.
Wetherill, Y.B., Fisher, N.L., Staubach, A., Danielsen, M., de Vere White, R.W.,
Knudsken, K.E., 2005. Xenoestrogen action in prostate cancer: pleiotropic
effects dependent on androgen receptor status. Cancer Res. 65, 54–65.
Xu, C., Chen, J.A., Qiu, Z., Zhao, Q., Luo, J., Yang, L., Zeng, H., Huang, Y., Zhang, L., Cao,
J., Shu, W., 2010. Ovotoxicity and PPAR-mediated aromatase downregulation
in female Sprague-Dawley rats following combined oral exposure to
benzo[a]pyrene and di-(2-ethylhexyl) phthalate. Toxicol. Lett. 199, 323–332.
Yang, M., Ryu, J.H., Jeon, R., Kang, D., Yoo, K.Y., 2009. Effects of bisphenol A on
breast cancer and its risk factors. Arch. Toxicol. 83, 281–285.
Yucra, S., Rubio, J., Gasco, M., Gonzales, C., Steenland, K., Gonzales, G.F., 2006.
Semen quality and reproductive sex hormone levels in Peruvian pesticide
sprayers. Int J. Occup. Environ. Health 12, 355–361.
Zachow, R., Uzumcu, M., 2006. The methoxychlor metabolite,
2,2-bis-(p-hydroxyphenyl)-1,1,1-trichloroethane, inhibits steroidogenesis in
rat ovarian granulosa cells in vitro. Reprod. Toxicol. 22, 659–665.
Zama, A.M., Uzumcu, M., 2010. Epigenetic effects of endocrine-disrupting
chemicals on female reproduction: an ovarian perspective. Front.
Neuroendocrinol. 31, 420–439.
Zama, A.M., Uzumcu, M., 2009. Fetal and neonatal exposure to the endocrine
disruptor methoxychlor causes epigenetic alterations in adult ovarian genes.
Endocrinology 150, 4681–4691.
Zhang, Y., Han, S., Liang, D., Shi, X., Wang, F., Liu, W., Zhang, L., Chen, L., Gu, Y., Tian,
Y., 2014. Prenatal exposure to organophosphate pesticides and
neurobehavioral development of neonates: a birth cohort study in Shenyang.
China. PLoS ONE 9, e88491.