PLOS ONE

RESEARCH ARTICLE

Developmental exposure to the brominated

flame retardant DE-71 reduces serum thyroid
hormones in rats without hypothalamic-
pituitary-thyroid axis activation or
neurobehavioral changes in offspring
Louise Ramhøj ID1*, Terje Svingen ID1, Karen Mandrup1, Ulla Hass1, Søren Peter Lund2,
Anne Marie Vinggaard ID1, Karin Sørig Hougaard2,3, Marta Axelstad1

1 National Food Institute, Technical University of Denmark, Kgs. Lyngby, Denmark, 2 National Research
a1111111111 Centre for the Working Environment, Copenhagen, Denmark, 3 Department of Public Health, University of
a1111111111 Copenhagen, Copenhagen, Denmark
a1111111111
* louram@food.dtu.dk
a1111111111
a1111111111

Abstract
Polybrominated diphenyl ethers (PBDEs) are legacy flame retardants for which human
OPEN ACCESS exposure remains ubiquitous. This is of concern since these chemicals can perturb develop-
Citation: Ramhøj L, Svingen T, Mandrup K, Hass ment and cause adverse health effects. For instance, DE-71, a technical mixture of PBDEs,
U, Lund SP, Vinggaard AM, et al. (2022) can induce liver toxicity as well as reproductive and developmental toxicity. DE-71 can also
Developmental exposure to the brominated flame
disrupt the thyroid hormone (TH) system which may induce developmental neurotoxicity
retardant DE-71 reduces serum thyroid hormones
in rats without hypothalamic-pituitary-thyroid axis indirectly. However, in developmental toxicity studies, it remains unclear how DE-71 expo-
activation or neurobehavioral changes in offspring. sure affects the offspring’s thyroid hormone system and if this dose-dependently relates to
PLoS ONE 17(7): e0271614. https://doi.org/
neurodevelopmental effects. To address this, we performed a rat toxicity study by exposing
10.1371/journal.pone.0271614
pregnant dams to DE-71 at 0, 40 or 60 mg/kg/day during perinatal development from gesta-
Editor: Jamie C. DeWitt, East Carolina University,
tional day 7 to postnatal day 16. We assessed the TH system in both dams and their off-
UNITED STATES
spring, as well as potential hearing and neurodevelopmental effects in prepubertal and adult
Received: March 9, 2022
offspring. DE-71 significantly reduced serum T4 and T3 levels in both dams and offspring
Accepted: July 1, 2022 without a concomitant upregulation of TSH, thus inducing a hypothyroxinemia-like effect. No
Published: July 19, 2022 discernible effects were observed on the offspring’s brain function when assessed in motor
activity boxes and in the Morris water maze, or on offspring hearing function. Our results,
Peer Review History: PLOS recognizes the
benefits of transparency in the peer review together with a thorough review of the literature, suggest that DE-71 does not elicit a clear
process; therefore, we enable the publication of dose-dependent relationship between low serum thyroxine (T4) and effects on the rat brain
all of the content of peer review and author
in standard behavioral assays. However, low serum TH levels are in themselves believed to
responses alongside final, published articles. The
editorial history of this article is available here: be detrimental to human brain development, thus we propose that we lack assays to identify
https://doi.org/10.1371/journal.pone.0271614 developmental neurotoxicity caused by chemicals disrupting the TH system through various
Copyright: © 2022 Ramhøj et al. This is an open mechanisms.
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.

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PLOS ONE Thyroid hormone disrupting effects of DE-71

Data Availability Statement: All relevant data are Introduction

within the manuscript and its Supporting
Information file. Disrupted thyroid hormone (TH) signaling during perinatal life can adversely affect the devel-
oping brain. This is because proper brain development is critically dependent on spatiotempo-
Funding: This study was funded by the Danish
Environmental Protection Agency, Ministry of
rally controlled thyroid hormone action [1, 2]. The TH system, however, comprise an intricate
Environment and Food of Denmark. KS Hougaard network of interconnected organs and signaling events. Thus, TH system disruption by chemi-
received support from FFIKA, Focused Research cal substances can occur by different mechanisms. This includes inhibition of thyroperoxidase
Effort on Chemicals in the Working Environment, (TPO), the sodium/iodide symporter (NIS), deiodinases, dehalogenases, and transporters such
from the Danish Government. The funders had no as the monocarboxylate transporter 8 (MCT8). It can also occur by displacement of TH from
role in study design, data collection and analysis,
the serum distributor protein transthyretin (TTR) or augmentation of liver metabolism
decision to publish, or preparation of the
manuscript. and excretion of TH through induction of metabolizing enzymes and excretion-transporters
[1, 3, 4].
Competing interests: The authors have declared
Exposure to compounds targeting any of the abovementioned TH system components can
that no competing interests exist.
have different effects on the TH system itself, as well as on the developing brain. One mecha-
nism and effect pattern that is well described in rats is TPO inhibition (reducing TH synthesis)
leading to activation of the hypothalamic-pituitary-thyroid axis (HPT-axis). In this scenario,
there is a dose-dependent correlation between reduced serum T4 concentrations and altered
brain development manifesting with both morphological and behavioral effects [5–12]. How-
ever, for some TH system disrupting chemicals not acting through this TPO-mediated mecha-
nism, it is unclear if there is the same correlation between low serum T4 concentrations and
TH-mediated brain effects. This includes environmental chemicals that presumably disrupt
the TH system by either binding to TTR or induce metabolizing enzymes in the liver, for
instance triclosan [13, 14], polychlorinated biphenyls (PCBs) [15], the perfluorinated com-
pound perfluorohexane sulfonate (PFHxS) [14, 16] and potentially also the polybrominated
diphenyl ether (PBDE) mixture DE-71 (PBDE) [17–19].
Now banned, PBDEs were for decades used as flame retardants in both industrial and con-
sumer products. Today, human exposure continues due to environmental contamination, per-
sistence and bioaccumulation. Thus, the primary exposure routes are food and house dust
[20]. DE-71 is a technical mixture of PBDEs and its congeners are still found in human serum,
breast milk and house dust [20, 21]. In humans and animals, exposure to PBDEs has been asso-
ciated with a range of effects including neurotoxicity, disruption of the reproductive and thy-
roid hormone systems.
In adult rats DE-71 markedly reduces serum thyroxine (T4) concentrations (>70% reduc-
tion), while TSH levels remain unaffected, or slightly increased [17, 22–24]. A similar effect
pattern is seen in perinatally exposed rat offspring, where DE-71 reduces serum T4 by more
than 70% without inducing TSH levels to any great degree [17, 18, 24]. One question is if such
marked reductions in T4, without a concomitant compensatory increase in TSH, can perturb
neurodevelopment and cause cognitive or other toxicological effects in exposed offspring simi-
larly to those caused by TPO-inhibitors. Based on a small number of developmental exposure
studies with DE-71, it may be assumed that it does not. However, no definite conclusions can
be drawn from the available data.
Previous toxicity studies with perinatal DE-71 exposure have reported marked reduction in
T4 (70–90% in high exposure groups), yet without consistent adverse behavioral effects in the
offspring [17–19]. In some of the studies, however, group sizes were relatively small which
could impair their ability to detect subtle effects. In addition, the studies performed cognitive
testing at different developmental ages using slightly different methodologies. Exposure win-
dows also varied slightly between studies, as did the rat strains used; Long-Evans [17], Spra-
gue-Dawley [18] or Wistar [19]. Finally, the potential effects on HPT-axis regulation remains
poorly examined. Three studies report on TSH levels in DE-71 exposed pups; one with a 30%

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PLOS ONE Thyroid hormone disrupting effects of DE-71

increase [18] and two others with no changes to TSH [17, 24], and there are no thorough histo-
logical examinations of pup thyroid glands. Together, this makes it difficult to conclude on
overall effect patterns on the TH system after perinatal exposure to DE-71.
To enable a more informed conclusion about any potential adverse effects on behavior fol-
lowing developmental exposure to DE-71, as well as address other unclear relationships as out-
lined above, we performed a robust in vivo toxicity study. The study design and endpoints are
aligned with our previous studies of TPO inhibitors propylthiouracil, methimazole and ami-
trole [6, 10] (refer to Table 1 for summary results).This was done to answer the outstanding
question whether there is a direct correlation between low serum T4 and neurobehavioral
effects of DE-71, since only few and sporadic effects have been found in other studies investi-
gating neurodevelopmental effects [17–19]. Furthermore, we wished to clarify if DE-71 can
activate the HPT-axis in a manner consistent with TPO inhibition and whether it is plausible
that DE-71 causes dose-dependent and correlated effects on serum T4 and TH-mediated brain
development.

Materials and methods

Chemicals
The test compound was DE-71 (a technical mixture of penta-brominated diphenyl ethers
(BDE), lot 7550OK20A), a commercial mixture of PBDEs (kind gift from Dr. Kevin Crofton at
the U.S. Environmental Protection Agency). A gas chromatography coupled with high-resolu-
tion mass spectrometry (GC-HRMS) analysis of the congener content in our DE-71 solutions
showed an approximate distribution of congeners in this lot of DE-71 as follows: ~29% tetra-
PBDE (BDE-47), 59% pentaPBDE (of which ~50% BDE-99 and ~9% is BDE-100) and ~9%
hexaPBDE (of which ~5% BDE-153 and ~4% is BDE-154). Corn oil (Sigma-Aldrich, St. Louis,
MO, USA) was used as control compound and vehicle for all treatments.

Animals and treatment

Two animal studies were conducted as depicted in Fig 1, using 40 and 66 time-mated pregnant
Wistar rats (HanTac: WH, Taconic Europe, Ejby, Denmark) that were received on gestation
day (GD) 3 of pregnancy (day of plug-detection designated GD1) and pseudo-randomly
divided into groups. The expected day of delivery, GD23, was designated PD1 irrespective of
actual day of delivery. A detailed description of experimental setup, reproductive toxicity out-
comes, postnatal growth and disruption of the reproductive hormone systems have been
reported previously [25]. In short, we found no evidence for overt toxicity in dams or pups,
but shorter anogenital distance and reduced prostate weight in male offspring, as well as
decreased mammary gland outgrowth in both sexes [25].
Study 1 comprised 4 treatment groups of 10 dams each, exposed to vehicle control (corn
oil), or DE-71 at 20, 40 or 60 mg/kg bodyweight (bw)/day from GD7 to PD14, except the day
of delivery. Study 2 was conducted in two balanced blocks and comprised three treatment
groups of 22 dams each, exposed to vehicle control (corn oil), or DE-71 at 40 or 60 mg/kg bw/
day from GD7 to PD16, except day of delivery. All exposure were administered by oral gavage
at a constant volume of 2 ml/kg bw/day. Dams were housed pairwise in semitransparent plastic
cages (15 × 27 × 43 cm) until GD17 and individually thereafter. Conditions were environmen-
tally controlled and with reverse light/dark cycles (light from 9 pm-9 am). The animals were
provided standard Altromin 1314 diet ad libitum (soy and alfalfa-free, Altromin GmbH, Lage,
Germany). The iodine and selenium contents were 1.52 mg/kg and 0.26 mg/kg, respectively.
Acidified tap water was provided ad libitum in PSU bottles (84-ACBTO702SU, Tecniplast,
Buguggiate, Italy).

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PLOS ONE Thyroid hormone disrupting effects of DE-71

Table 1. Developmental DE-71 exposure studies of the thyroid hormone system and brain development in rats.
Exposure to DE-71 TH/TSH Thyroid gland Developmental neurotoxicity and hearing Reference
Studies including only thyroid hormone system endpoints
0, 0.1, 1, 10 or 30 mg/kg/day to SD dams Dam PD21 tT4 and fT4# up to - - [34] (Bansal
GD6-PND21 (wafer, oral) (n = 6–9) ~50%, tT3$, TSH$ et al., 2014)
Pup PD21 tT4# up to 85%,
fT4# up to 75%, tT3$,
TSH$
0 or 18 mg/kg/day to SD dams GD6-LD18 Dam LD19 tT4#45%, tT3$, Dam thyroid gland weights were obtained - [24] (Ellis-
(intragastric) (n = 3–7) TSH$ but results not stated Hutchings
Pup PND18 tT4#75%, tT3$, et al., 2006)
TSH$
0, 1.7, 10.2 or 30.6 mg/kg/day to LE dams from Male pup: PND4 tT4# up to - - [4] (Szabo et al.,
GD6-PND21 (oral gavage) (n = 7–9) 50%, tT3$ 2009)
PND21 tT4# up to 75%, tT3$
0, 1, 10 or 30 mg/kg/day to LE dams Dam GD20: tT4# 48% in high - - [35] (Zhou
GD6-PND21 (oral gavage) (n = unclear, maybe dose, tT3$ et al., 2002)
38–48 dams per group for both GD20 and Dam PND22: tT4#45%, tT3$
PND22) Fetal GD20: tT4# (more than
15%, control group very close
to LOQ)
Pup PND4: tT4# up to 40%,
tT3$
Pup PND14: tT4# up to 66%,
tT3$
Studies also including developmental neurotoxicity endpoints
0, 0.3, 3.0 or 30 mg/kg/day to SD dams Dam PND21: tT4 and tT3 # Male pup PND21: increased epithelial cell Brain weight PND21, 50, 105, 250: $ [18] (Bowers
GD1-PND 21 (cookie, oral) (n = 20–22 litters, (data not shown) height in high dose except PND21 raw brain weight # in low et al., 2015)
TH: n = 9–10, TSH: n = 20–22, thyroid histo Male pup PND21: tT4# up to dose and relative brain weight " in high
n = 8–10) ~95%, tT3# up to 40% dose PND21 (both sexes),
Female pup PND21: tT4# up Motor activity PND 16, 55, 110 and 230: $,
to ~90%, tT3# up to 40% except small changes in rearing PND110
Pup PND21, TSH" ~30% in Acoustic startle PND20 and 90:
high dose potentiation on PND90 indicating delayed
effects on sensory reactivity
Beam test PND 33, 60: $
Emergency latency PND35, 80: $
Morris Water Maze PND 235: $
Nicotine-induced activity PND450: $
0 or 30 mg/kg/day to Wistar rat pups PND5-22 Male pup PND 23: tT4# ~50% Male pup PND23: thyroid gland weight$ Brain weight PND23$ [19] (De-
(oral gavage) (Open field: n = 17, radial maze: Female pup PND 23: tT4# Female pup PND23: thyroid gland Open field PND42 and 70: $ motor Miranda et al.,
n = 11, T4: n = 10) ~50% weight$ activity both sexes 2016)
RAM PND100: reference memory deficit,
females only
0, 1.7, 20.2 or 30.6 mg/kg/day to LE dams Dams PND22: tT4# up to - FOB including open field PND24, 60 and [17] (Kodavanti
GD6-PND21 (oral gavage) (n > 15 per group, ~40%, tT3$, TSH" ~130% in 273: $, except significant interaction et al., 2010)
n = 8–13 in FOB, n = 7–8 open field, TSH high dose dose� age interaction from PD24-60, but no
n = 5–12) Male pups PD4: tT4# 52% in overall treatment effect on PND60
high dose, TSH$ Motor activity in figure eight, PND 24 and
Male pups PD7: tT4# 60% in 60: $
high dose, TSH$ motor activity (motron) PND~110: $
Male pups PD14: tT4# up to
78%, TSH$
Male pups PD21: tT4# up to
74%, TSH$
Female pups PD4: tT4# 56%
in high dose, TSH$
Female pups PD7: tT4# 50%
in high dose, TSH$
Female pups PD14: tT4# up to
80%, TSH$
Female pups PD21: tT4# up to
76%, TSH$
(Continued )

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PLOS ONE Thyroid hormone disrupting effects of DE-71

Table 1. (Continued)

Exposure to DE-71 TH/TSH Thyroid gland Developmental neurotoxicity and hearing Reference
0, 40, 60 mg/kg/day to Wistar dams Dam GD15 tT4# up to 60%, Dam PD27 thyroid gland weight$ Motor activity PD21 and PD79: $ This study
GD7-PD16 (oral gavage) (n = 19–21) tT3# up to 25%, TSH$ Female pup PD16 thyroid weight$ Morris water maze at 4.5–6 months of age:
Pup PD8-PD27 tT4# ~60% all Male pup PD27 thyroid weight$ $
doses, PD16 tT3# ~25% all Male PD16 histology: no changes in Hearing function at 7.5–8 months: $
doses, PD27 tT3#~15% all follicular epithelium, follicular
doses morphology, stroma or c-cells but
increased minimal vacuolation of follicular
colloid in high dose
For comparison: studies with exposure to TPO-inhibitors in same or similar
studies
0, 0.8, 1.6 or 2.4 mg/kg/day PTU to Wistar Dam GD15: tT4# with up to Pups PND16: thyroid gland weight" Motor activity: PND 14#, PND17$, [10] (Axelstad
dams GD7-PD17 (oral gavage) (n = 18–21) ~60% Pup histology PND16: marked changes PND23", 16 weeks: " et al., 2008)
Pup PND16: tT4# with up to with hyperplasia and hypertrophy of Morris water maze, weeks 8–9: $
~85% epithelial cells, papillary projections into RAM, males 5–6 months: # learning and
lumen and reduced lumen size memory
Hearing, adult offspring: #
0 or 30 mg/kg/day PTU to Wistar rat pups Male pup PND 23: tT4# PND23: thyroid gland weight", both sexes Brain weight, relative PND23: ", both sexes [19] (De-
PND5-22 (oral gavage) (Open field: n = 17, >95% Open field PND42 and 70: "motor activity, Miranda et al.,
radial maze: n = 11, T4: n = 10) Female pup PND 23: tT4# both sexes 2016)
>95% RAM PND100: working and reference
memory learning deficits, both sexes
0, 8 mg/kg/day MMI, 16 mg/kg/day MMI, 25 Dam GD15: tT4# up to ~60%, Dam PD22: thyroid gland weight " Motor activity, offspring PND21:" [6] (Ramhøj
or 50 mg/kg/day amitrole SD dams GD7-PD22 tT3#23% in high dose Male pup PD16: thyroid gland weight" et al., 2022)
(oral gavage) (n = 12). Effects are similar in amitrole, TSH" up to ~800% Female pup PD17: thyroid gland weight"
both MMI and amitrole unless otherwise Dam PD22: tT4# up to ~60% Male pup PD16 histology (n = 4):
stated. in MMI and 90% in amitrole, hyperplasia/hypertrophy, irregular
tT3"30% in low dose MMI, follicles, reduced lumen size and colloid
TSH" up to ~800% depletion
Male pup PD16: tT4# up to
~80–90% in high dose,
tT3#25–35% in high dose,
TSH"330–425% in high dose
Female pup PD17: tT4# up to
~80–90% in high dose,
tT3#25–30% in high dose,
TSH"250–360% in high dose

Abbreviations: f: free, LD: lactation day, LE: Long-Evans, MMI: Methimazole, PND: postnatal day, PD: pup day, PTU: propylthiouracil, RAM: radial arm maze, SD:
Sprague-Dawley, T4: thyroxine, T3: 3,3´,5-tri-iodothyronine, TH: thyroid hormones, t: total, TSH: thyroid stimulating hormone
All doses given in mg/kg bodyweight/day.

https://doi.org/10.1371/journal.pone.0271614.t001

In Study 2, one male and one female pup per litter were weaned (PD27) and housed pair-
wise with an animal of the same treatment group (or with a sibling if none were available).
These offspring were then used for motor activity assessments on PD79, testing in the Morris
water maze for 8 days at 4.5–6 months of age and testing of hearing function on one day at
approx. 7–8 months of age, as described below.
Animal experiments were conducted at the DTU National Food Institute’s facilities (Mørkhøj,
Denmark). Ethical approval was obtained from the Danish Animal Experiments Inspectorate,
with authorization number 2012-15-2934-00089 C4. The experiments were overseen by the
National Food Institute’s in-house Animal Welfare Committee for animal care and use.

Organ weights, tissue and serum sample collection

In both studies all animals, as specified below, were killed by decapitation under CO2/O2 anes-
thesia and trunk blood collected.
Study 1 was terminated on PD14 and livers from the dams excised and weighed. Tongue
blood samples were taken from live dams on GD15. Depending on the number of pups in the

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PLOS ONE Thyroid hormone disrupting effects of DE-71

litters, blood samples were pooled from 2–4 pups on PD3 and PD8. On PD14, plasma was
pooled separately for male and female pups.
In Study 2, remaining pups (except those weaned) and dams were terminated on PD27.
From dams liver and thyroid glands were excised and weighed. Necropsies were performed on
one male and one female pup per litter on PD16 and PD27. Livers were weighed and on PD16
a piece was fixed in 10% formalin and subsequently processed and embedded in paraffin for
histology. Thyroid glands were excised and weighed from PD16 female pups and PD27 male
pups. For PD16 males, the thyroid glands were excised attached to a piece of the thyroid carti-
lage and fixed in 10% formalin, then tissue processed and embedded in paraffin. Tongue blood
samples were taken from dams on GD15. Blood was collected from one male and one female
pup per litter on PD16. On PD27 blood samples were pooled separately for males and females
and included all remaining pups not weaned for later in life tests.
All blood samples were collected in heparinized Eppendorf/vacutainer tubes and stored on
ice until centrifugation for 10 min at 4˚C and 4000 rpm. The plasma was collected and stored
at -80˚C until analysis.

Hormone analysis
Serum total T4 and 3,3´,5-tri-iodothyronine (T3) concentrations were measured at different
times in the two studies, as detailed in Fig 1. Total T4 and T3 concentrations were analyzed by
electrochemiluminescence-immunoassay (ECLIA)–photoncount using a Cobas 8000 E-modul
at the Department of Clinical Biochemistry, Rigshospitalet, Copenhagen, Denmark.
In Study 2 serum TSH were assessed in dam tongue blood on GD15 and in blood from
PD16 pups. TSH concentrations were assayed according to manufacturer’s instructions using
a TSH rat ELISA kit (DEV9977) together with a Rat Control (DEV99RC) both from Demedi-
tec Diagnostics GmbH (Kiel, Germany).

Fig 1. Study design for two developmental toxicity studies with the DE-71 technical mixture of brominated flame retardants. Study 1
assessed early postnatal thyroid hormone disruption and toxicity while Study 2 was used for examination of later postnatal thyroid hormone
system disruption, liver and later in life effects on behavior, learning and hearing function. Bw: body weight, GD: gestation day, PBDE:
polybrominated diphenyl ethers (DE-71), PD: postnatal day. T3: 3,3´,5-tri-iodothyronine, T4: thyroxine. TSH: thyroid stimulating hormone. The
toxicity data and reproductive effects are reported elsewhere [25]. Timeline not drawn to scale.
https://doi.org/10.1371/journal.pone.0271614.g001

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PLOS ONE Thyroid hormone disrupting effects of DE-71

Histological evaluation of thyroid glands and liver

Two sections each from PD16 thyroid glands and livers were stained with hematoxylin and
eosin (H&E) following standard protocols. Thyroid glands of male offspring were assessed for
follicle size and shape (presence of papillary projections), vacuolation of follicular colloid, type
of follicular epithelium (flattened to columnar and position of nucleus), hyperplasia of follicu-
lar epithelium, c-cell hyperplasia, vascularization and fibrosis.
Livers from male and female offspring were assessed for hypertrophy (severity and localiza-
tion) and vacuolation (severity) of hepatocytes. Vacuolation was defined as large vacuoles dis-
placing the nucleus of hepatocytes.
All histological evaluations were performed by a veterinary pathologist and scoring was
done blinded to exposure group.

Motor activity and habituation

Motor activity and habituation was assessed in prepubertal male and female pup offspring on
PD21, and in the weaned adult offspring on PD79. Testing protocols were as previously
described [6]. Briefly, each pup was placed in sound and light insulated activity boxes that rec-
ords horizontal activity via interruptions of adjacent photo beams. Testing took place for 30
min during which the number of photo beam interruptions was registered (via computer in
adjoining room) in 10 periods of 3 min. Total counts during the 30 min represents total motor
activity levels. Habituation was assessed by the average counts during the initial (0–9 min),
middle (10–21 min) and last part of the test (22–30 min). In addition, activity counts were
averaged during the first (0–15 min) and second half of the test (16–30 min).

Learning in the Morris water maze

Learning was assessed in the weaned adult offspring distributed into three new blocks and
with testing performed in the Morris water maze with animals ages PD132-PD176 for a period
of 5+3 days (separated by two days without testing), in a pool with a diameter of 220 cm. The
animals were challenged to learn to find the location of a platform hidden just below the water
surface in the pool surrounded by optical cues. The platform was transparent, circular and
placed on a solid stand 1 cm below the water surface. Every animal was in four trials per day
placed in the pool at four different starting points and allowed to search for the platform. The
trial was completed when the animal climbed onto the platform. If the platform had not been
located within 60s, the animal was led to the platform by hand and allowed to sit on the plat-
form for 20 seconds before being dried and returned to the cage. Endpoints included latency
to locate the platform, path lengths and swimming speeds which where all registered by a cam-
era equipped with a tracking system (Viewpoint video-tracking system, Sandown Scientific,
Middlesex, England).

Auditory function
After the maze testing, a subset of 12 males and females (maximum one of each sex from every
litter) per group were subjected to test of hearing function over the course of 4 days between
PD216-233. Animals were anesthetized with 1.8 ml/kg bw of a mixture of zolazepam 12.9 mg,
tiletamine 12.9 mg, xylazine 1.8 mg, fentanyl 10.3 μg in 0.9% NaCl per 1 ml. Animals roused
after approximately 2 hrs and were killed on the same or the following day. Testing procedures
and equipment were as previously described [10, 26]. Briefly, hearing was assessed by measur-
ing the distortion product otoacoustic emissions (DPOAE). DPOAE was measured as the
amplitude of the cubic distortion product (CDP) from two tones generated by a two-channel

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PLOS ONE Thyroid hormone disrupting effects of DE-71

tone generator with phase control (HP 8904) and the probe microphone output feeding into a
FFT spectrum analyzer (HP 35670A). The first tone (f1) was always 10 db higher than the sec-
ond tone (f2) and there was a fixed ratio between them of f2/f1 = 16713 = 1.23. Distortion
product diagrams (DP-grams) were generated by measuring CDP at fixed levels of primary
tones across frequencies (2 to 70 kHz), based on 64 time-averaged recordings. DPOAE input/
output curves were made at f2 = 4096Hz and 32384 Hz by measuring CDP amplitude at vary-
ing levels of primary tones.

Statistical analysis
Data from continuous endpoints with normal distribution and homogeneity of variance were
tested by ANOVA, with appropriate covariates included in the statistical analysis (e.g. body
weight for organ weights and motor activity). Dunnett’s post-hoc test was applied to account
for multiple testing with the statistical significance level set at 0.05. Litter effects were
accounted for by only analyzing one pup per litter or by including the litter as an independent
random and nested factor in the analysis. Hearing data was analyzed by ANOVA with expo-
sure level and sex as factors, assuming unequal variance between groups.
A 2-sided Fisher’s exact test was used for histology data. First a rxk Fisher’s exact test was
used (including more than 2 groups and more than 2 scores) to detect differences in the distri-
bution of scores in all the groups. To determine if one group differed from controls, a 2xk Fish-
er’s exact test was performed. For dichotomous data, a 2x2 Fisher’s test was used to compare
two groups.
SAS Enterprise guide 4.3 (2010) (SAS Institute Inc, Cary, NC, USA) and GraphPad Prism 5
(Graphpad Software, San Diego, CA; USA) was used for statistical analysis. Statistical analysis
of hearing data was performed in SYSTAT Software Package v. 9.

Results
Dam thyroid hormone system disruption
We assayed dam TH system disruption in the pregnant animals. This was done because the
fetus is entirely dependent on maternal transfer of T4 to the fetal circulation during early
development. On GD15, dam T4 and T3 levels were dose-dependently reduced by DE-71
exposure (<50% and <85% of controls, respectively, for T4 and T3) (Fig 2A and 2B). There
were no statistically significant effects on TSH concentrations, albeit values appeared more
variable in exposed group compared to controls (Fig 2C).

Pup thyroid hormone system disruption

Postnatally, brain development depends on the offspring’s own TH production. All doses of
DE-71 markedly reduced postnatal T4 concentrations to 25–45% of control levels (Fig 3A).
On PD16 and PD27, T3 levels were reduced to ~75–85% of controls by DE-71 exposure (Fig
3B). DE-71 exposure was discontinued on PD16, but there was only slow recovery in T4 and
T3 levels between PD16 and PD27 (Fig 3A and 3B).

Effects on the liver

TH system disruption can arise from different mechanisms, one of which is liver enzyme
induction (microsomal enzyme inducers). We, therefore, looked for effects in dam and pup
livers. Dam liver weights were increased dose-dependently in all exposure groups on PD14 in
Study 1 (~20% in high dose) (Fig 4A). The increase was observed for both absolute liver weight

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PLOS ONE Thyroid hormone disrupting effects of DE-71

Fig 2. Dam serum T4, T3 and TSH concentrations on GD15 after 7 days of exposure to DE-71. (A) Dam serum T4
concentrations as % of control in Study 1 and Study 2. Study 1: n = 8–9 except PBDE-20 with n = 6. Study 2: n = 19–21. Mean
+ SEM. (B) Dam serum T3 as % of control (Study 2). Mean + SEM. n = 19–21. (C) Dam serum TSH concentrations (Study
2). Mean + SEM. Individual data points with mean and whiskers indicating SEM. n = 9–11. �� p<0.01. GD: gestation day,
PBDE: polybrominated diphenyl ethers (DE-71). T3: tri-iodothyronine, T4: thyroxine. TSH: thyroid stimulating hormone.
https://doi.org/10.1371/journal.pone.0271614.g002

analyzed with body weight as covariate and for relative weights (data in S1 File). There was no
effect on body weights. By PD27, in Study 2, liver weights were similar between groups.
Pup liver weights were markedly increased (up to ~50%), particularly on PD16, at the end
of exposure (Fig 4B). By PD27 the increase was less pronounced, demonstrating some recovery
after exposure ceased. Histological evaluation of pup livers revealed increased hypertrophy
and vacuolation of hepatocytes with increasing dose of DE-71 (Fig 4C–4E). Hypertrophy was
seen in all exposed animals, and severity increased with increasing dose (Fig 4C). One control
male displayed marginal signs of hypertrophy. Hypertrophy of hepatocytes was localised to the
centrilobular areas. No microvesicular vacuolation was observed. Vacuolation of hepatocytes
was only seen in exposed pups and the severity of vacuolation increased with dose (Fig 4E).

Fig 3. Pup serum T4 and T3 concentrations during postnatal development. (A) Pup serum T4 (% of control) in Study 1
and 2 covering PD8 to PD27 T4 was statistically significantly decreased at all doses at all points in time. Study 1: n = 6–8,
Study 2: n = 19–21 litters. Mean + SEM. (B) Pup serum T3 concentrations PD16 and PD27 (Study 2). n = 19–21. Mean
+ SEM. �� p<0.01. PBDE: polybrominated diphenyl ethers (DE-71), PD: postnatal day, T3: tri-iodothyronine, T4: thyroxine.
https://doi.org/10.1371/journal.pone.0271614.g003

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PLOS ONE Thyroid hormone disrupting effects of DE-71

Fig 4. Effects of DE-71 exposure from GD7-PD14/16 on dam and pup liver weights and histology. (A) Dam liver weights were increased dose-dependently
in Study 1 on PD14, while there were no effects on PD27 in Study 2. n = 7 in Study 1, n = 19–21 in Study 2. Mean + SEM. (B) Male and female pup liver weights
PD16 and PD27. n = 17–21. Mean + SEM. (C) Severity of hepatocytic hypertrophy in male and female offspring PD16. Centrilobular hypertrophy was observed
in all exposed animals. Number of assessed offspring shown in each bar. n = 36–40. (D) Male PD16 liver from control (top), high-dose presenting with severe
centrilobular hepatocytic hypertrophy (middle) and high dose with moderate hepatocytic vacuolation (bottom). Arrows show examples of large macrovesicular
vacuoles in hepatocytes (E) Severity of hepatocytic vacuolation in male and female offspring PD16. Significantly increased incidence of vacuolation was seen in
exposed offspring. Number of assessed offspring shown in each bar. n = 36–40. � p<0.05, �� p<0.01. Scalebar = 100 μm. PBDE: polybrominated diphenyl ethers
(DE-71), PD: postnatal day.
https://doi.org/10.1371/journal.pone.0271614.g004

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PLOS ONE Thyroid hormone disrupting effects of DE-71

Fig 5. Effects on the hypothalamic-pituitary-thyroid axis in rat offspring exposed to DE-71 during development. (A) TSH concentrations in PD16 male
and female offspring. Individual data points shown with mean and SEM whiskers. n = 7–8. (B) Thyroid gland weights in female offspring PD16, male PD27
and dams PD27. Females: n = 19–21, males: n = 15–17, Dams: n = 19–21. (C) Vacuolation of thyroid gland follicular colloid in male PD16 offspring. Left:
incidences of vacuolation and severity, number of assessed male pups shown inside bars. Upper right: Thyroid gland with no vacuolation of follicular colloid.
Lower right: thyroid gland with minimal vacuolation (arrows) of follicular colloid. � p< 0.05. Scalebar = 50 μm. PBDE: polybrominated diphenyl ethers (DE-
71), PD: postnatal day, TSH: thyroid-stimulating-hormone.
https://doi.org/10.1371/journal.pone.0271614.g005

The hypothalamic-pituitary-thyroid axis

TH system disruption by microsomal enzyme inducers appears to cause two different effect
patterns within the HPT-axis. Some enzyme inducers, such as phenobarbital, reduce serum T4
concentrations with a concurrent activation of the HPT-axis, resulting in increased TSH levels
to stimulate TH production in the thyroid gland [27, 28]. This leads to increased thyroid gland
weight and histological changes. In this study, DE-71 exposure reduced T4 without affecting
TSH levels or thyroid gland weights, either in PD16 and PD27 pups (Fig 5A and 5B) or PD27
dams (Figs 2C & 5B). No changes in the follicular epithelium, follicular morphology, stroma
or c-cells were evident in thyroid glands of PD16 male pups. However, we observed a dose-
dependent increase in vacuolation of follicular colloid with a statistically significant higher
incidence in the high dose-group compared to controls (Fig 5C). The vacuolation was negligi-
ble and no vacuolation of the follicular epithelium was observed in relation to vacuolation of
colloid. This could be signs of increased activation of the thyroid follicles. However, the lack of
effects on epithelium and follicular morphology indicates that the activation was minor, if any.

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PLOS ONE Thyroid hormone disrupting effects of DE-71

Taken together, our results indicate that the DE-71 mixture is a microsomal enzyme inducer
which does not cause an activation of the HPT-axis in rat offspring.

Offspring neurobehavior
Motor activity levels and habituation were tested in prepubertal (PD21) and adult (PD79) off-
spring. We found habituation (decreasing activity levels over the course of the test) at both
ages in males and females and observed the expected sex difference in adulthood, i.e. higher
activity levels in females than in males. There were no significant treatment-related effects of
developmental DE-71 exposure on general activity (Fig 6), nor on habituation in males (Fig 6)
or females (data in S1 File) at any age. Learning was tested in the Morris water maze at approx-
imately 5 months of age. We found improved performance over the course of the test, demon-
strating a functional assay. However, we found no effects of developmental exposure on
distance, latency to reach platform or swimming speed (Fig 6C). This indicates that DE-71
exposure did not affect learning as assessed by this simple behavioral assay.

Offspring hearing function

We have previously shown how developmental hypothyroidism results in increased hearing
thresholds and decreased Cubic Distortion Product (CDP) with loss of activity in the outer
hair cells spread equally over the basilar membrane in the cochlea [10]. We performed the
same tests on the DE-71 exposed animals but found no differences compared to control ani-
mals (Fig 7).

Discussion
One of the main aims of this study was to clarify if there is a dose dependent correlation
between low serum T4 levels caused by developmental exposure to DE-71 and neurobeha-
vioral changes in the rat offspring. Based on our results, and those of previous studies, the
answer appears to be no. This is despite the fact that perinatal exposure to DE-71 markedly
reduces plasma T4 and T3 concentrations throughout development. Notably, however, the
HPT-axis does not seem to be activated by this marked reduction in T4 as, for instance, is the
case with hypothyroidism induced by propylthiouracil (PTU) [10]. In PTU-exposed animals,
TSH levels increase in response to a drop in T4. Regardless, a severe reduction in T4 did not
result in adverse effects on behavior or hearing. It thus appears that thyroid hormone system
disruption by DE-71 does not result in the severe hypothyroidism and associated effects that
can be ascribed to PTU and other potent TPO-inhibiting compounds [6–8, 10].
As discussed in the introduction, several studies have examined the potential effects of DE-
71 on the thyroid hormone system and neuroendocrine development. However, it is challeng-
ing to draw general conclusions since study designs vary considerably. This is also the conclu-
sion reached by a systematic review of developmental neurotoxicity by PBDEs in animal
studies [29]; nevertheless the evidence for neurotoxicity in humans is quite strong [30–33]. In
Table 1, we have summarized the key rat studies of DE-71 and reported effects in order to inte-
grate our new results in a more holistic analysis. From this synthesis it is evident that DE-71
induces very few effects on behavioral endpoints across developmental toxicity studies.
There is also an obvious decoupling between low serum T4 levels and changes to brain
development as assessed in standard neurobehavioral tests. The dose-dependent correlations
seen with TPO inhibitors simply do not occur with DE-71 exposure.
One of the primary target organs of PBDEs is the liver. We found a pronounced increase in
liver weights and histological changes in DE-71 exposed animals, which is consistent with
microsomal enzyme induction reported by others. More specifically, DE-71 can induce

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PLOS ONE Thyroid hormone disrupting effects of DE-71

Fig 6. Neurobehavior in rat pups exposed to PBDEs during development. (A) Total motor activity (total counts in
30 min) levels in PD21 male and female offspring and habituation (average activity counts per 3 min period) in male
offspring. Control and PBDE-40: n = 18–19, PBDE-60: n = 14–15. (B) Total motor activity levels in PD79 adult male
and female offspring and habituation in male offspring. Control and PBDE-40: n = 17–20, PBDE-60: n = 15. (C) Mean
distance travelled and time spent to reach hidden platform in the Morris water maze (8 days of testing at 4.5–6 months
of age). n = 18–20. Mean + SEM. PBDE: polybrominated diphenyl ethers (DE-71), PD: postnatal day.
https://doi.org/10.1371/journal.pone.0271614.g006

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PLOS ONE Thyroid hormone disrupting effects of DE-71

Fig 7. Hearing function in adult 7.5–8 months old male and female offspring exposed to PBDEs during
development. (A) Distortion product diagram of cubic distortion product at all tested frequencies (2 to 70 kHz). Data
represent group means, black curve represents background noise ± 95% confidence interval. (B) Average cubic
distortion product for f2 at 4 and 32 kHz. PBDE: polybrominated diphenyl ethers (DE-71). n = 11–12.
https://doi.org/10.1371/journal.pone.0271614.g007

enzymes related to TH metabolism and excretion [4, 35–37]. This suggests that increased
hepatic clearance of THs contributes to the reduction in serum T4. Notably, PBDEs also pos-
sess the ability to bind and displace T4 from the serum thyroid hormone transport protein

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PLOS ONE Thyroid hormone disrupting effects of DE-71

TTR, with hydroxylated metabolites of PBDEs having particularly high affinity [38–40]. In
vivo these two mechanisms may act together to increase excretion of thyroid hormones and
produce the phenotype of low serum T4 concentrations. How this relates to the lack of HPT-
axis activation, however, remains unexplained.
DE-71 exposure severely reduced serum T4 concentration throughout postnatal develop-
ment. Comparing this to other studies with developmental exposure to DE-71 (Table 1) shows
that there is concordance between studies and strong evidence to conclude that DE-71 consis-
tently reduces serum T4 in perinatally exposed pups. It also correlates with T4 effects in similar
developmental toxicity studies with TPO inhibitors. With regard to T3, however, few studies
have found, or even measured, any effect on serum concentrations in pups exposed to DE-71.
Three studies with small group sizes report no effect on T3 levels [4, 24, 35], while one report
reduced serum T3 in both male and female pups on PD21 [18] (Table 1). We also found
reduced serum T3 levels in PD16 pups measured across relatively large group sizes of 19–21.
This strengthens the evidence for DE-71 causing a reduction in pup T3 concentrations, yet the
available data is inconsistent.
The thyroid gland is the site of TH synthesis and the target of TSH and thus natural to
assess for potential thyroid hormone system disruption and HPT-axis activation. That is, thy-
roid gland weight and histology should be scrutinized even though DE-71 does not appear to
increase TSH levels in the majority of studies. With regard to TSH, our study, together with
two others, observed no effect on pup serum TSH [17, 24] while a fourth study reported a 30%
increase [18]. This latter study also found follicular epithelium cell height to be increased in
PD21 pups, but did not investigate other adverse effects in the thyroid glands [18] (Table 1). In
fact, the only other assessment of thyroid glands is a study that found no effects on pup thyroid
gland weight after DE-71 exposure [19] (Table 1). Thus, assessments of thyroid gland weight
and histology are scarce and our study is the first to report on a full histological assessment of
DE-71-exposed pup thyroid glands. This assessment revealed no marked effects, which is in
contrast to what is reported in PD16 pups developmentally exposed to PTU, MMI or amitrole
[6, 10, 41, 42]; however, we did find minimal effects on colloid vesicles. Taken together, expo-
sure to DE-71 does not appear to activate the HPT-axis in perinatally exposed rat pups, and
only induces marginal, if any, changes to thyroid gland morphology.
Growing evidence suggests that rodent models for hypothyroxinemia-like effect patterns
are not sufficiently sensitive to determine if a TH system disrupting chemical possess develop-
mental neurotoxicity potential [1]. That is, the effects on the brain, if present, are not pro-
nounced enough to be detected by standard neurotoxicity assays, but the TH system
disrupting potential is great enough to be of concern to human health. The fact is that our cur-
rent experimental conditions limit our ability to measure and determine if there is an effect or
not due to endpoint sensitivity. Firstly, dose-response studies of effects of TH deficiency
induced by TPO inhibitors show that serum and brain THs need to be severely reduced in foe-
tuses and postnatal pups before statistically significant effects on brain development are
observed [1, 6, 9, 10, 12, 42, 43]. Thus, current endpoints for neurotoxicity are not sensitive
enough to detect developmental TH system disruption. Secondly, there likely is a discrepancy
between serum and brain TH concentrations in rodents subject to chemically induced
hypothyroxinemia-like conditions. Here, brain hormones can be less affected despite pro-
nounced effects on serum hormones [14], opposite to the relationships seen with PTU expo-
sure [9, 44]. Whether this also applies to DE-71 exposed animals should be investigated in
future studies. Regardless, the simple neurobehavioral tests used herein, and in other studies,
cannot identify adverse neurological effects of DE-71 induced TH-system disruption in rat
offspring.

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PLOS ONE Thyroid hormone disrupting effects of DE-71

While our neurobehavioral assays, and potentially also the rodent brain, may be relatively
insensitive to TH deficiency, there is no question that the situation is very different for the
human brain. In humans, severe lack of TH during development has profound consequences
for brain development. All the while there is also a substantial body of literature showing that
even slightly reduced serum T4 with no effects on TSH, so-called hypothyroxinemia, is enough
to impair child brain development. This is evident from epidemiological studies, that find
decreased IQ, altered white-to-grey matter ratio, altered motor and language development,
and increased risk of developing neurobehavioral disorders such as autism, ADHD and
schizophrenia [45–51]. Thus, while it is challenging to test rodent brain function for effects of
TH system disruption, the evidence from humans is that hypothyroxinemia does negatively
impact human brain development. Accordingly, the widespread exposure of humans to TH
system disrupting chemicals is of great concern if we are to protect human cognition and
health for future generations.

Concluding remarks
Perinatal exposure to DE-71 reduces serum T4 markedly in postnatal rat pups, but without a
concomitant increase in serum TSH, i.e. a hypothyroxinemia-like response. Based on data
herein and from historical studies, there is no clear correlation between significantly lower
serum T4 levels and adverse effects as assessed by standard neurobehavioral tests in rats. This
contrasts with the correlations seen for TH system disruption by TPO inhibitors, where low
serum T4 correlates with TH-mediated effects on the brain. To address this conundrum,
future studies should aim to measure TH levels also in target tissues, such as the brain, and
include endpoints that are specific to TH system disruption. Only then can we answer the out-
standing question if the absence of adverse neurobehavioral effects in DE-71 exposed pups are
due to decoupling of systemic TH concentrations and local TH concentrations in the brain.
Importantly, however, it should be noted that a reduction in serum THs is in itself of great
concern to human health. From what we know, the human brain is most likely very sensitive
to any alteration in thyroid status of pregnant women and their developing foetuses. In
humans TH system disruption of any kind–hypothyroidism, hypothyroxinemia, or otherwise–
is cause for concern as regards brain development.

Supporting information
S1 File. Datasets.
(XLSX)

Acknowledgments
We would like to thank Tommy Licht Cederberg (National Food Institute, Technical Univer-
sity of Denmark) for the DE-71 congener analysis.
We thank Mette Voigt Jessen, Heidi Broksø Letting, Lillian Sztuk, Dorte Lykkegaard Kors-
bech, Birgitte Møller Plesning, Ulla El-Baroudy, Sarah Grundt Simonsen, Lene Ravn, Eva Ter-
rida and Lasse Laub-Ekgreen for excellent technical assistance. We also thank Anne Ørngreen
and staff for animal care and husbandry.

Author Contributions
Conceptualization: Louise Ramhøj, Ulla Hass, Marta Axelstad.

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PLOS ONE Thyroid hormone disrupting effects of DE-71

Formal analysis: Louise Ramhøj, Karen Mandrup, Søren Peter Lund, Karin Sørig Hougaard,
Marta Axelstad.
Funding acquisition: Ulla Hass, Marta Axelstad.
Investigation: Louise Ramhøj, Karen Mandrup, Søren Peter Lund, Marta Axelstad.
Visualization: Louise Ramhøj, Karen Mandrup.
Writing – original draft: Louise Ramhøj, Terje Svingen, Marta Axelstad.
Writing – review & editing: Louise Ramhøj, Terje Svingen, Karen Mandrup, Ulla Hass, Søren
Peter Lund, Anne Marie Vinggaard, Karin Sørig Hougaard, Marta Axelstad.

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