paper

1. Endocrine Disrupting Chemicals:
Overview of recent published
literature
April 2012 – June 2012
June 2013

2. 2
The Institute of Environment and Health (IEH) was established at Cranfield University in November
2005. The research and consultancy activities of the Institute are principally funded through specific
grants, contracts and awards by UK Government Departments and Agencies.
This document is a report by the Institute of Environment and Health for Defra.
Prepared by Minako Takamiya Allen, Louise Youngs & Lini Ashdown
Edited by Ruth Bevan & Derrick Crump
Institute of Environment and Health, 2013
Institute of Environment and Health
Cranfield Health
First Floor, Vincent Building
Cranfield University
Bedfordshire
MK43 OAL
UK
www.cranfield.ac.uk/health/ieh

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Introduction
This report is an overview of key recent publications concerning the health and environmental
effects of Endocrine Disrupting Chemicals (EDCs). The term EDCs is normally applied to
describe chemicals that are synthetic agents that mimic or block hormones resulting in
the potential disruption of critical endocrine processes including reproduction, growth
and development. The review is based on the peer-reviewed papers, abstracts and
conference proceedings that have been published in the period April – June 2012.
The papers were selected because they address research areas that are considered of direct
relevance to the health and environmental effects of EDCs. The papers are presented under
six subject areas that are outlined below. When considered appropriate, some papers appear
under more than one of the subject areas.
Section 1 – HUMAN EXPOSURE MEASUREMENT AND MODELLING: Papers
relating to the measurement or modelling of consumer and/or environmental exposure to
EDCs and/or mixtures of EDCs; the development of human biomarkers of exposure to or
effect of EDCs and/or mixtures of EDCs.
Section 2 - HEALTH EFFECTS: Papers on the influence of EDCs and/or mixtures of EDCs
on health, disease and dysfunction; assessment of the influence of genetic and epigenetic
factors on human susceptibility to the effects of EDCs and/or mixtures of EDCs:
a.) Reproductive/developmental health
b.) Carcinogenic effects
c.) Other effects.
Section 3 – BIOLOGICAL MECHANISMS and TOXICITY TESTING: Papers on the
biochemical and toxicological mechanisms underlying the effects of EDCs and/or mixtures of
EDCs, including oestrogenic, androgenic and thyroid effects; other mechanisms including
free radical generation.
Section 4 – ENVIRONMENTAL EFFECTS: Papers relating to the effects following
environmental exposure to EDCs and/or mixtures of EDCs that are specific to terrestrial and
aquatic organisms:
a.) Aquatic effects
b.) Terrestrial effects.
Section 5 – RISK ASSESSMENT, RISK MANAGEMENT and RISK
COMMUNICATION: Papers relating to risk assessment and risk management of EDCs
and/or mixtures of EDCs and risk communication issues.
Section 6 – MISCELLANEOUS: Other papers considered of general interest or potential
relevance to the study of EDCs and/or mixtures of EDCs that do not relate to the above
categories.
The papers presented herein were identified using a series of structured searches of the
following on-line databases: Medline, Toxline, Biological Sciences, Scopus and
Environmental Sciences and Pollution Management. The search terms used were “endocrine
disrupt” and “human or environment or exposure”; thus ensuring any relevant abstracts
containing these terms in their title, abstract or descriptors were identified. The paper

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abstracts were reviewed and categorised by an experienced IEH Scientist to confirm their
relevance before inclusion in this report.

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Review of papers
1. HUMAN EXPOSURE MEASUREMENT AND
MODELLING
During this reporting period a number of reviews have been published looking at potential
sources of Endocrine Disrupting Chemicals (EDCs) and pathways of exposure that may
impact on human health. Asimakopoulos et al. (2012) summarised currently available
literature on the analytical methodologies, sample preparation technique, exposure
assessments and epidemiological biomonitoring studies of bisphenol A (BPA), 4-t-
octylphenol (tOP), and 4-nonylphenol (NP) in human populations. Mass spectrometry-based
methods have detected the aforementioned EDCs in non-occupational human matrices at < 1
ng/ml, highlighting the efficacy of the technique for exposure assessments. The authors did
not detail the literature search strategy inclusion/exclusion criteria adopted. Jung et al., (2012)
reviewed in vitro and in vivo biomarkers of oestrogenicity; cabindin-D9k was highlighted as a
potential biomarker for the assessment of oestrogenic activity of EDCs. Veeramachaneni
(2012) suggested the use of transmission electron microscopy for morphological evaluation of
spermatozoal organelle features.
The use of sunscreens with UV-filters is increasing globally; however, the protective effect of
sunscreens against malignant melanoma remains uncertain1
. Krause et al. (2012) reviewed the
potential side effects of selected UV-filters (e.g. benzophenone-3, 2-ethylhexyl 4-methoxy
cinnamate, homosalate). The authors concluded that a large number of animal studies report
perturbation of the endocrine system consequent to UV-filter exposure; however, some
studies failed to find such effects. Additionally, Krause et al. noted rapid skin absorption
rates, enabling distribution of the UV-filters and systemic exposure.
In light of the perceived rise in testicular dysgenesis syndrome-associated conditions in some
European countries, Nordkap et al. (2012) investigated a link between male reproductive
disorders and specific environmental toxicants. The authors were not able to establish a
significant correlation between disease development and specific toxicants, most likely as a
result of multiple exposures and life-style factors. Furthermore, the use of semen quality as a
marker of male reproductive health was discussed. Unüvar & Büyükgebiz (2012) suggested
the ubiquity of environmental risk and EDC exposure based on published epidemiological
and toxicological findings, recommending that local authorities perform preventive exposure,
particularly with regard to pregnant women and children.
A number of studies pertaining to the bioaccumulation and biomonitoring of EDCs in humans
have been reported. Polybrominated biphenyl ether (PBDE) flame retardants are lipophilic
and thus have the propensity to bioaccumulate in organisms and persist in the environment.
Aleksa et al., (2012) developed a GC/MS assay to quantify the distribution of eight BDE
congeners (BDE-28, BDE-47, BDE-99, BDE-100, BDE-153, BDE-154, BDE-183 and BDE-
209) in hair (10 – 40 mg) from children of 0 – 15 years old (12 newborn and 12 children).
PBDE’s were detected in all hair samples; tΣPBDE ranged from 0.038 - 1.01 pg/mg and
0.208 - 2.695 ng/mg in newborn and child hair, respectively. The most abundant PBDE
congener in newborn hair was BDE-153, whereas the variable PBDEs of BDE-47 and BDE-
99 were most commonly detected in child hair. The highest molecular weight congener BDE-
1
Green, A.C., Williams, G.M., Logan, V. et al. (2011) Reduced melanoma after regular sunscreen use:
randomized trial follow-up. Journal of Clinical Oncology, 29(3), 257-263.

6. 6
209, was detected in 10/24 paediatric hair samples (LOQ: 0.0625 pg BDE-209/mg). The
efficiency of extraction was between 70 and 90%, demonstrating the sensitivity of the
technique for quantifying PBDE congeners. The authors suggest the use of the GC/MS assay
developed to further examine the association between in utero PBDE exposure and
cryptorchidism.
Urinary concentrations of di-(2-ethylhexyl) phthalate (DEHP) metabolites (monoethyl
phthalate (MEP); mono-n-butyl phthalate (MBP); mono-iso-butyl phthalate; and, monobenzyl
phthalate (MBzP)) and BPA were characterised by Braun et al. (2012). 1001 urinary
samples were obtained from 137 women (18 – 45 years of age) participating in a prospective
birth cohort as part of the Boston fertility clinic Environment and Reproductive Health Study.
The study subjects provided spot urine samples before (n ≥ 2) and during (n ≥ 2) pregnancy.
Absolute concentrations of phthalate metabolites and BPA before and during pregnancy were
comparable; however, variability of urinary BPA and MBzP were higher during pregnancy.
No significant variation of the other studied phthalates was detected during pregnancy; MEP
(intraclass correlation coefficient (ICC) = 0.50) and MBP (ICC = 0.45) were less variable
than BPA (ICC = 0.12), MBzP (ICC = 0.25), and ΣDEHP metabolites (ICC = 0.08). The
authors noted that the urinary concentrations of phthalate metabolites and BPA were variable
both before and during pregnancy, but the magnitude of variability was biomarker specific
and replication in other pregnancy cohorts was recommended.
Nahar et al. (2012) measured total urinary BPA concentrations in spot samples collected from
urban (n = 28) and rural (n = 29) Egyptian girls (10 – 13 years of age). Similar concentrations
or urinary BPA were detected in Egyptian girls of urban and rural descent, with unadjusted
median values of 1.00 and 0.60 ng/ml, respectively. Egyptian girls’ median urinary BPA
concentration (0.70 ng/mL unadjusted) was significantly lower than age-matched American
girls (2.60 ng/ml unadjusted) based on the US National Health and Nutrition Examination
Survey (NHANES) 2009 - 2010 data. However, in the specific gravity adjusted total urinary
BPA, the difference between Egyptian and American was not statistically significant. A
collation of anthropometric and questionnaire data led the authors to identify a significant
association between food storage in plastic containers and urinary BPA concentrations,
suggesting that diet is an important route of BPA exposure.
Geens et al. (2012) determined BPA, triclosan (TCS) and NP concentration in paired adipose,
liver and brain tissue samples collected during autopsy from 11 unspecified patient volunteers
(9 – 62 years of age) in Belgium. The measurements were based on their pentafluorobenzoyl-
derivatives (PFB) in the tissues using gas chromatography coupled with mass spectrometry
(GC-ECNI/MS). BPA was detected in almost all tissue samples, with the highest
concentrations found in the adipose tissue (mean 3.78 ng/g), followed by the liver (1.48 ng/g)
and brain (0.91 ng/g). The highest concentration of TCS was detected in the liver (3.14 ng/g)
and adipose tissue (0.61 ng/g); however, TCS was only detected in one brain sample. NP
levels were below the level of detection (i.e. 0.003 – 0.004 ng/g) in most tissue samples. The
authors suggest that the high concentrations of BPA detected may be as a result of external
contamination, due to the fast elimination of BPA, although no quantitative measurements
have been reported to support this argument.
The prevalence of congenital cryptorchidism in Denmark is higher than that predominantly
reported in Finland2
. Environmental and life style factors have been associated with mild
types of cryptorchidism3
. In order to assess the relationship between exposure to EDCs and
congenital cryptorchidism, Krysiak-Baltyn et al. (2012) measured polychlorinated biphenyls
2
Boisen, K.A., Kaleva, M., Main, K.M., et al., (2004) Difference in prevalence of congenital
cryptorchidism in infants between two Nordic countries. Lancet, 363(9417), 1264-1269.
3
Main, K.M., Skakkebaek, N.E., Virtanen, H.E., et al,. (2010) Genital anomalies in boys and the
environment. Best Practice & Research Clinical Endocrinology & Metabolism, 24(2), 279-289.

7. 7
(PCBs), polybrominated diphenyl-ethers (PBDEs), dioxins (OCDD/PCDFs), phthalates,
polybrominated biphenyls and organochlorine (OC) pesticides in breast milk samples from
Danish and Finnish single-birth mothers. Breast milk samples were collected from 1 month
until 2 (Finland) or 3 months (Denmark) post partum. Of the 130 mothers, 68 of the newborns
were healthy controls and 62 were born with cryptorchidism. Computational analysis showed
that exposure to PBDE119, PBDE85, PBDE138 and OCDF (the 50th
percentiles of 9.00E-04
ng/g lipid, 4.70E-03 ng/g lipid, 3.00E-03 ng/g lipid and 2.39E-01 pg/g lipid, respectively),
was significantly higher in boys with cryptorchidism in the Danish cohort, but not in the
Finnish cohort. Furthermore, the data suggested a protective effect of PCBs within the Danish
cohort, which was supported by molecular evidence recovered through systems biology
(computer screening system to find known genetic association partners). The authors
concluded that there is a stronger correlation between exposure to persistent environmental
chemicals and cryptorchidism in the Danish cohort, than in the Finnish boys studied.
In a prospective pilot study in Argentina, Cecchi et al. (2012) quantified organophosphate
(OP) pesticides exposure in 97 pregnant women (15 – 36 years of age) living in a rural area of
the Rio Negro, in which OPs were intensively applied throughout six months of the year.
Blood samples were obtained and biomarkers of OPs exposure (cholinesterases and β-
glucuronidase), cortisol (CT), progesterone (PG) levels and glycemia were determined. Liver
injury was evaluated by aspartate aminotransferase (AST) and alanine aminotransferase
(ALT) concentration, while liver function was evaluated by serum albumin level. The results
showed that the plasma cholinesterase (PCh) and erythrocyte cholinesterase (AChE)
significantly decreased (p < 0.01) during the pesticide spraying period (SP), suggesting that
the study population was exposed to OPs. CT values increased significantly (p < 0.01) in the
first trimester of pregnancy during spraying, relative to the pre-spraying period (PreS).
Individual values above the upper limit of the CT and PG reference range were found both in
PreS and SP, which could be indicative of alterations of hormone metabolism by OP pesticide
exposure. During the second trimester of pregnancy there was a slight but significant increase
in the serum ALT levels and the AST/ALT ratio in SP, suggesting subtle hepatic injury.
However, the authors note that the results were within reference values and should be
considered with caution.
In order to assess an association between active smoking and male reproductive hormone
profile, serum levels of follicle-stimulating hormone (FSH), luteinising hormone (LH),
prolactin, total testosterone, inhibin B and oestradiol were measured in a group of 136 male
Mexican flower growers (18 - 50 years of age) using an enzyme-linked immunoabsorbent
assay (Blanco-Muñoz et al., 2012). Non-smokers were used as controls and after adjusting for
potential confounders (age, body mass index, alcohol consumption, residence or serum level
of dichlorodiphenyltrichloroethane [DDE]), β-coefficient regression for current smokers of
five or more cigarettes/day showed significantly higher levels of LH (β = 0.33, p = 0.01),
prolactin (β = 0.18, p = 0.03) and testosterone (β = 0.21, p = 0.02). However, no alteration in
serum FSH, inhibin B or oestradiol was observed. Current smokers of less than five
cigarettes/day also showed higher levels of prolactin (β = 0.12, p = 0.03) and testosterone (β =
0.18, p < 0.01) than controls. Hormone levels of ex-smokers were similar to those of controls,
suggesting temporality of the observed endpoints. The authors concluded that the reported
results are compatible with the hypothesis that tobacco consumption may act as an EDC on
the male hormone profile. However, most of the hormone concentrations were within the
normal range, thus the clinical significance needs to be assessed.
It has previously been reported that urinary concentrations of DEHP metabolites were weakly
associated with serum levels of hormones in two disparate US populations; fertile (n = 425,

8. 8
mean age 32.2 ± 6.2 years of age)4
and infertile men (n = 425, mean age 36 ± 5.3 years of
age)5
. Mendiola et al. (2012) pooled these two populations to examine the relationship
between urinary phthalate metabolites (mono(2-ethylhexyl) phthalate [MEHP], mono(2-ethyl-
5-hydroxyhexyl) phthalate [MEHHP], mono(2-ethyl-5-oxohexyl) phthalate [MEOHP],
monoethyl phthalate [MEP], mono-n-butyl phthalate [MBP], and monobenzyl phthalate
[MBzP]) with serum levels of FSH, LH, testosterone (T), inhibin B, and oestradiol (E2) and
sex hormone-binding globulin (SHBG). Statistical analysis showed that the urinary
concentrations of MEHP, MEHHP and MEOHP were inversely associated with the free
androgen index (FAI = T/SHBG) and calculated free testosterone. The urinary concentrations
of MEHHP and MEOHP were positively associated with SHBG, and MEHP was inversely
associated with E2. No other phthalate metabolites were associated with serum hormones.
The authors concluded that DEHP exposure was robustly associated with some changes in
circulating levels of male sex steroid hormones, consistent with the known anti-androgenic
effect of the chemical.
Fortenberry et al. (2012) explored the relationship between urinary concentrations of 3, 5, 6-
trichloro-2-pyridinol (TCPY) with serum thyroid parameters (total thyroxine [T4] and thyroid
stimulating hormone [TSH]) in US adults (n = 3249) participating in the US NHANES 1999
– 2000 and 2001 – 2002. The authors identified a significant positive relationship between
urinary TCPY with serum T4 in a group of males (12 - 40 years of age), and a significant
inverse relationship with TSH in males at 18 – 40 years of age and at > 60 years of age.
Conversely, a significantly positive relationship between TCPY and TSH was observed in
females (> 60 years of age). The authors suggest that the reported results add to the existing
evidence that exposure to organophosphate insecticides and their metabolites can disrupt the
hypothalamic-pituitary-thyroid (HPT) axis; however, further research is needed to confirm
these findings.
Shy et al. (2012) measured the PBDE congeners in breast milk and hormonal levels of thyroid
hormones (triiodothyronine [T3], T4, TSH, and free T4 [FT4]) and insulin-like growth factor
1 (IGF-1) in the umbilical cord blood of healthy pregnant Taiwanese women (n = 149). The
study participants consisted of two cohorts: 2000 – 2001 participants from central Taiwan (n
= 42, mean 29.0 ± 3.56 years of age) and 2007 – 2009 participants from southern Taiwan (n =
107, mean 30.2 ± 4.40 years of age). The average and median levels of Σ(14)PBDEs6
were
5.34 and 3.38 ng/g lipid in 2000 - 2001 and 5.22 and 3.13 ng/g lipid in 2007 - 2009,
respectively. Breast milk Σ(14)PBDE was not significantly correlated with either thyroid
hormones or IGF-1 in cord blood. Adjusted multiple stepwise linear regression demonstrated
the significance of a slight correlation between logT4 cord blood concentration and BDE-154
in breast milk (B = 0.113, p = 0.017). The log FT4 concentration was significantly associated
with a decrease in log BDE-99 levels (B = -0.137, p = 0.043) and an increase in the log of
BDE-154 level (B = 0.158, p = 0.008). Additionally, the log IGF-1 level was significantly
associated with an increase in the log BDE-196 concentration (B = 0.532, p = 0.028) and a
decrease in the log BDE-85 concentration (B = -0.235, p = 0.018). The authors stress the need
to further examine the relationship between PBDE’s and thyroid hormones and IGF-1 leves in
both animals and humans.
Bermudez et al. (2012) evaluated oestrogen receptor (ER) transcriptional activation levels
using an in vitro T47D-KBluc transcriptional activation assay. T47D-KBluc cells naturally
4
Mendiola, J., Jørgensen, N., Andersson, A.M., et al. (2011) Associations between urinary
metabolites of di(2-ethylhexyl) phthalate and reproductive hormones in fertile men. International
Journal of Andrology, 34(4), 369-378.
5
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. Journal of Andrology, 30(3), 287-297.
6
BDE-28, PDE-47, PDE-49, PDE-85, PDE-99, PDE-100, PDE-153, PDE-154, PDE-183, PDE-196,
PDE-197, PDE-206, PDE-207 and PDE-209.

9. 9
expresses ERα and a small amount of ERβ, and are stably transfected with a triplet oestrogen
responsive element promoter and luciferase reporter gene construct. Seven oestrogens
(oestrone [E1]; oestradiol-17α [E2α]; oestradiol-17β [E2]; oestriol [E3]; ethinyl oestradiol -
17α [EE2]; mestranol [MES]; equilin [Eq]) were individually tested for their oestrogenic
response levels. Log EC50 and Hillslope values for individual oestrogens were: E1, -11.92,
1.283; E2α, -9.61, 1.486; E2, 11.77, 1.494; E3, -11.14, 1.074; EE2, -12.63, 1.562; MES, -
11.08, 0.809; and Eq, -11.48, 0.946, respectively. Oestrogen response of fixed ray oestrogen
mixtures, which mirrored the oestrogens found in swine, poultry, dairy concentrated animal
feedlot operations (CAFO) effluent, and hormone replacement therapy mixture and/or oral
contraceptives (ternary mixture of Eq, MES and EE2), were also evaluated. The authors
stated that the observed response from each of these mixtures correlated with predicted
concentration addition (CA) and oestrogen equivalence (EEQ) models.
James-Todd et al. (2012) reported discrepancy between hair product use between African-
American and African-Caribbean women in the New York metropolitan relative to
Caucasians, leading the authors to suggest that these ethnic groups may be exposed to higher
concentrations of hormonally-active chemicals commonly found in hair products. However,
information pertaining to the hormonally-active chemicals present in hair product labels was
limited. Nelson et al,. (2012) explored the relationship between urinary concentrations of
BPA (n = 4739), serum concentrations of four polyfluoroalkyl chemicals (PFCs;
perfluorooctanoic acid [PFOA], perfluorooctane sulfonic acid [PFOS], perfluorononanoic
acid [PFNA], and perfluorohexane sulfonic acid [PFHxS]) (n = 3953), and population
disparities in exposure to these chemicals in participants of the US NHANES (2003 – 2004
and 2005 – 2006). Amongst the socioeconomic position assessed (family income, food
security status, emergency food, education and occupation), family income with adjustment
for family size was the strongest predictor of BPA and PFC levels. Urinary BPA was
inversely associated with family income, whereas all four serum PFCs were positively
associated with family income, suggesting people with lower incomes had higher body
burdens of BPA; the reverse was true for PFCs.
The presence of selected polybrominated diphenyl ethers (PBDEs) and BDE-153 in effluent
and sewage sludge samples collected from a wastewater treatment plant (WWTP) in South
Africa, were analysed using a high-capillary gas chromatograph equipped with an electron
capture detector (Daso et al., 2012). The sum of the eight PBDE congeners (BDE congeners
28, 47, 99, 100, 153, 154, 183 and 209) ranged from 369 to 4370 ng/l, 19.2 to 2640 ng/l, and
90.4 to 15,100 ng/l for raw water, secondary effluent, and final effluent, respectively. A
similar result was observed for the sewage sludge samples, which ranged between 13.1 and
652 ng/g dry weight (dw). The results obtained for BB-153 ranged from none detected
(detection limit unspecified) to 18.4 ng/l and ND to 9.97 ng/g dw for effluents and sewage
sludge, respectively. The authors concluded that the reuse of the treated effluent could
enhance the possibility of these contaminants entering into the food chain, thus causing
undesirable health problems in exposed subjects. The presence of four phenolic EDCs (NP,
nonylphenol monoethoxylate [NP1EO], BPA and triclosan [TCS]) and four nonsteroidal anti-
inflammatory drugs (NSAIDs; ibuprofen (IBF), ketoprofen (KFN), naproxen (NPX) and
diclofenac (DCF) was investigated in a Greek river receiving treated municipal wastewater
(Stasinakis et al., 2012). Four point samples were taken from the river and the outlet of a
sewage treatment plant (STP), during six sampling campaigns. The GC-MS analysis detected
phenolic EDCs in almost all samples (92/96 river samples and 22/24 STP samples). NSAIDs
were mainly detected in wastewater and recieving waters. Among monitored compounds, the
highest mean concentrations were for NP (1,345 ng/l) and DCF (432 ng/l). Calculation of
daily loads suggested STP’s as the major source of NSAIDs contamination, whereas other
sources contributed significantly to the detection of EDCs.

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2. HEALTH EFFECTS
a) Reproductive/developmental health
This reporting period has seen a number of publications pertaining to the influence of EDCs
on health, disease and dysfunction. Spencer et al. (2012) report that exposure to EDCs can
cause infertility and cancer in exposed individuals and their offspring via developmental
programming. Fowler et al. (2012) reviewed the evidence pertaining to an association
between environmental chemicals and female reproductive dysfunction. The authors noted
that maternal exposure to EDCs can lead to an inhibition of trophoblast differentiation and
invasion. However, the currently available data is inadequate to fully support a rigorous
conclusion and the need for further research was highlighted. Gourounti (2012) conducted a
meta-analysis of 13 epidemiological studies pertaining to the association between
endometriosis and organochlorine (OC) exposure; no significant associations were reported.
Béranger et al. (2012) conducted a systematic review of human and animal data to identify
chemical agents associated with ovarian follicular toxicity. Amongst 20 chemicals which may
potentially damage the ovarian reserve, in utero ethylene glycol methyl ether, 2,2-
bis(bromomethyl)-1,3-propanediol, benzo[a]pyrene, and dimethylbenzanthracene exposure
was associated with premature ovarian failure (POF). Davis (2012) concluded that there is no
evidence to support an association between EDCs and endometrial polyp7
formation in adult
rats, on the premise that there is no evidence that uterine endometrial polyps in women and
uterine stromal polyps in rodents are morphologically or biologically comparable. In an
overview of the current literature, Dunbar et al. (2012) assess immune function in relation to
the human female reproductive tract and conclude that exposure to EDCs could substantially
impact normal immune function.
In a review of male reproductive health, Lagos-Cabre and Moreno (2012) suggest that
premature release of germ cells (sloughing), disruption of the blood-testis barrier and germ
cell apoptosis are the most commonly reported effects of bisphenol A (BPA), nonylphenol
(NP), di-(2-ethylhexyl) phthalate (DEHP) and mono(2-ethylhexyl) phthalate (MEHP) on male
spermatogenesis. The authors also highlight the membrane-anchored metzincin of the
adamalysin subfamily, ADAM (A Distintegrin And Metalloprotease) as a novel element in
the germ cell apoptosis model. Virtanen and Adamsson (2012) review the association
between congenital cryptorchidism and exposure to EDCs. The authors conclude that prenatal
exposure to diethylstilbestrol (DES) and parental exposure to pesticides are associated with
increased risk of cryptorchidism in humans. Furthermore, an association between the anti-
androgenicity of EDC’s present in breast milk (phthalates and polybrominated biphenyl ether
[PBDE]) and infant reproductive hormone levels was reported.
Cederroth et al. (2012) review the available human and animal literature pertaining to the
effects of soy and phytoestrogens on reproductive health. No significant associations could be
drawn from the limited human data, however, animal studies suggest that perinatal exposure
to isoflavones disrupts fertility in females. Furthermore, an association between postnatal
consumption of soy or phytoestrogens and altered androgenicity of males was reported.
However, Cederroth et al. note a lack of consistency in the literature and stress the need for
comprehensive large-scale prospective human studies. Additionally, Jefferson et al. (2012)
conclude that developmental exposure to phytoestrogens can permanently reprogram adult
tissue responses, consequent to a review of human and animal studies. The authors emphasise
the importance of monitoring dietary or therapeutic phytoestrogen intake during critical
periods of development. The incidence of gastroschisis, a congenital abnormality
characterised by protruding abdominal contents due to a defect in the abdominal wall, is
rising. Lubinsky (2012) reports an association between gastroschisis and atrazine exposure,
7
A polyp refers to an abnormal growth of tissue from a mucous membrane.

11. 11
suggesting a role for oestrogenic chemicals in the aetiology of gastroschisis. In a review of
female reproductive tract development in pigs, Bartol et al. (2012) lighlight a lactorine-driven
link between the neonatal uterine developmental programme and subsequent reproductive
health in adulthood. The authors report that such events are supported by lactorine signalling
(colostrum/milk) as a consequence of nursing and also influenced by macro- (i.e. climate,
diet, disease and EDCs) and micro-environment (i.e. endocrine, paracrine and autocrine
conditions).
This reporting period has seen the critical evaluation of the relationship between disease
aetiology and EDC exposure. Gaspari et al. (2012) explored the association between in utero
exposure to environmental contaminants and the development of male genital anomalies in a
Brazilian birth cohort (2710 male newborns < 3 months old). Congenital defects reported
included 56 cases of genital malformation (2.07%), 23 cryptorchidism (0.85%), 15
hypospadias (0.55%) and 18 micropenis (0.66%). All the cases demonstrated normal
testosterone production and none presented androgen receptor (AR) or 5α-reductase gene
mutation in the peripheral blood leucocytes. Based on parent-administered questionnaires in
relation to habitat before and during pregnancy, in addition to occupations and lifestyle
factors, Gaspari et al. suggest that 92% of investigated newborns were exposed to EDCs in
utero. Their mothers reported daily domestic use of pesticides (i.e.
dichlorodiphenyltrichloroethane [DDT]) and other EDCs. Proportions of 80.36% of the
mothers and 58.63% of the fathers reported occupational exposure to pesticides and other
EDCs before/during pregnancy of the mothers and around the time of fertilisation. The
authors conclude that prenatal EDC exposure may be a risk factor for male genital
malformation. The incidence of micropenis in the Brazilian cohort was significantly higher
than that of the USA8
, Egypt9
, and France10
; however, the statistical power of these studies
was not assessed.
McAuliffe et al. (2012) explored the role of environmental risk factors on sex-chromosome
disomy in spermatozoa collected from American men (n = 192). In genetics, disomy refers to
an extra chromosome in the haploid state that is homologous to an existing chromosome.
Semen samples from subfertile men (20 – 54 years of age) were evaluated for chromosomal
abnormalities using fluorescence in situ hybridization (FISH) and polychlorinated biphenyl
(PCB) and p,p´-DDE accumulation. The authors reported a significant positive correlation
between adjusted incidence rate ratios (IRRs) and p,p´-DDE for XX disomy (p < 0.001), XY
disomy (p = 0.001) and total sex-chromosome disomy (p < 0.001). A significant positive
correlation between adjusted IRRs with increasing PCB concentration was also reported for
XY disomy (p < 0.001) and total sex-chromosome disomy (p < 0.001); however, there was a
significant inverse association for XX disomy (p < 0.001). McAuliffe et al. stress that these
findings need to be confirmed in other study populations.
Mieritz et al. (2012) examined the association between pubertal gynaecomastia and exposure
to anti-androgenic phthalate metabolites in a cross-sectional study in Denmark. A significant
correlation between chronological age and urinary concentrations of phthalate metabolites
(DEHP, di-isononylphthalate [DiNP], monoethyl phthalate [MEP]) and isomers (mono-n-
butyl phthalate [MBP](i+n)) (r = -0.139; p < 0.01) was reported in healthy boys (n = 555, 6 – 19
years of age) recruited as part of the Copenhagen Puberty Study during the period of 2006 –
2008. However, the urinary levels of phthalate metabolites were not associated with age at
8
Nelson, C.P., Park, J.M., Wan, J., et al. (2005) The increasing incidence of congenital penile
anomalies in the United States. Journal of Urology, 174(4 Pt 2), 1573-1576.
9
Mazen, I., El-Ruby, M., Kamal, R., et al. (2010) Screening of genital anomalies in newborns and
infants in two egyptian governorates. Hormone Research in Paediatrics, 73(6), 438-442.
10
Gaspari, L., Paris, F., Jandel, C., et al. (2011) Prenatal environmental risk factors for genital
malformations in a population of 1442 French male newborns: a nested case-control study. Human
reproduction, 26(11), 3155-3162.

12. 12
pubertal onset, serum testosterone levels or gynaecomastia. The authors suggest that any
possible perinatal or long-term postnatal effects of phthalates need to be evaluated in
longitudinal studies. Using the same Danish databank, Frederiksen et al. (2012) evaluated the
relationship between urinary phthalate concentration and pubertal development in healthy
girls (n = 725, 5 – 19 years of age). Consequent to stratification of urinary phthalate excretion
into quartiles, a significant correlation between delayed pubarche and increasing urinary
phthalate metabolite (∑MBP(i+n) and monobenzyl phthalate [MBzP]) was identified;
furthermore, the correlation was more pronounced in combined exposure cases. Further
assessment of females with precocious puberty recruited from an outpatient clinic between
2008 and 2009 (n = 25), did not show any differences in urinary phthalate metabolite levels
relative to controls. The authors conclude that urinary phthalate concentrations are associated
with significantly delayed pubarche in healthy girls, indicative of an anti-androgenic mode of
action. No association between phthalates and breast development was observed. Suzuki et al.
(2012) reported an association between corrected urinary MEHP concentration and anogenital
index (AGI) (adjusted, p = 0.047) in Japanese pregnant women (n = 111, mean 33 ± 4 years
old) between 9 – 40 weeks of gestation. High intakes of naturally occurring oestrogenic soy
isoflavones during pregnancy did not appear to have any significant impact on the AGI of
newborns in this study.
An association between occupational exposure to non-persistent pesticides during early
pregnancy and impaired reproductive function and congenital cryptorchidism has previously
been reported in a Danish cohort (n = 113)11
. In a follow-up study, Wohlfahrt-Veje et al.
(2012a) re-examined 94 boys of the Andersen et al. 1996 – 2000 cohort (6 – 11 years of age)
to assess the long-term effects of prenatal pesticide exposure on reproductive development.
Multiple regression analysis identified significantly lower adjusted testicular volumes in the
boys with maternal pesticide exposure, relative to controls (p = 0.05), and effects on testis
size appeared to persist at school age. Furthermore, exposed boys had on average 24%
smaller testes and 9.4% shorter penile length. There was a significant association between in
utero pesticide exposure and male reproductive tract abnormalities (cryptorchidism or
hypospadias were reported in 8 of 59 exposed boys, relative to 0/35 unexposed boys; (p =
0.047). The authors conclude that prenatal pesticide exposure has long-term effects on male
reproductive function.
The association between maternal pesticide exposure and female reproductive development
has been evaluated in a Danish cohort (1996 – 2000) (Wohlfahrt-Veje et al., 2012b). Girls,
born to mothers occupationally exposed to pesticides in trimester 1, were examined at 3
months of age (n = 90) and again at age 6 – 11 (n = 83) for anthropometry12
and pubertal
stages. Relative to controls, the mean age at breast development was significantly earlier in
the exposed group (p = 0.05), with significantly higher serum androstenedione levels (p <
0.05). Furthermore, serum anti-Müllerian hormone (AMH) levels in the exposed group were
significantly lower (p < 0.05), which may be indicative of a reduced antral ovarian follicle
pool. The authors conclude that the positive association between maternal pesticide exposure
and earlier breast development in female progenies may be linked to higher androgen levels,
which may indirectly increase oestrogen levels through aromatisation.
In an Indian hospital-based case control study, Sharma et al. (2012) measured OC pesticides
in maternal and umbilical cord blood of mothers who bore children with foetal growth
restriction (FGR) (n = 50, mean age 23.02 ± 3.2). FGR was categorised by a birth weight < 10
11
Andersen, H.R., Schmidt, I.M., Grandjean, P., et al. (2012) Impaired Reproductive Development in
Sons of Women Occupationally Exposed to Pesticides during Pregnancy. Environmental Health
Perspectives, 116(4), 566–572.
12
The branch of anthropology concerned with comparative measurements of the human body and its
components.

13. 13
percentile for gestational age, according to Lubchenco’s growth chart13
. Age matched (23.78
± 2.5) unexposed mothers of healthy term neonates were used as controls (n = 50). Adjusted
mean levels of OC pesticides (β-hexachlorocyclohexane [β-HCH], γ-HCH, p,p’-DDT) were
significantly higher (p < 0.05) in cases relative to controls, both in the maternal (9.02 ± 5.2
ppb vs 3.97 ± 3.9 ppb for β-HCH; 7.06 ± 6.7 ppb vs 2.58 ± 3.9 ppb for γ-HCH; 1.67 ± 1.4 ppb
vs 0.73 ± 1.1 ppb for p,p’-DDT) and cord blood (5.00 ± 4.1 ppb vs 2.67 ± 2.4 ppb for β-HCH;
3.59 ± 3.8 ppb vs 1.44 ± 2.1 ppb for γ-HCH; 0.83 ± 0.71 ppb vs 0.36 ± 0.64 ppb for p,p’-
DDT). Furthermore, serum α-HCH levels were significantly higher in foetal growth
restriction births (p = 0.042). Interestingly, the frequency of the phase II metabolising
enzyme, glutathione S-transferase GSTM1-/GSTT1- (null) genotype was significantly
higher in cases maternal blood relative to controls (OR = 6.42 [95% CI 1.56 – 26.46] p =
0.01). Gene-environment interaction analysis identified a significant association between
adjusted β-HCH concentration and GSTM1- genotype in the cord blood (p = 0.037), with a
213 gram reduction in birth weight. Sharma et al. conclude that maternal exposure to high
levels of OC pesticides may be an important aetiological factor for 'idiopathic14
' FGR and that
genetic polymorphisms of GST may modify the impact of OC exposure.
Louis et al. (2012) examined the strength of the relationship between persistent POP
(persistent organic pollutant) exposure and endometriosis, with varying diagnostic
methodologies. For this purpose, an operative cohort of 473 menstruating women (18 – 44
years of age) scheduled for laparoscopy or laparotomy at US hospitals between 2007 – 2009,
were compared to a cohort of 127 (mean 32.1 ± 7.8 years old) menstruating women,
diagnosed by magnetic resonance imaging. Omental fat and serum samples were collected
from both cohorts and analysed for POP concentrations. In models adjusted for age, body
mass index, breast-feeding, serum cotinine and lipids, γ-hexachlorocyclohexane was
significantly associated with endometriosis (odds ratio [OR] = 1.27; 95% CI of 1.01 - 1.59) in
omental fat samples of the operative cohort. Whereas, β-HCH was a significant predictor in
serum samples of the population cohort (OR = 1.72; 95% CI of 1.09 - 2.72) no other
significant associations were identified in either cohorts. The authors conclude that the
observed association was cohort and biological-medium specific, underlining the importance
of methodological considerations when interpreting findings.
The effects of BPA on foetal mammary gland development were examined in the rhesus
monkey, Rhesus macaque, as a non-human primate model (Tharp et al., 2012). Pregnant
macaques (n = 5 for control, n = 4 for the exposed group) were fed BPA in a fruit vehicle (0
or 400 µg BPA/kg bw/day) during gestational days (GD) 100 – 165. At necropsy, the
neonatal mammary glands were removed for histopathology. Measurement of maternal serum
identified 0.68 ± 0.312 ng unconjugated BPA/ml, which is comparable to that found in
humans (≈ 1 ng/ mL15
). The density of neonatal mammary buds was significantly increased in
the treated monkeys (p = 0.027) and the overall development of mammary glands was more
advanced relative to control macaques. However, no significant differences were observed in
ERα or ERβ gene expression. The authors conclude that BPA affects several mammary gland
developmental parameters in rhesus monkeys, some of which are relevant to the assessment
of breast cancer risk in humans (e.g. epithelial density).
The effect of in utero and postnatal sewage sludge exposure, consequent to pasture fertiliser
application (2.25 tonnes dry matter/ha twice per year), has been explored in adult male sheep
13
Lubchenco, L.O., Hansman, C., Dressler, M., et al. (1963) Intrauterine growth as estimated from
liveborn birth-weight data at 24 to 42 weeks of gestation. Pediatrics, 32, 793-800.
14
The term idiopathic is primarily used to describe afflictions arising spontaneously or from an
obscure or unknown cause.
15
Vandenberg, L.N., Chauhoud, I., Heindel, J.J., et al. (2010) Urinary, circulating and tissue
biomonitoring studies indicate widespread exposure to bisphenol A. Environmental Health
Perspectives, 118, 1055 – 1070.

14. 14
(Bellingham et al., 2012). The authors note a significant association between sludge exposure
and spermatogenic abnormalities (p < 0.001); including reductions in germ cell numbers per
testis. There were no major treatment-related effects on Sertoli cell number, hormonal profiles
or concentrations of EDCs (phthalates, polycyclic aromatic hydrocarbons [PAHs], PBDEs
and PCBs) in the liver. Thus, Bellingham et al. conclude that developmental exposure to
environmentally relevant levels of EDC mixtures can result in a major reduction in male
sheep germ cell numbers, indicative of impaired sperm production in exposed males.
Furthermore, variation in individual susceptibility may indicate adverse consequences of EDC
exposure in some exposed human male populations.
Hejmej et al. (2012) examined the effects of gestational and neonatal flutamide exposure on
cell-cell adhesion molecules in the testes of adult boars, Sus scrofa domesticus, crossed from
a Large White and Polish Landrace. Pregnant gilts were subcutaneously exposed to flutamide
from GD 20 - 28 or GD 80 – 88. Furthermore, a group of male piglets were exposed to
flutamide on postnatal day (PND) 2 – 10, via subcutaneous injection. Flutamide was
suspended in corn oil and administered in five doses (50 mg/kg bw each) every second day, to
antagonise testosterone action within the testes without producing toxic effects on the sow
and neonates. The expression of zonula occludens-1 (ZO-1), N-cadherin and β-catenin were
significantly decreased in both at mRNA and protein levels in all the testes of the treated
animals, whereas there was no change in the occluding expression levels. The seminiferous
tubules of postnatally exposed boars were severely damaged, and mislocalisation of ZO-1, N-
cadherin and β-catenin was observed. Changes in junction protein expressions were
accompanied by disturbed intratesticular androgen-oestrogen balance, although AR
expression was not altered. The authors conclude that a blockade of androgen action by
flutamide during perinatal periods affects the expression of junction proteins in the adult pig
testes. Additionally, the toxicological mechanism of flutamide and the association between
junction protein expression and insufficient testosterone production and/or excessive
oestradiol synthesis consequent to impaired Leydig cell function is discussed.
Muczynski et al. (2012) examined the effects of MEHP (mono-(2-ethylhexyl) phthalate)
exposure on the male reproductive function using organotypic culture systems. Human foetal
testes were obtained from legally aborted male foetus during the first trimester of pregnancy
(7 – 12 weeks). Gonad explants were cultured with or without 10-5
M MEHP for three days.
Immunohistological analysis of cleaved caspase-3 expression showed that the rate of germ
cell apoptosis significantly increased in the treated culture. In parallel experiments using a
C57B1/6 mouse model, comparable effects for the germ cell apoptosis were observed both in
vitro (with 2 × 10-5
M MEHP) and in vivo (720 mg/kg MEHP). The concentration of MEHP
employed in this study was slightly higher than the mean concentration found in the human
foetal cord blood samples16
, and the highest concentrations in human fluids during pregnancy
and lactation reviewed by the authors. However, approximately 30% of MEHP was absorbed
into plastic elements of the culture system, and the validation process employed in the
experiments support that the intratesticular MEHP levels corresponded to the concentrations
in the culture system.
Paro et al. (2012) investigated toxic effects of the fungicide mancozeb (0, 0.001, 0.01, and 1
µg/ml) in the Swiss CD1 mouse and human ovarian somatic granulosa cells (GCs). Cells
were evaluated for morphology, induction of apoptosis, and p53 expression levels. The
treated mouse GCs (< 36 hours) demonstrated time- and dose-dependent modification of
morphology, and the cells acquired the ability to migrate into the wound, but not to
proliferate. Exposure to 1 µg/ml mancozeb did not trigger the apoptosis in mouse GCs. The
gene and protein expression of p53 decreased in a dose-dependent manner both in mouse and
human GCs. The authors conclude that low doses of mancozeb affect the somatic cells of
16
Latini, G., De Felice, C., Presta, G., et al. (2012) In utero exposure to di-(2-ethylhexyl)phthalate and
duration of human pregnancy. Environnmenal Health Perspectives, 111(14), 1783–1785.

15. 15
mammalian ovarian follicles, by inducing a premalignant-like status, and subsequently
mancozeb should be regarded as a reproductive toxicant.
In this reporting period, a number of toxicological studies using rodent models have
investigated the effects of EDCs on the reproductive and developmental health. Christiansen
et al. (2012) assessed the developmental toxicity of a mixture of chemicals in pregnant Wistar
rats. The 13 component mixture consisted of; phthalates (di-n-butyl phthalate [DBP]; DEHP),
pesticides (vonclozolin; prochloraz; procymidone; linuron; epoxiconazole), UV-filters (octyl
methoxycinnamate [OMC]; 4-methyl-benzylidene camphor [4-MBC]), p,p’-DDE, BPA, the
preservative (butyl paraben [BP]), and the drug paracetamol (PM). The point of departure
index (PODI) was employed to calculate the dose levels for the study. A PODI of 0.016 was
calculated for high end human exposures to the selected chemicals. Animals were orally
administered the mixture from GD7 to GD21, and again postnatally from PND1 to PND 22.
Developmental toxicity was observed at the dose level, which was 450-fold higher than high
end human intake estimates, in terms of increased nipple retention (NR) and reduced ventral
prostate weight. Furthermore, the 150-fold dose group exhibited significantly increased NR.
The authors conclude highly exposed population groups, especially women of reproductive
age, may not be protected sufficiently against the combined effects of the EDCs.
De Castro and Maia (2012) studied the developmental effects of the fungicide, epoxiconazole,
in Wistar rats. Animals were orally exposed to epoxiconazole (50, 100 and 150 mg/kg) by
gavage between GD 1 – 6 or GD 6 – 15. Following parturition, maternal toxicity, pup growth
and maturational milestones were assessed. Irrespective of window of exposure, maternal
exposure to epoxiconazole significantly reduced mean time to vaginal opening and delayed
time to testes descent in pups at the highest dose level (p < 0.0001). The authors conclude that
maternal exposure to epoxiconazole may lead to alterations in developmental patterns in
nursing pups, consistent with the known effects of epoxiconazole on steroid hormone
synthesis 17
. Chlormephos (S-chloromethyl O,O-diethyl phosphorodithioate) is an OP
insecticide introduced to the EU market in 1973 to control soil-dwelling pests18
; however, its
use was withdrawn in 2006. Active ingredients of chlormephos are classified as class 1a:
"Extremely Hazardous" by the World Health Organization according to their acute toxicity19
.
Čeh et al. (2012) investigated the effects of chronic exposure to low doses of chlormephos, on
reproductive and developmental parameters in BALB/c mice. The adult mice were exposed to
0.35 or 3.5 µg/ml of Chlormephos in drinking water from mating until weaning of offspring.
Offspring were monitored daily for sperm production, number of apoptotic cells in the testes
and immunoexpression of antimüllerian hormone and 3β-hydroxysteroid dehydrogenase (3β-
HSD). However, no statistically significant differences were found in any of the observed
parameters. The authors further examined the effect of chlormephos on brain development,
using anxiety-like behaviour as an endpoint in the elevated plus maze test. The male and
female offspring exposed to chlormephos (3.5 µg/ml) spent significantly less time in the open
arms of the elevated plus maze than the control (p < 0.05). No changes in the brain
ultrastructure, including blood-brain barrier, were reported. The authors conclude that
chlormephos may affect neurodevelopment, but that in utero chlormephos exposure does not
affect testes or reproductive tract development, under the conditions of the study.
17
Kjaerstad, M.B., Taxvig, C., Andersen, H.R., et al. (2010) Mixture effects of endocrine disrupting
compounds in vitro. International Journal of Andrology, 33(2). 425-433.
18
Hrženjak, R. Zidar, P., Celestina, T.V., et al. (2009) Toxicity of the organophosphorous insecticide
chlormephos to the earthworm Eisenia andrei and the terrestrial isopod Porcellio scaber. Journal Acta
Biologica Slovenica, 52(2), 85-94.
19
World Health Organization (WHO). (2010) The WHO recommended classification of pesticides by
hazard and guidelines to classification 2009, Stuttgart, Germany. Available at:
http://www.who.int/ipcs/publications/pesticides_hazard_2009.pdf.

16. 16
Pillai et al. (2012) investigated gestational and lactational exposure to metals in F1 generation
Charles Foster rats. Pregnant rats were subcutaneously treated with 0.05 mg/kg bw/day of
sodium acetate (control), lead acetate, cadmium acetate, or a mixture of lead acetate and
cadmium acetate throughout gestation until PND 21; male F1 offspring were sacrificed on
PND 56. A significant reduction in steroidogenic enzyme activity (17β-HSD and 3β-HSD)
and serum testosterone levels were observed in metal-treated groups, in addition to a
significant depletion in cholesterol, ascorbic acid and reduced glutathione. Reductions in
catalase and superoxide dismutase activities in the treated groups were accompanied by
concomitant increases in the levels of thiobarbituric acid reactive substance, indicating
suppression of the antioxidant defence system. Sperm content and sperm motility patterns
were significantly altered in all metal-treated groups (p < 0.05), suggesting the spermotoxic
effects of lead and cadmium. Overall, the observed changes were more pronounced in the
cadmium treated group. The authors conclude that gestational and lactational exposure to
endocrine active metals can alter epididymal and testicular functions.
Bisphenol AF (BPA-F) is a BPA analogue widely used in the production of polymers.
Recently, concerns regarding the toxicity and endocrine-disrupting potential of BPA-F have
been raised. Feng et al. (2012) investigated the effects and biological mechanisms of BPA-F
on testosterone production using adult male Sprague Dawley rats. Using a corn oil/ethanol
vehicle, BPA-F (2, 10, 50, and 200 mg/kg bw/day) was administered by oral gavage for 14
days. BPA-F administration led to decreased serum cholesterol levels at 50 and 200 mg/kg
bw/day. Furthermore, high dose (200 mg/kg bw/day) animals showed significant reductions
in the serum testosterone (p < 0.01) and increases in luteinizing hormone (LH; p < 0.05) and
follicle-stimulating hormone (FSH) levels (p < 0.05). The concentrations of BPA-F in testes
tissues increased in a dose-dependent manner. High BPA-F exposure also led to a decline in
the gene and protein expression of cholesterol transport and steroid biosynthesis associated
markers. A reduction in the mRNA expression levels of inhibin B, ERα and LH receptor
(LHR) were also reported. The authors conclude that inhibition of testosterone production
consequent to BPA-F exposure primarily results from the alteration of genes and proteins
involved in the testosterone biosynthesis pathway. In another study using the Sprague Dawley
rodent model, Simon et al. (2012) investigated the effects of neonatal exposure to anti-
androgens in the absence of oestrogen. Dissolved in saline solution or olive oil, the following
anti-androgenic chemicals were subcutaneously injected into male rats: antide (10 mg/kg
bw/day), flutamide (50 mg/kg bw/day), a mixture of antide, flutamide, and diethylstilbestrol
(DES) (0.1 mg/kg bw/day), in the presence or absence of AR agonist dihydrotestosterone
(DHT) (20 mg/kg bw/day). Exposure occurred from PND 1 – 6, with an exception of the
mixture group, where animals were injected on PND 1, 2, 3 and 6 - animals were necropsied
on PND 7, 12 or at adulthood. Penile length and weight significantly decreased in all
treatment groups (p < 0.05). Fat accumulation was induced in the antide, mixture and DES
treated groups. Furthermore, the males treated with a mixture showed significant loss of
smooth muscle cells and cavernous spaces in the body of the penis, appearing to be infertile
(p < 0.05). DHT co-administration mitigated penile deformities in the DES group, but not in
the mixture group. Testicular testosterone levels reduced by 70-95% on PND 7 and 12 in all
treated groups (p < 0.05), with the exception of flutamide treated group on PND 7, where
testosterone levels were threefold higher than controls (p < 0.05). The authors conclude that
neonatal exposure to anti-androgens, in the absence of exogenous oestrogen exposure, results
in infertility and permanent malformation of the penis.
The trace element selenium (Se) is essential for several fundamental cellular biological
processes, including cellular antioxidant defence, protection and repair of DNA and
apoptosis. Alteration in physiological concentrations of Se leads to abnormal
spermatogenesis20
. Erkekoglu et al. (2012) investigated the effects of DEHP, a known
20
Olson, G.E., Winfrey, V.P., Hill, K.E., et al. (2004) Sequential development of flagellar defects in
spermatids and epididymal spermatozoa of selenium-deficient rats. Reproduction, 127(3), 335-342.

17. 17
testicular toxicant, on the expression pattern of vimentin filaments in Sertoli cells and germ-
cell apoptosis in the testes of pubertal Sprague Dawley rats, in conjunction with different Se
status. Animals were fed with various Se concentrations (≤ 0.05, 0.15, 1 mg/kg Se) for five
weeks. During the last 10 days of treatment, the animals received 1000 mg/kg of DEHP by
intragastric gavage and subjected to vimentin immunolabelling and the TUNEL assay. Se
deficiency caused a mild decrease in the intensity of Sertoli cell vimentin immunoreactivity
and significantly enhanced germ-cell apoptosis (p < 0.0033). DEHP exposure caused
disruption and collapse of vimentin filaments and significantly induced apoptotic death of
germ cells (p < 0.0033). In DEHP-exposed Se-deficient animals, collapse of vimentin
filaments was more prominent. The authors conclude that Se deficiency exacerbated the
toxicity of DEHP on Sertoli cells and spermatogenesis, whereas Se supplementation provided
protection.
Perobelli et al. (2012) examined the effects of prebubertal exposure to flutamide in male
Wistar rats. To correspond with prepubertal development, immature male rats were
administered flutamide (0, 25 mg/kg bw) in a corn oil vehicle by oral gavage from PND 21 to
PND 44 and necropsied on PND 50 or PND 80. Flutamide exposure significantly reduced
absolute and relative organ weights of the epididymis, prostate, vas deferens and seminal
vesicle on PND 50, and the absolute seminal vesicle on PND 80 (p ≤ 0.0001). A decrease in
fertility potential was observed in the flutamide-treated adults in the artificial in utero
insemination group (p < 0.05), but not in the natural mating group. Flutamide accelerated
sperm transit time through the epididymis (p < 0.05), impairing sperm motility and storage. A
quantitative analysis of the sperm cauda plasma membrane proteome revealed a significant
change in CYP5B, CALM, and PB11A protein expressions. No statistically significant effects
on serum hormone levels were observed. The authors conclude that prepubertal exposure to
flutamide compromises epididymal function in addition to epididymal sperm quality.
Romano et al. (2012) investigated the efficacy of the herbicide, glyphosate, no observed
adverse effect level (NOAEL; 50 mg/kg bw/day) in female Wistar rats. Pregnant rats were
exposed to the equivalent concentration of Roundup Transorb, a glyphosate-based herbicide
by oral gavage from GD 18 to PND 5. Reproductive examination of exposed male offspring
on PND 60 identified an increase in sexual partner preference scores (p < 0.01) and latency
time to the first mount (p < 0.05). An increase in serum concentrations of testosterone (p <
0.01), oestradiol (p < 0.01) and lutenising hormone (LH) (p < 0.05) was observed in treated
males; in addition to elevated lutenising hormone mRNA expression and protein content in
the pituitary gland. Treated males demonstrated significant increases in sperm production (p <
0.05), alterations in seminiferous epithelium morphometry and early onset of puberty. The
authors conclude that perinatal exposure to glyphosate disturbed the masculinisation process
of the test animals. However, the relevance of the exposure level employed in this study to
humans remains uncertain.
The effects of perinatal exposure of BPA and DES on mammary gland differentiation and
milk protein expression were investigated in female Wistar rats (Kass et al., 2012). Pregnant
rats (F0) were orally administered with BPA (0, 0.7, 64 μg/kg bw/day) or DES (6 μg/kg
bw/day) via drinking water from GD 9 to weaning. The F1 offspring were cohabitated with
F0 lactating dams until weaning on lactation day (LD) 21. Randomly selected 90-day old F1
females were then bred to unexposed males and the mammary gland differentiation was
examined on GD 18 or GD 21. A decrease in the mRNA and protein expression levels of α-
lactalbumin and β-casein levels in the mammary gland of all treated F1 females on GD 18 (p
< 0.05) was reported, which was accompanied by a significant reduction in prolactin receptor
and Stat5a/b mRNA expressions (p < 0.05). A statistically significant delay in histological
mammary gland differentiation was observed on GD 21 in all treated groups (p < 0.05). β-
Casein levels remained decreased in the mammary gland on GD21 (p < 0.05) and in milk
samples on LD 14 (p < 0.05). Both of the BPA- and DES-exposed groups demonstrated
altered milk yield patterns during lactation. The authors conclude that low dose xenoestrogen

18. 18
exposure leads to long delays in mammary gland differentiation, altered milk yield and
modified milk composition. In another study investigating mammary gland development,
Mandrup et al. (2012) investigated the effect of ethinyl oestradiol exposure on Wistar rat
mammary gland development. Pregnant rats were exposed to ethinyl oestradiol (0, 5, 15, 50
µg/kg bw/day) in corn oil from GD 7 to GD 21 (the day before expected birth) and again from
PD 1 and PD21/22, by oral gavage. Male pups were necropsied on PD 21, whereas female
pups on PD 22. Mammary gland development was significantly accelerated both in males and
females; changes in the overall density was a sensitive marker in both males (p < 0.05 at 50
µg/kg) and in females (p < 0.05 at 15 µg/kg but not at 50 µg/kg). Furthermore, a correlation
between the number of terminal end buds (TEBs) in zone c and in the TEBs in the whole
mammary gland was present in both sexes. The authors recommend mammary gland
development as a novel endpoint in OECD Test Guidelines (TGs) such as TG 443.
Dean et al. (2012) demonstrated that the female reproductive tract is susceptive to virilisation
by exogenous androgen (dihydrotestosterone) exposure prior to and during the
masculinisation programming window in Wistar rats. In contrast, masculinisation of the male
foetuses was not affected by the treatment of exogenous additional androgens. Kobayashi et
al. (2012) investigated the effects of low-dose exposure to BPA on sexual maturation and
function in rats. Pregnant Sprague Dawley rats (F0) were fed a diet containing BPA at 0, 0.33,
3.3 or 33 ppm between GD 6 and PND 21. The F1 offspring were fed with a normal diet and
necropsied at 5 weeks or 3 months of age. The absolute epididymis weight was significantly
lower in 3-month-old F1 males at 33 ppm. Anogenital distance (AGD), the ratio of AGD
(mm)/3
√ body weight, and relative ovary weight were significantly lower in 5-week-old F1
females at 3.3 ppm and above (p < 0.05); however, the difference was not significant in 3-
month-old females. No treatment-related effects on body weight, reproductive outcomes,
major reproductive organ weights, sperm motility, or plasma steroid hormone levels were
observed in F1 animals. The authors conclude that in utero and early life to low-dose BPA
does not adversely affect reproductive development in F1 rat offspring.
Thimerosal is an organic compound, containing methyl mercury, commonly found in flu
vaccines for pregnant women and infants in the USA and developing countries. Sulkowski et
al. (2012) evaluated the neurodevelopmental effects of thimerosal using spontaneously
hypersensitive rats (SHR) and Sprague Dawley (SD) rats. Pregnant rats (F0) were
subcutaneously injected with 200 µg/kg bw/day of thimerosal during GD 10 – 15 and again
PND 5 – 10. Exposure to thimerosal delayed startle response in male and female SD F1
neonates (p < 0.05) and a decrease in motor learning was observed in SHR F1 males and SD
F1 males and females (p < 0.05). A significant increase in cerebellar levels of the oxidative
stress marker 3-nitrotyrosine in SHR F1 females (p < 0.05) and SD F1 males (p < 0.05) was
also reported. The activity of cerebellar type 2 deiodinase, responsible for local intra-brain
conversion of thyroxine to the active hormone, 3',3,5-triiodothyronine (T3), was significantly
decreased in the treated SHR F1 males (p < 0.01), coinciding with elevated gene expression
of Odf4 (p < 0.05), which is negatively regulated by T3, indicative of local intracerebellar T3
deficiency. The authors conclude that perinatal thimerosal exposure results in a delayed
auditory maturation and impaired motor leaning in rat pups in a strain- and sex-dependent
manner.
Kouidhi et al. (2012) investigated the susceptibility of the submandibular salivary gland
(SSG) to EDCs with oestrogenic activity (genistein), anti-androgenic activity (vinclozolin),
and the mixture of genistein and vinclozolin in female Wistar rats. Pregnant animals were
orally administered with 1 mg/kg bw/day of genistein, vinclozolin, or a mixture in corn oil
between GD 1 and PND 21. The selected dose level of genistein was close to normal human
dietary exposure and the dose level of vinclozolin was notably higher than estimated levels in
humans. Female offspring were necropsied on PND 35 and both SSG were removed for the
examination of morphogenesis and immunohistochemical analysis. A significantly lower
number of striated ducts were observed in genistein (p < 0.05), vinclozolin (p < 0.01) and

19. 19
genistein + vinclozolin mixture (p < 0.01) treatment groups, relative to controls. Striated duct
number was associated with an increase in area and lower acinar proliferation (Ki-67-positive
nuclei). The mixture treatment demonstrated the most potency, particularly regarding the
repression of epidermal growth factor (EGF) (p < 0.05), nerve growth factor (p < 0.01), and
transforming growth factor α (p < 0.01) expression. The authors conclude that the SSG are
targeted by both oestrogenic and anti-androgenic compounds, in both single and mixed
exposures. Since the SSGs are the major source of circulating EGF, the repression of EGF
gene expression level may be predictive of a disturbance later in adulthood.
In the viable yellow agouti (Avy) mouse, maternal diet affects the coat colour distribution of
offspring by perturbing the establishment of methylation at the Avy metastable epiallele.
Hence, the Avy mouse can be a sensitive epigenetic biosensor to assess the effects of dietary
environmental manipulations on locus-specific DNA methylation21
. Anderson et al. (2012)
investigated global and candidate gene methylation patterns using Avy mice consequent to
dietary perinatal BPA exposure (0, 50 ng BPA/kg, 50 µg BPA/kg, and 50 mg BPA/kg).
Following two weeks of dietary exposure, the virgin dams were mated with Avy/a males;
dams remained on the assigned diets throughout pregnancy and lactation until sacrifice at
PND 22. Total genomic DNA was isolated from tail tissue for methylation analysis. The
results showed hypermethylation in tail tissue of a/a and A(vy)/a offspring across all dose
groups (p < 0.02). Maternal exposure to 50 ng or 50 μg BPA/kg resulted in altered coat colour
distributions (p = 0.04 and 0.02, respectively). However, no DNA methylation effects at the
Agouti gene were noted. DNA methylation at the CDK5 activator-binding protein (CabpIAP
)
metastable epiallele shows hypermethylation in the 50 μg BPA/kg offspring (p = 0.02). The
authors conclude that perinatal BPA exposure affects epigenetic regulation across multiple
human relevant doses, suggesting the need to evaluate dose effects in human populations.
In order to understand the dynamics of epigenetic reprogramming consequent to exposure to
the fungicide vinclozolin (VCZ), Lee and Oh (2012) conducted a genome-wide analysis of
DNA methylation in C57BL/6 mice. Pregnant mice were intraperitoneally administered 100
mg/kg bw/day of vinclozolin during GD 8 – 15. F1 male offspring were then mated with
untreated females to generate F2 offspring. A loss of normal spermatogeneiss and abnormal
seminiferous tubule morphology were observed in the treated F1 males; subsequently, the
fertility ratio of treated F1 males was reduced by 18%. Although statistically insignificant, the
number of abnormal F2 foetuses was 25% higher in the VCZ exposed group relative to
control (p = 0.06), suggesting the effect of VCZ on reproductive performance of F1 males.
Microarray analysis of F1 male sperm DNA identified the alteration of 293 hyper-methylated
and 783 hypo-methylated target genes (p < 0.05), although the results were heterogeneous.
In another epigenetics study, Tainaka et al (2012) investigated the effects of BPA exposure on
the testicular gene expression profile, using a testis-specific microarray (Testis2). Pregnant
ICR mice were subcutaneously injected with BPA (0, 5, and 50 mg/kg in corn oil) on GD 7
and GD 14. Pups were weaned on PND 21 and sacrificed at 6 weeks old. The dissected testes
tissues were subjected to microarray analysis. The gene expression profiles were annotated
for the cellular components and biological functions. Five Sertoli cells-related genes (Msi1h,
Ncoa1, Nid1, Hspb2, Gata6) were down-regulated consequent to 50 mg/kg BPA. The
analysis of gene expression profiles indicated that the mechanisms underlying the testicular
toxicity may differ between low and high dose of prenatal BPA treatment. There was a
significant decrease in the sperm quality (sperm count, morphology and mortility p < 0.05)
and number of Sertoli cells (p < 0.001) both at 5 and 50 mg/kg groups. A histopathological
examination identified a shedding of immature germ cells in the seminiferous tubules,
indicative of disrupted sperm differentiation consequent to BPA exposure. The authors
21
Dolinoy, D.C., Huang, D., Jirtle, R.L. (2007) Maternal nutrient supplementation counteracts
bisphenol A-induced DNA hypomethylation in early development. Proceedings of the National
Academy of Sciences of the United States of America, 104(32), 13056-13061.

20. 20
conclude that in utero exposure to BPA changes the gene expression patterns in the spermatic
and histological abnormalities.
Manikkam et al. (2012) studied transgenerational effects consequent to ancestral
environmental mixture exposure in Sprague Dawley rats. During embryonic gonadal sex
determination (embryonic day (ED) 8 – 14), pregnant rats (F0) were given daily
intraperitoneal injections of a pesticide mixture (150 mg/kg bw/day of permethrin and 40
mg/kg bw/day of N,N-diethyl-meta-toluamide [DEET]), a plastic mixture (50 mg/kg bw/day
of BPA, 66 mg/kg bw/day of DBP, and 750 mg/kg bw/day of DEHP), dioxin (100 ng/kg
bw/day 2,3,7,8-tetrachlorodibenzo-p-dioxin [TCDD], and a hydrocarbon mixture (500 mg/kg
bw/day of jet fuel JP8) in sesame oil. The assessment of F3 progeny showed that the early-
onset female puberty was promoted following ancestral (F0) exposure to plastics (p < 0.05),
dioxin (p < 0.01), and jet fuel (p < 0.05). Spermatogenic cell apoptosis significantly increased
in all the jet fuel-treated F3 males. Ovarian primordial follicle pool size significantly
decreased in all treated F3 females (p < 0.001). Examination of the F3 generation sperm
promoter epigenome using microarray identified differential DNA methylation regions
(DMR) in the all exposure lineage males accompanied by low density cytosine-phosphate-
guanine (CpG) content, which were consistent within a specific exposure lineage but different
between the exposures. The same research group further reported the results by setting
additional exposure groups (100 mg/kg bw/day of vinclozolin, and a half dose of plastic
mixture) (Nilsson et al., 2012). There was a significant increase in the ovarian cysts in some
of the treated F1 females (vinclozolin, low pesticides and jet fuel, p ≤ 0.05) and all treated F3
females (p ≤ 0.05). There was also a significant decrease in the ovarian primordial follicle
pool size in all treated F1 females (p ≤ 0.005) and all treated F3 females (p ≤ 0.005). These
findings suggest a role of environmental epigenetics in the aetiology of ovarian disease. The
granulosa cells of F3 generation were found to have transgenerational effects on the
transcriptome and epigenome (differential DNA methylation). The authors suggest that the
identified exposure-specific epigenetic biomarkers could be used for the assessment of
ancestral environmental exposures associated with adult onset diseases.
Animal studies indicate that postnatal exposure to BPA may affect neocortex development in
embryos by accelerated neurogenesis and promoting neuronal migration defects22
. Komada et
al. (2012) exposed pregnant C57BL/6J mice to 200 µg/kg BPA, in a corn oil vehicle, by oral
gavage from ED 8.5 to 13.5. Histopathological examinations of foetuses on ED 14.5,
immunolabelling and cell-cycle-specific labelling of the BPA-treated embryos using
thymidine-analogues identified a significant increase in the total thickness of the dorsal
telencephalon in the (Tuj1-stained region (neuron-specific class III β-tubulin; pan-neuronal
marker; p < 0.05). Cortical plate was hyperplastic and there was a decrease in the number of
neural progenitor cells, particularly intermediate progenitor cells (IPCs). Exposure to BPA
was associated with the promotion of the cell cycle exit in radial glial cells (RGCs, neural
stem cells) and IPCs, and decreased the proliferation resulting from the prolong cell cycle
length of IPCs in the subventricular zone (SVZ). The authors conclude that maternal oral
exposure to BPA affects the normal cell cycle in neural IPCs and neurogenesis in the
developing neocortex.
Animal evidence suggests that some mild analgesics might be a risk factor for the
development of male reproductive disorders, such as cryptorchidism, through potent anti-
androgenic effects23
. Kristensen et al. (2012) investigated the effects of mild analgesics in a 3-
22
Nakamura, K., Itoh, K., Yaoi, T., et al. (2006) Murine neocortical histogenesis is perturbed by
prenatal exposure to low doses of Bisphenol A. Journal of Neuroscience Research, 84(6), 1197-1205.
23
Kristensen, D.M., Hass, U., Lesné, L., et al. (2011) Intrauterine Exposure to Mild Analgesics is a
Risk Factor for Development of Male Reproductive Disorders in Human and Rat. Human
Reproduction, 26(1), 235-244. (The study was cited in Endocrine Disrupting Chemicals: Overview of
recent published literature January 2011 – March 2011)

21. 21
day ex vivo organotypic model system, where sections of GD 14.5 Sprague Dawley rat foetal
testes were cultured with 0.1 – 100 µM of paracetamol or aspirin for three days. Indomethacin
was used as a positive control for prostaglandin (PG) inhibition. The results showed that
testosterone production was inhibited by paracetamol (0.1 - 100 μM), aspirin (1 - 100 μM),
and indomethacin (10 μM). The production of the other Leydig cell hormone, insulin-like
factor (Insl3), was not disrupted by paracetamol. No significant effect of the analgesics was
observed on the gross testes morphology, Leydig cell counts or rate of gonocyte apoptosis.
Parallel experiments suggested that the effects of paracetamol and aspirin on PG-D2 and
testosterone were not connected. The authors conclude that mild analgesics exert direct and
specific anti-androgenic effects in the rat foetal testes, but the mechanism of action is
probably not related to inhibition of PG synthesis.
b) Carcinogenic effects
Jenkins et al. (2012) review the role of structurally similar EDCs: BPA; DES; TCDD; soy
component (genistein); and, red grape phytoalexin [resveratrol], in the aetiology of breast
cancer. The authors conclude that BPA, DES and anti-oestrogenic TCDD (2,3,7,8-
tetrachlorodibenzo-p-dioxin) increased chemically-induced mammary carcinogenesis through
retardation of mammary gland maturation. While genistein and resveratrol protected against
chemically-induced and spontaneous mammary cancers. In a review of the health effects of
DES24
, a known carcinogen for human and experimental animals, Harris and Waring (2012)
conclude that humans appear to be less susceptible to DES than rodents. Although the long-
term effects of DES may depend on the timing of exposure, the authors suggest that very high
doses of potent pseudo-oestrogens over long periods of time appear to have relatively little
effect on the human population. In a conference abstract, Birnbaum (2012) suggests that early
life exposures to endocrine active substances affect mammary gland architecture, oestrogen
and progesterone receptor function, as well as susceptibility to tumours.
Adams et al. (2012) estimated the association between dietary cadmium intake and risk of
invasive postmenopausal breast cancer in the Vitamins and Lifestyle (VITAL) cohort in the
USA. Postmenopausal women (n = 30543, aged 50 – 76) were monitored for incidence of
breast cancer during a 7.5 year follow-up study. Dietary cadmium consumption was estimated
based on a self-administered questionnaire, relative to figures published by the US Food and
Drug Administration. There was no evidence for an association between dietary cadmium
intake and invasive breast cancer risk. The adjusted hazard ratio (aHR) for the highest (>
13.30 µg/day) to lower quartile cadmium was 1.00 (95% CI 0.72 – 1.41; p = 0.95). No
association was found in the stratified analysis by tumour oestrogen receptor status. The
authors conclude that the study does not support the hypothesis that dietary cadmium intake is
a risk factor for breast cancer. However, caution regarding the accuracy of the exposure
assessment was stressed. In a conference abstract, Bidgoli (2012) reported an association
between breast cancer and various risk factors in Iranian women with premenopausal breast
cancer (PBR; n = 50) and their aged- and hospital record-matched controls (n = 100).
Overexpressions of aryl hydrocarbon receptor (AhR) were detected in 87% of epithelial cells
of young breast cancer patients. Living near factories that generate PAHs and dioxins was
significantly associated with PBR (OR = 4.80, p = 0.001). History of idiopathic infertility was
also associated with PBR (OR = 3.50, p = 0.002), which is believed to be affected by
endogenous oestrogen levels. Consumption of the oral contraceptive pills, for more than 5
years was also identified as an associated risk factor (OR = 2.27, p = 0.006). Adiposity and
abnormal weight gain after 18 years old appeard to be the major background factor. Breast
24
The International Agency for Research on Cancer (IARC) evaluated that there was sufficient
evidence in humans and experimental animas for the carcinogenicity of DES and the substance is
classified to be carcinogenic to humans (Group 1); IARC. (2012) ‘Diethylstilbestrol’ IARC Monograph
on the evaluation of carcinogenic risk to humans, volume 100A-16, pp. 175–218.

22. 22
cancer affects Iranian women at least one decade earlier than their counterparts in developed
countries25
and the authors suggests that this may be partly related to the interactions between
hormonal and environmental factors.
Lee et al. (2012) investigated the effects of BPA and the organochlorine pesticide
methoxychlor (MXC) on cell proliferation facilitated by oestrogen receptor (ER) signalling in
the human breast cancer cells (MCF-7), which are known to be ERα-positive and to be a
highly 17β oestradiol (E2)-responsive cancer cell line26
,27
. The cells were co-treated with
agonists (propyl pyrazoletriol [PPT] and diarylpropionitrile [DPN]) or an antagonist (ICI
182,780) of ER signalling to reduce ERα gene expression via siRNA before the treatment
with EDCs. BPA and MXC induced the cancer cell proliferation by the up-regulation of genes
that promote the cell cycle and the down-regulation of anti-proliferative genes, especially
those affecting the G1/S transition via ERα signalling. The authors propose the necessity of
further studies to determine whether EDCs promote carcinogenesis in vivo.
c) Other effects
In this reporting period, Andra and Makris (2012) reviewed the literature pertaining to human
exposure to exogenous thyroid disrupting chemicals (TDCs); including BPA, phthalates and
PBDEs, and their effects on endogenous thyroid hormones. The authors conclude that a lack
of consistency and specificity of reported qualitative assessment prevents formulation of a
conclusive statement. Janesick and Blumberg (2012) discuss the role of ‘obesogenic’ EDCs
(such as tributyltin) on fat accumulation and obesity, concluding that exposure during
sensitive windows of development might pre-program an individual to store more fat,
resulting in lifelong health problems. Additionally, vom Saal et al. (2012) review the
developmental effects of EDCs on adipocyte differentiation and function in relation to
obesity. The authors highlighted animal evidence where BPA was found to accelerate
postnatal growth and alter insulin secretion and glucose sensitivity, while further expanding
the discussion to placental blood flow, nutrient transport to foetuses and abnormalities in the
homeostatic control systems.
Lee et al. (2012) measured baseline plasma concentrations of 21 persistent organic pollutants
(POPs) in a cross-sectional cohort in Sweden (n = 970, aged 70 years), participants were
followed-up 5 years later (n = 511). The cross-sectional analyses identified an association
between serum concentrations of low chlorine PCBs, OC pesticides (e.g. p,p'-DDE) and
dioxin with persistent abdominal obesity (measured waist circumference). In contrast,
concentrations of highly chlorinated PCBs showed a strong negative correlation with
abdominal obesity. The association between POPs and risk of abdominal obesity persisted in
the prospective analyses, although the association was weaker. The authors conclude that
abdominal obesity is positively associated with low-dose exposure to low-chlorine PCBs,
p,p'-DDE, and dioxin. Highly chlorinated PCBs were inversely associated in the elderly
population, despite the previous observation of higher incident diabetes associated with these
25
Harirchi, I., Karbakhsh, M., Kashefi, A., et al. (2004) Breast cancer in Iran: results of a multi-center
study. Asian Pacific Journal of Cancer Prevention, 5(1), 24-27.
26
Hu, Q., Kwon, Y.S., Nunez, E., et al. (2008) Enhancing nuclear receptor-induced transcription
requires nuclear motor and LSD1-dependent gene networking in interchromatin granules. Proceedings
of the National Academy of Sciences of the United States of America, 105, 19199–19204.
27
Keshamouni, V.G., Mattingly, R.R. & Reddy, K.B. (2002) Mechanism of 17-beta-estradiol-induced
Erk1/2 activation in breast cancer cells. A role for HER2 AND PKC-delta. The Journal of Biological
Chemistry, 277(25), 22558-22565.

23. 23
same PCBs28
. In another epidemiology study associating EDC’s to obesity, Rundle et al.
(2012) explored the relationship between prenatal PAH exposure and children’s obesity.
Children born to women who underwent personal air monitoring for PAH during pregnancy
(n = 702), as part of a longitudinal birth cohort of African-American and Hispanic children in
New York City (USA), were followed at 5 years (n = 422) and 7 years (n = 341) of age. In the
prospective assessment, 21% of the children at 5 years of age and 25% of those followed at 7
years of age were obese. After adjustment, higher prenatal PAH exposures were significantly
associated with higher childhood body size. In the adjusted analyses, relative to children of
mothers with the lowest PAH exposure (tertile 1 < 1.73 ng/m3
), children of high exposure
mothers (tertile 3 ≥ 3.08 ng/m3
) had a 0.39-unit higher body mass index z score and a relative
risk (RR) for obesity of 1.79 (95% CI 1.09 - 2.96) at 5 years of age. The association between
PAH and obesity persisted at 7 years of age; a 0.30-unit higher body mass index z score, a
1.93-unit higher percentage of body fat, and a RR of 2.26 (95% CI 1.28 -4.00) for obesity.
The authors conclude that prenatal exposure to PAHs leads to increased fat gain during
childhood and a higher risk of childhood obesity.
Some evidence suggests that BPA exposure promotes experimental asthma in mice29
. Spanier
et al. (2012) examined the relationship between prenatal BPA exposure and wheeze in early
childhood in a prospective birth cohort (n = 365 mother-infant pairs). Parent-reported child
wheeze was assessed every 6 months, from birth to 3 years of age. The LC-MS analysis
identified BPA in 99% of maternal urine samples during pregnancy (the geometric mean of
prenatal urinary concentration = 2.4 μg/g creatinine). In a multivariable analysis, adjusted
mean urinary BPA concentration was not significantly associated with child wheeze from
birth to 3 years of age. However, there was an interaction of BPA concentration with time (p
= 0.003); prenatal BPA measurements above the median were positively associated with
wheeze, relative to low BPA groups, at 6 months of age (adjusted OR = 2.3; 95%CI 1.3 - 4.1),
but not at 3 years. In secondary analyses, where associations of each prenatal BPA
concentration were evaluated separately, urinary BPA concentrations measured at 16 weeks
of gestation were associated with wheeze (adjusted OR = 1.2; 95%CI 1.0 - 1.5), but BPA
concentrations at 26 weeks of gestation or at birth were not, suggesting that early pregnancy
may be the most sensitive time window of exposure. The authors conclude that prenatal BPA
exposure is associated with increased wheeze in early life, but that associations diminish over
time.
Batista et al. (2012) investigated the effects of low-dose BPA on the development of type II
diabetes using adult male Swiss albino mice. Animals were subcutaneously injected with
BPA (0 or 100 µg/kg/day) in a corn oil vehicle for 8 consecutive days. Mice treated with BPA
presented signs of insulin resistance and had increased glucose-stimulated insulin release (p <
0.05). A decrease in food intake, lower body temperature and locomotor activity was
observed in the treated mice. Furthermore, exposed animals showed impaired insulin-
stimulated tyrosine phosphorylation of the insulin receptor-β subunit in skeletal muscle (p <
0.05), and to a lesser extent, the liver (p < 0.05). The skeletal muscle impairment was
associated with a reduction in insulin-stimulated Akt phosphorylation of the Thr308 residue
(p = 0.05). Treated animals displayed up-regulation of IRS-1 protein in both skeletal muscle
and liver (p < 0.05). The mitogen-activated protein kinase (MAPK) signaling pathway was
also impaired in the skeletal muscle of the treated animals (p < 0.05). The authors conclude
that BPA is a risk factor for the development of type II diabetes, as supported by a delay in
28
Lee, D.H., Lee, I.K., Song, K., et al. (2006) A strong dose-response relation between serum
concentrations of persistent organic pollutants and diabetes: results from the National Health and
Examination Survey 1999-2002. Diabetes Care, 29(7), 1638-1644.
29
Midoro-Horiuti, T., Tiwari, R., Watson, C.S., et al. (2010) Maternal bisphenol A exposure promotes
the development of experimental asthma in mouse pups. Environmental Health Perspectives, 118, 273–
277.

24. 24
whole body energy metabolism and impaired insulin signaling in exposed murine peripheral
tissues.
Testa et al. (2012) assessed the association between autism spectrum disorders (ASDs) and
phthalate exposure in a matched case-control study in Italy. Study participants consisted of 48
children with ASD (mean 11 ± 5 years old) and 45 healthy controls (mean 12 ± 5 years old).
Urine samples were collected from the study participants for measurement of DEHP
metabolites (MEHP; mono-(2-ethyl-6-hydroxyhexyl) 1,2-benzenedicarboxylate [6-OH-
MEHP]; mono-(2-ethyl-5-hydroxyhexyl) 1,2-benzenedicarboxylate [5-OH-MEHP]; mono-(2-
ethyl-5-oxohexyl) 1,2-benzenedicarboxylate [5-oxo-MEHP]). There was a significant
increase in urinary concentrations of 5-OH-MEHP (p = 0.02) and the 5-oxo-MEHP
metabolite (p = 0.005) in ASD cases, relative to controls. Furthermore, a significant positive
correlation between 5-OH-MEHP and 5-oxo-MEHP was identified (rs = 0.668, p < 0.0001).
Amongst the measured phthalates, 5-oxo-MEHP showed the highest specificity (91.1%) in
identifying ASD patients. The authors conclude that prenatal and postnatal phthalate exposure
is associated with ASDs, thus the screening of urinary DEHP metabolites in high-risk
populations could have an important social impact.
This reporting period has seen the publication of studies pertaining to transgenerational
effects of EDC exposure. Jang et al. (2012) evaluated the transgenerational effects of BPA on
hippocampal neurogenesis and neurocognitive function in C57BL/6 mice. Pregnant females
(F0) were intraperitoneally injected with BPA (0, 0.1, 1, 10 mg/kg/day in corn oil) from GD 6
to GD 17. Consequent to weaning (3 months), treated F1 females were mated with F1 control
males to produce the F2 generation; cerebral cortices and hippocampi were excised from F2
females. Exposure of F0 mice to 10 mg/kg of BPA decreased proliferation of newly generated
cells in hippocampi (p < 0.01). Passive avoidance testing suggested that high doses of BPA
(1, 10 mg/kg) decrease cross-over latency time in F2 mice (p < 0.05), indicative of a BPA-
mediated neurocognitive deficit in memory retention. The gene expression levels, of phospho-
ERK, brain-derived neurotrophic factor (BDNF) and phospho-CREB in hippocampi, were
significantly lower in F2 mice. The effects of BPA on hippocampal neurogenesis were
associated with altered DNA methylation. The authors conclude that maternal exposure to
high doses of BPA can affect hippocampal neurogenesis and cognitive function, by
modulating the ERK (extracellular signal-regulated kinases) and BDNF (brain-derived
neurotrophic factor)-CREB (cyclic adenosine monophosphate [cAMP] response element
binding) signalling cascades. In another neurodevelopment study, Crews et al. (2012) tested
the effects of vinclozolin on neural and behavioural development and chronic restraint stress
(CRS) in Sprague Dawley (SD) rats. Female F0 rats were injected with vinclozolin (100
mg/kg/day) or vehicle (dimethyl sulfoxide [DMSO]) during ED 8 – 14. The F1 generation
were bred to generate the F2, and then the F2 were bred to generate F3 generation. One day
after weaning (PND 22) F3 animals were pair-housed (1:1) and subject to chronic restraint
stress treatment for 21 days. Vinclozolin treated F3 males demonstrated altered physiology,
behaviour and metabolic activity. The transcriptome in discrete brain nuclei was altered in the
treated males and vinclozolin treatment altered CRS response. The authors conclude that
adolescent CRS response is altered by ancestral exposure to vinclozolin.
The effects of perinatal exposure to BPA on learning were explored by Jones and Watson
(2012). Pregnant female Long-Evans rats were orally treated with BPA (0, 5, 50, 500, or 5000
µg/kg bw/day in corn oil) from GD 7 to PND 14. Pups were weaned on PND 21 and
subjected to a course of behaviour tests between the ages of 90 and 150 days. No treatment
effects of BPA were observed in the Morris Water Maze test. Conversely, 5 μg/kg BPA
eliminated sex differences in the Elevated Plus Maze and the Forced Swim tests. However,
sexual dimorphism was not always observed in other groups, including the control. The
authors conclude that maternal exposure to BPA has the potential to alter the normal
development of affective behaviours, eliminating the typical sex differences and altering

25. 25
learning strategy under the condition of the study, although this only occurred at an
environmentally relevant dose in males.
The effects of BPA on the hippocampal neurons were also examined using an in vitro test
system (Tanabe et al., 2012). Hippocampal slices were dissected from the brain of decapitated
adult male Wistar rats and incubated with BPA (0, 1, 10, 100, or 10,000 nM) for up to 2
hours. Spinogenesis as spine density was significantly enhanced by 10 nM of BPA within 2h
of treatment. In particular, the density of middle-head spine (with head diameter of 0.4 - 0.5
μm) was significantly increased (p < 0.01). BPA-induced enhancement of the spine density
was blocked by hydroxytamoxifen, an antagonist of both oestrogen-related receptor gamma
(ERRγ) and oestrogen receptors (ERα/ERβ), but not by an antagonist of ERα/ERβ (ICI
182,780). The BPA-induced enhancement of spinogenesis was also suppressed by MAP
(mitogen-activated protein) kinase inhibitor (PD98059) and the NMDA ((N-methyl-D-
aspartate) receptors antagonist (MK-801). However, the effects observed consequent to BPA
exposure appeared to be reversible. The authors conclude that low concentrations of BPA
could induce significant effects on the synaptic plasticity of the hippocampus in mammals.
Lasserre et al. (2012) investigated the effects of PCB 153 and atrazine on subcellular
compartments in the MCF-7 human breast cancer cell line. Cells were treated with atrazine
(200 ppb), PCB 153 (500 ppb), E2 (positive control, 10nM) or DMSO (0.1%, negative
control) for 36 hours. Proteins from cell membrane and cytosol were isolated for proteome
analysis using 2D-DiGE; analysis identified a total of 36 differentially regulated proteins (>
1.5-fold change, p < 0.05) in the membrane fraction and 22 proteins in the cytosol. Identified
proteins were mainly involved in cell structure, stress response, and xenobiotic metabolism.
Consequent to atrazine exposure 67% and 50% of membrane and cytosol proteins were
present, respectively. Conversely, nearly 100% of membrane and 45% of cytosol proteins
were down-regulated following PCB 153 exposure. Western blots of HSBP1, FKBP4 and
STMN1 proteins confirmed the proteome analysis results. The authors conclude that
environmental concentrations of atrazine and PCB 153 have potential endocrine toxicity,
highlighting cell structure, stress response and xenobiotic metabolism as sensitive pathways.
The potential effects of perfluorooctanesulfonic acid (PFOS) on cellular response has been
investigated in human umbilical vein endothelial cells (HUVECs) (Liao et al., 2012).
HUVEC cells were cultured with PFOS (0, 50, 100 mg/L) in DMSO for 24 - 48 hours.
Exposure to PFOS (100 mg/L) significantly up-regulated the gene expression of PPARγ (24,
48 hours of exposure) and ERα (48 h). The gene expressions of all the monitored
inflammatory-related genes (IL-1β, IL-6, COX-2, NOS3, ICAM-1, P-Selectin) were
significantly up-regulated after 24 or 48 hours of exposure. Furthermore, when HUVECs
were treated with 100 mg/l of PFOS for up to 40 hours, the amount of reactive oxygen species
(ROS) significantly increased, in a time dependent manner. In a parallel experiment, the
adhesion of the human acute monocytic leukemia cells (THP-1) onto HUVECs was
significantly increased in the 100 mg/L treatment group after 48 hours of exposure (p <
0.001). The authors conclude that PFOS exposure may play an important role in the vascular
inflammatory disorders and endothelial dysfunctions.

26. 26
3. BIOLOGICAL MECHANISMS and TOXICITY
TESTING
During this reporting period, a large number of reports pertaining to biological mechanisms of
Endocrine Disrupting Chemicals (EDCs) have been published. Alonso-Magdalena et al.
(2012) reviewed the evidence pertaining to the oestrogenic activity of bisphenol A (BPA),
emphasising its potency through non-classical oestrogen activated pathways. Bazer et al.
(2012) summarise the critical role of uterine endometrium and their secretions for normal
conceptus development, discussing the vulnerability of uterine adenogenesis to environmental
oestrogens and progestins. Boas et al. (2012) provide an overview of the current knowledge
regarding thyroid disrupting chemicals (TDCs), such as polychlorinated biphenyls (PCBs),
phthalates, BPA, brominated flame retardants and perfluorinated chemicals. Hu et al. (2012)
evaluated the evidence for a role of EDCs in increased prostate cancer risk. The authors
discuss the use of human prostasphere and chimetric prostate models as novel test systems for
the assessment of the reprogramming of human prostate stem/progenitor cells. McLachlan et
al. (2012) explored the origins and distribution of oestrogen signalling, illuminating the
various biological mechanisms of environmental agents on reproductive development. In a
review, Roth and Sathyanarayana (2012) conclude that the metabolic pathways involved in
the regulation of growth, body weight and sexual maturation are regulated by hormones,
genes and epigenetic programming and thus, susceptible to EDCs. William (2012) details the
metabolic events and exogenous factors which up-regulate P450 aromatase for hormonal
secretions, focussing on the possible effects that stress, xeno-oestrogens and poor dietary
choices may have on these events.
Guerrero-Bosagna and Skinner (2012) reviewed epigenetic transgenerational inheritance of
phenotype and disease, stressing the importance of investigative systems biology. Raldúa et al.
(2012) highlight the use of zebrafish eleuthero-embryos for the assessment of potential
thyroid disrupting chemicals (TDCs), emphasising a higher degree of conservation with upper
vertebrates in the thyroid follicles. Miyata and Ose (2012) discuss the OECD and EPA test
guidelines30
which employ larval African clawed frogs (Xenopus laevis) for screening of
potential EDCs on frog metamorphosis. The authors stress the sensitivity and importance of
thyroid gland histopathological examinations as an incorporated test parameter.
Yuan et al. (2012) examined the effects of phthalates on androgen biosynthesis in Leydig
cells. Amongst the tested phthalates (dipropyl phthalate [DPrP], dibutyl phthalate [DBP],
dipentyl phthalate [DPP], bis(2-butoxyethyl) phthalate [BBOP], dicyclohexyl [DCHP]
phthalate), BBOP and DCHP at 1 mM significantly inhibited activity of the enzymes 3β-HSD
(3-β-hydroxysteroid dehydrogenase) and 17β-HSD3 both in human and rat testis tissues (p <
0.001). Phthalates with 1-2 or 7-8 carbon atoms in ethanol moieties had no effect on the
investigated enzyme activities even at concentrations up to 1 mM. The authors conclude that
there are clear structure-activity responses for phthalates in the inhibition of HSD activities.
The determination of the potential inhibitory effects of HSDs in different length carbon chains
in the ethanol moieties of phthalates is recommended.
According to a Chinese study, BDE-47 is found to be the most predominant polybrominated
biphenyl (PBDE) congener in maternal blood and breast milk31
. Wang et al. (2012c)
investigated whether or not low concentrations of BDE-47 cause hormesis effects in human
30
OECD Guideline for the Testing of Chemicals No. 231: The Amphibian Metamorphosis Assay
(2009) and Endocrine Disruptor Screening Program Test Guidelines OPPTS 890.1100: Amphibian
Metamorphosis (Frog) (2009).
31
Bi, X., Qu, W., Sheng, G., et al. (2006) Polybrominated diphenyl ethers in South China maternal
and fetal blood and breast milk. Environmental Pollution, 144(3): 1024-1030.