Developmental Exposure to DecabrominatedDiphenyl Ether (BDE-209): Effects on SpermOxidative Stress and Chromatin DNA Damage inMouse Offspring

Li-Ho Tseng,1 Ping-Chi Hsu,2 Chia-Wei Lee,2 Shinn-Shyong Tsai,3 Min-Hsiung Pan,4

Mei-Hui Li5

1Department of Occupational Safety and Hygiene, Tajen University, Pingtung, Taiwan

2Department of Safety, Health, and Environmental Engineering, National Kaohsiung FirstUniversity of Science and Technology, Kaohsiung, Taiwan

3Department of Veterinary Medicine, National Pingtung University of Science and Technology,Pingtung, Taiwan

4Department of Seafood Science, National Kaohsiung Marine University, Kaohsiung, Taiwan

5Environmental Toxicology Lab, Department of Geography, National Taiwan University, Taipei,Taiwan

Received 15 December 2010; revised 27 March 2011; accepted 30 March 2011

ABSTRACT: Polybrominated diphenyl ethers (PBDEs) are used as brominated flame retardants and havebeen found in human milk in recent years. This study investigates whether prenatal exposure to decabro-minated diphenyl ether (BDE-209) induces sperm dysfunction in male offspring. Pregnant CD-1 mice weregavaged once daily with corn oil (control), 10, 500, and 1500 mg kg21 body weight of BDE-209 from day 0of gestation to day 17. The outcomes of male reproductive parameters were assessed on postnatal day71. Anogenital distance, sperm-head abnormalities, and testicular histopathology were significantlyaffected in male offspring prenatally exposed to 1500 mg kg21. Significant increases in the tendency forsperm DNA denaturation (aT) induction and the DNA fragmentation index (DFI) were found in thoseexposed to 10, 500, and 1500 mg kg21 (P\ 0.05). We observed a significant increase of sperm hydrogenperoxide (H2O2) generation in the 10 and 1500 mg/kg/day groups compared to the control group (P \0.05). Although our findings suggested that the mechanisms underlying BDE-209-induced sperm DNAdamage and H2O2 generation might not be represented as a dose-response relationship, we found thatthe greater the excess production of sperm H2O2, the greater the sperm aT (r 5 0.65, P 5 0.0155) and DFI(r5 0.53, P 5 0.002). In conclusion, developmental exposure to BDE-209 induced sperm-head abnormal-ity, oxidative stress, chromatin DNA damage, and testicular histopathological changes. These findingssuggest that BDE-209-induced male reproductive effects might involve the formation of sperm H2O2

which attacks nucleic acids via H2O2 generation. # 2011 Wiley Periodicals, Inc. Environ Toxicol 28: 380–389, 2013.

Correspondence to: Dr. Ping-Chi Hsu; e-mail: pchsu@nkfust.edu.tw

Published online 27 May 2011 in Wiley Online Library

(wileyonlinelibrary.com). DOI 10.1002/tox.20729

Contract grant sponsor: National Science Council of Taiwan

Contract grant number: NSC 96-2314-B-327-001 MY3, NSC 97-2221-

E-327-009-MY3

�C 2011 Wiley Periodicals, Inc.

380

Keywords: polybrominated diphenyl ethers (PBDEs); decabrominated diphenyl ether (BDE-209);prenatal exposure; oxidative stress; sperm DNA damage

INTRODUCTION

Brominated flame retardants (BFRs) have been widely used

in many products for fire prevention purposes since the late

1970s (WHO, 1994). The most commonly used BFRs

are the polybrominated diphenyl ethers (PBDE), hexabro-

mocyclododecane (HBCD), and tetrabromobisphenol A

(TBBPA). Decabrominated diphenyl ether (BDE-209), the

second most used BFR after TBBPA, is used in the produc-

tion of electronic enclosures and upholstery textiles and

foams (Hardy, 2002). During the manufacturing process,

use, or disposal of such products, PBDEs may leach into

the environment (Sjodin et al., 2001).

These persistent and bioaccumulative compounds

(Fangstrom et al., 2005; Streets et al., 2006) have been

found not only in the environment (Ikonomou et al.,

2002; Kelly et al., 2008) but also in the blood of preg-

nant women (Bradman et al., 2007) and in breast milk

(Kalantzi et al., 2009). Elevated concentrations of BDE-

209 have also been found in waterbird egg samples (Lam

et al., 2007), wild harbor seals (Shaw et al., 2008), and

plasma from polar bears and glaucous gulls (Verreault

et al., 2005).

The effects on human health of increased of PBDE

levels in the environment on the health of humans is a

cause for concern (Zoeller, 2005; Main et al., 2007).

Although the EU banned the manufacture and use of

penta-BDE and octa-BDE (EU, 2003) and both products

were subsequently withdrawn from the US market (Betts,

2008), the environmental and occupational pollution of

PBDEs remain a major concern. There is, therefore, a

continuing need to study their effects. Workers disman-

tling electronics (Muenhor et al., 2010), computer techni-

cians (Jakobsson et al., 2002), and laborers involved in

the manufacturing or handling of rubber (Thuresson

et al., 2005) have been found to be exposed to BDE-209.

Recently, a survey in the general population indicated

that certain higher brominated PBDEs appear to affect

the menstruation characteristics of reproductive-age

females (Chao et al., 2010). However, the toxicity of

BDE-209 is not clearly understood.

A study from Indiana found a close association between

maternal and fetal cord blood PBDE concentrations

(Mazdai et al., 2003). Another study, from the EU, found a

similar relative distribution of PBDEs, especially highly

brominated PBDEs, between maternal and fetal serum

(Antignac et al., 2009). These studies showed that PBDEs

may be able to cross the placental barrier and enter fetal cir-

culation. PBDEs are potential endocrine disruptors and

may have an effect on the reproductive system. In a study

of the reproductive effects of PBDEs on mice, Kuriyama

et al. (2005) reported that prenatal exposure to a single low

dose of PBDE 99 on gestation day (GD) 6 resulted in sig-

nificantly decreased sperm counts in offspring. Lilienthal

et al. (2006) have also reported that prenatal exposure to

PBDE 99 resulted in reduced testosterone and anogenital

distance (AGD) in adult male rat offspring. To the best of

our knowledge, however, very few studies have investi-

gated the effects of BDE-209 exposure during pregnancy

on the relationship between sperm oxidative stress and

DNA damage in adult offspring.

In this study, we examined the effects of prenatal expo-

sure to BDE-209 on sperm and testis dysfunction in the off-

spring of mice. To do this, we:

1. evaluated infant development, including body and repro-

ductive organ weights, AGD, anogenital index (AGI,

weight-adjusted AGD), and testis index (TI),

2. characterized sperm function—including sperm count,

motility, motion, morphology, sperm chromatin struc-

ture analysis (SCSA), and sperm reactive oxygen species

(ROS) generation,

3. assessed testicular functions, including testosterone hor-

mone levels and histopathology.

The results of this study may provide additional useful

information on the extent of BDE-209-related adverse

effects on the environmental toxicology and help make pos-

sible the assessment of health risk.

MATERIALS AND METHODS

Overview of Study Design

Adult male (12-weeks old) and female (10-weeks old) CD-

1 mice were provided by the Animal Center of National

Cheng Kung University Medical Center (Tainan, Taiwan).

The animal chamber was supplied with UV sterilized air

and maintained at 248C 6 28C with a constant humidity of

60% 6 10%. The mice were maintained on a 12-h light/

dark cycle. Food (Laboratory Rodent Diet 5001, LabDiet,

Richmond, IN) and distilled water were provided ad libi-tum. After 2 weeks of acclimation, the breeding process

was conducted by placing one female mouse into a male

mouse’s cage. The day of vaginal plug was defined as GD

0. Twenty pregnant females were randomly divided into

four groups, each containing with five mice. Each mouse

was housed individually. Animals from each group were

gavaged once daily with corn oil (control), 10, 500, and

381DEVELOPMENTAL EXPOSURE TO DECABROMINATED DIPHENYL ETHER (BDE-209)

Environmental Toxicology DOI 10.1002/tox

1500 mg kg21 of body weight of BDE-209 from GD 0 to

GD 17. Dosages were adjusted daily on the basis of

changes in body-weight. On postnatal day (PND) 71, three

male offspring from each litter were randomly selected, to

make a total of 15 from each group. Their AGD and AGI

were then measured. AGD was defined as the distance from

the center of the anus to the anterior base of the penis. AGI

was estimated after adjusting for body weight. The animals

were euthanized by CO2. The testis, epididymis, and semi-

nal vesicles were removed and weighed. Blood samples

were taken by heart puncture for testosterone analysis. The

left cauda epididymis was used to perform SCSA. The right

cauda epididymis was prepared for sperm suspension in

order to measure sperm count, motility, morphology, and

ROS generation.

Chemicals

BDE-209 (98%, CAS no. 1163-19-5) was obtained from

Sigma-Aldrich (St. Louis, MO). The BDE-209 solution

was prepared by mixing the BDE-209 compound with

corn oil and sonicating the mixture at room temperature

for 30 min. The desired doses (10, 500, or 1500 mg

kg21) were prepared weekly. The suspensions were vor-

texed during dosing for at least 30 min. Hydroethidine

(HE) and 20,70-dichlorofluorescin diacetate (DCFH-DA)

were purchased from Molecular Probes (Eugene, OR).

Corn oil, phosphate-buffered saline (PBS), Ham’s F12

medium, eosin Y, propidium iodide (PI) stain, and all

other reagents were obtained from Sigma-Aldrich (St.

Louis, MO).

Sperm Count, Motility, Motion Analysis

The right cauda epididymis was removed and placed in a

medium composed of 1 mL Ham’s F12 and maintained

at 348C in an environment saturated with 5% CO2. After

5 min, cauda epididymis was minced with curved scissors

and the sperm was dispersed. After 5 min, the sperm was

collected and transferred to a fresh tube. Sperm suspen-

sion (10 lL) was placed on a prewarmed Makler cham-

ber (10 lm depth; Sefi-Medical Instruments, Haifa,

Israel). Spermatozoa were counted and the ratio between

the number of motile spermatozoa and total number was

used to express sperm motility. Sperm motion parameters

were analyzed from videotaped images using computer-

assisted sperm analysis (CASA) obtained for velocity

indices with a Hamilton Thorn Research motility analyzer

(version HTMIVOS Specification, Beverly, MA). Motion

parameters, including curvilinear velocity (VCL, lm

s21), angular progressive velocity (VAP, lm s21),

straight-line velocity (VSL, lm s21), lateral head ampli-

tude (ALH, lm), and beat-cross frequency (BCF, Hz)

were also measured.

Sperm Morphology Analysis

Morphological abnormalities were classified based on a

modification of a method reported in Wyrobek and Bruce

(1975). Briefly, the sperm suspension was diluted with

phosphate buffered saline (PBS) and then mixed with 1%

aqueous eosin Y (10:1), smeared onto glass slides, and air-

dried. Slides were briefly rinsed in methanol to remove

excess stain. They were air dried again and cover-slipped

with mounting medium. Two samples were made for each

mouse. At least 600 cells from each mouse were examined

for morphological abnormalities under a light microscope

(4003) (Zeiss, Axioskop2, Germany). Head was defined as

abnormal when it lacked the usual hook, or had a banana-

shaped, triangular, or giant head.

Flow Cytometry (FCM) Analysis for SpermChromatin DNA Damage

The SCSA described by Evenson et al. (2002) is used to

detect the susceptibility of sperm to in situ acid denatura-

tion of DNA. The sample was analyzed using FCM (BD

Immunocytometry Systems, San Jose, CA). A total of

10 000 spermatozoa were analyzed. AO is a metachromatic

fluorochrome used to distinguish stain double-stranded

from single-stranded nucleic acids. AO intercalates into

double-stranded DNA and has green fluorescence, and it

binds to single-stranded nucleic acids and had has red fluo-

rescence. In SCSA analysis, the metachromatic shift from

green to red fluorescence was expressed as aT, a ratio

between red and total fluorescence [red/(red 1 green)].

Each sample was calculated and results were expressed as

the mean of the aT distribution (X aT) reflecting the level

of sperm with DNA damage. Spermatozoa with abnormal

chromatin structure or DNA damage were represented by

DNA fragmentation index (DFI), formally called COMP aT(the percentage of cells outside the main population of aT).

Sperm ROS Assay

Modifying a method reported by Fisher et al. (2005), we

measured sperm hydrogen peroxide (H2O2) levels. Briefly,

sperm cells (1 3 106 cells mL21) were added into 2.5 mMDCFH-DA to a final concentration of 12.5 lM. DCFH-DA

is a stable compound that passively diffuses into cells and

is hydrolyzed by intracellular esterase to yield DCHF,

which is trapped inside cells. H2O2 produced by cells oxi-

dizes DCHF to the highly fluorescent compound, 20,70-dichlorofluorescein (DCF), which is fluorescent at 530 nm.

Sperm superoxide anion (O22l) levels were measured using

a modification of a previously described method (Marchetti

et al., 2002). Sperm cells at a concentration of 1 3 106 cells

mL21 were added into 0.33 mM HE to a final concentration

of 2 lM. The mixture was set aside and maintained at 348Cfor 30 min. Sperm H2O2 and O22l levels were measured

382 TSENG ET AL.

Environmental Toxicology DOI 10.1002/tox

using a FCM with excitation and emission set at 488 and

530 nm, respectively.

Assessment of TI and Serum TestosteroneAssay

TI was calculated for each mouse (testicular length 3 tes-

ticular width/body weight). Testosterone was measured

using Coat-a-Count radioimmunoassay kits obtained from

Diagnostic Products Corporation (Los Angeles, CA) and

performed following manufacturer’s directions. The assays

were performed in the Department of Nuclear Medicine at

National Cheng Kung University Medical Center (Tainan,

Taiwan).

Histopathology of Testis

The left testis was fixed in Bouin’s solution for at least

48 h. To prepare them for histological studies, the testes

were embedded in paraffin and sections (3 to 4 lm) were

stained with hematoxylin and eosin. The damage to semi-

niferous tubules was evaluated in cross sections. Sections

were observed under 4003 magnification with an Olympus

microscope (Milton Keynes, Buckinghamshire, UK) con-

nected to a computer on which the digital images were

saved. Each germ cell stage in the seminiferous epithelium

was categorized according to a classification system used

by Oakberg (1956).

Statistics

Results were expressed as mean 6 standard error of the

mean (SEM). For the analysis of male offspring, the litter

was the statistical unit. The body weights, reproductive

organ weights, AGD, TI, sperm count, motility, velocity,

morphology, SCSA, generation of H2O2 and O22l of the

groups exposed to BDE-209 and the control group were

compared using one-way analysis of variance (ANOVA),

followed by the Tukey-Kramer honestly significant differ-

ence (HSD). The serum testosterone data were not normally

distributed, so log transformed testosterone levels were per-

formed. A P-value of\0.05 was considered significant. All

statistical operations were performed using the JMP 5.0 sta-

tistical package (SAS Institute, Gary, NC).

RESULTS

Body and Reproductive Organ Weights ofMale Offspring

We found no significant differences between any of the

exposed groups or between those groups and the controls

with regard to body weight, absolute or relative weights of

testis, epididymis, cauda epididymis, and seminal vesicles

at PND 71 (data not shown).

Sperm Count, Motility, Motion, andMorphology Analysis

As can be seen in Table I, a summary of average sperm

count, motility, motion, and morphology for each of the

treatment groups, there was a significantly greater increase

in mean percentages of sperm morphological abnormalities

in the 1500 mg kg21-treated offspring than in the controls

(P\ 0.05), though there were no significant differences in

sperm count and motility among the treatment groups.

Based on our CASA motion analysis of cauda epididymal

TABLE I. Sperm count, motility, morphology, and velocity of CD-1 male mice prenatally exposed to decabrominateddiphenyl ether (BDE-209)

Parameters

Treatment of BDE-209 (mg kg21)

P valueControl 10 500 1500

Sperm count (106/mL) 15.5 6 1.9 13.1 6 1.1 13.9 6 0.7 16.2 6 1.2 0.3304

Sperm motility (%) 65.6 6 1.5 64.3 6 4.6 66.1 6 4.4 67.1 6 1.3 0.8826

Abnormal sperms head (%)a 10.3 6 2.5 14.9 6 1.4 15.5 6 0.4 18.2 6 1.3* 0.0207

Velocity of motionVCL (lm s21)b 145.8 6 7.6 147.9 6 7.3 149.3 6 7.7 148.5 6 9.0 0.9908

VAP (lm s21)c 80.3 6 4.9 82.6 6 3.0 79.6 6 3.3 80.9 6 4.8 0.9597

VSL (lm s21)d 59.8 6 5.1 63.7 6 3.0 59.4 6 3.0 62.3 6 4.1 0.8453

ALH (lm)e 6.46 6 0.2 6.7 6 0.4 6.8 6 0.5 7.0 6 0.9 0.8285

All data was expressed in means 6 SEM.

*P\0.05 as compared with control group.a Based on scoring 600 sperm per animal (mean 6 SEM).b VCL: curvilinear velocity.c VAP: Average path velocity.d VSL: straight line velocity.e ALH: amplitude of lateral head displacement.

383DEVELOPMENTAL EXPOSURE TO DECABROMINATED DIPHENYL ETHER (BDE-209)

Environmental Toxicology DOI 10.1002/tox

sperm from male offspring, there were no significant differ-

ences in sperm motion parameters, including VCL, VAP,

VSL, ALH, and BCF.

SCSA and ROS Generation

The sperm function of male offspring for each treatment

group is shown in Table II. Measurement of sperm chroma-

tin structure after in situ denaturation by flow cytometric

methods represents a relatively stable and sensitive indica-

tor of conventional sperm quality characteristics (concen-

tration, motility, and morphology). Epididymal sperm was

analyzed by FCM SCSA to investigate whether there was

any induced change in sperm chromatin DNA integrity af-

ter in utero exposure to BDE-209. We found a significant

increase in the two indicators of sperm chromatin DNA

damage, X aT and DFI, in the 10, 500, and 1500 mg kg21

day21 groups compared to the control group (P \ 0.05)

(Table II). To investigate sperm oxidation stress, we next

studied sperm H2O2 and O22l generation. We observed a

significant increase of sperm H2O2 generation in the 10 and

1500 mg kg21 day21 groups compared to the control group

(P \ 0.05) (Table II). However, there were no dose-

dependent effects in sperm DNA damage and H2O2

generation among BDE-209-treated groups and controls.

However, the percentage of sperm with excessive H2O2

generation was found to be significantly correlated with

DFI (r 5 0.53; P 5 0.002) [Fig. 1(A)] and X aT (r 5 0.65,

P 5 0.0155) [Fig. 1(B)].

AGD, AGI, and TI Measurement and SerumTestosterone Hormone Levels

The mean AGD and AGI measured in male offspring on

PND 71 were significantly reduced in male mice in utero-exposed to the highest dose (1500 mg kg21) compared to

the controls [Fig. 2(A)]. We found no significant group dif-

ferences in TI [Fig. 2(B)]. Although there was a marked

reduction for all BDE-209 doses of testosterone levels,

according to the statistical analysis, they did not result as

significant changes as compared to the control group

(Table II).

Histopathology of Testis

On PND 72, control offspring mice were found to have nor-

mal testicular morphology and spermatogenesis [Fig. 3(A)].

In the BDE-209-treated offspring, however, pathological

lesions, mainly in the interstitial cells and/or seminiferous

tubules were found. The cytoyplasm of the interstitial cells

of male offspring treated with 10 mg kg21 BDE-209

showed moderate vacuolization [Fig. 3(B)]. Those treated

with 500 mg kg21 group had similar but less severe

changes of vacuolization [Fig. 3(C)]. Those treated with

1500 mg kg21 BDE-209 had many interstitial cells and

seminiferous tubules with severe vacuolization and had

almost lost all spermatozoa and spermatids. The vacuoliza-

tion in the tubules was probably Sertoli cells suggesting

direct toxicity and apoptosis of sperm, whereas vacuoliza-

tion in Leydig cells indicated an accumulation of hormone

precursors, though basement membrane remained intact

and a few spermatogonia remained [Fig. 3(D)]. Histopatho-

logic findings are summarized in Table III.

DISCUSSION

Studies have documented the transfer of PBDEs through

the placenta to the fetus (Mazdai et al., 2003; Schecter

et al., 2007). In this study, we hypothesized that maternal

exposure to BDE-209 might have adverse effects on the

reproductive system of male offspring. The adverse effects

are summarized in Table IV.

In this study, we observed that the highest BDE-209

dose (1500 mg kg21 day21) significantly decreased the

AGD and AGI of male offspring. The reduction of these

TABLE II. Sperm DNA damage, reactive oxygen species (ROS) generation, and serum testosterone levels frommalemice in prenatal exposed to decabrominated diphenyl ether (BDE-209)

Parameters

Treatment of BDE-209 (mg kg21)

P valueControl 10 500 1500

X aTa 199.2 6 1.2 217.4 6 1.5* 208.2 6 3.3* 215.0 6 2.3* 0.0001

DFI (%)b 3.3 6 0.3 8.9 6 1.0* 7.5 6 1.8* 8.9 6 0.9* 0.0081

Sperm H2O2 Generationc 38.1 6 3.5 57.7 6 3.4* 43.9 6 2.2 57.8 6 4.5* 0.0017

Sperm O22� Generationc 248.4 610.6 296.2 6 14.9 305.9 6 13.9 290.3 6 21.6 0.0899

Log serum testosterone levels (ng mL21) 0.95 6 0.19 0.35 6 0.10 0.46 6 0.24 0.32 6 0.18 0.1007

All data was expressed in means 6 SEM.

*P\0.05 as compared with control group.a X aT is the level of sperm with DNA damage.b DNA fragmentation index (DFI) is the % of sperm with chromatin DNA damage.c Fluorescence intensity.

384 TSENG ET AL.

Environmental Toxicology DOI 10.1002/tox

two values indicates antiandrogenic effects during develop-

ment (Rhees et al., 1997). Similar results have been

reported for BDE-99 and BDE-71. Prenatal exposure to

BDE-99 from GD 10 to GD 18 is reported to result in a

reduction of AGD and testosterone levels in adult male off-

spring (Lilienthal et al., 2006). Developmental exposure to

BDE-71 is reported to affect AGD in early postnatal male

offspring (Kodavanti et al., 2010). Furthermore, low testos-

terone levels have been associated with increased sperm

chromatin alterations (Krishnamurthy et al., 2000). This

study found that serum testosterone concentration seemed

to decrease (21% for 10 mg kg21 and 23% for 1500 mg

kg21 group) in offspring, although the difference was not

statistically significant. The results could be due to the

small number of animals tested at each dosage. These find-

ings suggest that BDE-209 might potentially affect the

homeostasis of sex hormones. Further studies of much

larger samples are needed to explore this possibility.

The high-dosage group in our study had a significant

increase of sperm head abnormalities, which suggests

genetic damage (Wyrobek et al., 1975; Bucci and Meis-

trich, 1987). Our SCSA results showed a significant

increase in DFI and the level of sperm with DNA damage

(X aT) not only in the group given a high dosage of PBDE

group but also in the groups receiving PBDEs in low and

medium-doses. This suggests that SCSA may be a more

sensitive indicator of DNA damage than the observation of

sperm morphology. We also found a significant induction

in the generation of sperm H2O2 in the 10 and 1500 mg

kg21 day21 treatment groups. Although it appears that this

is not a dosage-dependent effect, this finding is consistent

with our previously study of mice prenatally exposed to 10,

500, or 1500 mg kg21 of BDE-209, which showed that se-

rum total triiodothyronine was significantly decreased in

offspring in the 10 and 1500 mg kg21 treatment groups but

not in 500 mg kg21 treated groups (Tseng et al., 2008).

Our findings suggested that the mechanisms underlying

Fig. 1. Relationships between reactive oxygen species(ROS) and sperm chromatin structure assay (SCSA) insperm assay. Increase in H2O2 in relation to DNA fragmenta-tion index (DFI). DFI (%)520.561 0.16 (sperm H2O2 gener-ation) (r 5 0.53, P 5 0.002) (A). Raise H2O2 in relation to XaT. X aT 5 185.85 1 0.49 (sperm H2O2 generation) (r 5 0.65,P5 0.0155) (B).

Fig. 2. Anogenital distance (AGD) and anogenital index(AGI, weight-adjusted AGD) (A), and testis index (TI) (B)in male offspring following prenatal exposure to 10, 500,1500 mg kg21 of BDE-209 or corn oil on postnatal day(PND) 71. Error bars represent the SEM, P\ 0.05 as com-pared with control group.

385DEVELOPMENTAL EXPOSURE TO DECABROMINATED DIPHENYL ETHER (BDE-209)

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BDE-209-induced oxidative stress may not be represented

as a linear response. Shiner et al. (2006) found that cellular

oxidative stress had a biphasic U-shaped effect on the mac-

rophage antioxidant emzymatic activity, possibly because

toxins might lead to opposite effects when used at low and

high dosages, as has been found in the effects of other envi-

ronmental fluctuations (Arumugam et al., 2006; Ji et al.,

2006). Another study has found tissue and whole-body

elimination of PBDE 47 to be biphasic (Staskal et al.,

2005). Because it is not clear whether BDE-209 has bipha-

sic effects, however, further investigation is required.

An epidemiological study by Akutsu et al. (2008)

reported strong inverse correlations between the serum

2,20,4,40,5,50-hexabromodiphenyl ether levels and sperm

concentrations and testes sizes in Japanese. That study did

not provide clear the pathological evidence of the relation-

ship, however. We found the pathological lesions in the in-

terstitial cells and/or seminiferous tubules in BDE-209-

treated offspring. We also found vacuolization levels had a

tendency to be U-shaped in testicular interstitial cells in our

histopathological assays. Only the 1500 mg kg21 BDE-

209-treated group showed severe vacuolization of seminif-

erous tubules associated with almost complete loss of sper-

matozoa and spermatids. We did not, however, find signifi-

cant changes in epididymal sperm count in the 1500 mg

kg21 BDE-209-treated group. Additional large numbers of

animals tested at each dosage are needed to confirm the

results of the present study.

Fig. 3. Tissue sections of offspring testis stained with H&E were obtained from controland BDE-209-treated mice whose dams were exposed to BDE-209 from gestation day(GD) 0 to GD17. Control group shows normal testicular morphology (A). The interstitialcells of the testis treated with 10 mg kg21 BDE-209 show moderate vacuolar degeneration(B). Similar, but less severe, changes were found in the 500 mg kg21 BDE-209-treatedmice (C). In 1500 mg kg21 BDE-209 treated mice; the seminiferous tubules show severevacoualization associated with complete losses of spermatozoa and spermatids, but stillretained both basement membrane and a few spermatogonia. *, vacuolization of seminif-erous tubules; arrowhead, vacuolization of interstitial cells; Scale bar 5 100 lm. [Colorfigure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

386 TSENG ET AL.

Environmental Toxicology DOI 10.1002/tox

The low dose of BDE-209 administered to the dams was

�1.38 times higher than the human cord serum BDE-209

concentrations reported for women in France, who were

found to have a maximal level of about 363 ng g21 of body

fat weight for BDE-209 in serum lipid (Antignac et al.,

2009). Assuming that 20% of the body weight of an adult

mouse is composed of adipose tissue, such a concentration

could be achieved by administering a single dose of 0.073

mg BDE-209/kg BW. BDE-209 is poorly absorbed, rapidly

eliminated and marginally distributed to adipose tissue,

thus oral absorption has been reported to only be about 1%

of the BDE-209 dose in rats (Morck et al., 2003). Accord-

ing to our calculations, a dose of 10 mg kg21 was 1.38

times higher than maternal exposure from cord serum. We

administered 10 mg kg21 at the lowest dose to the pregnant

female mice in our study. The hypothesis of the high dos-

age of BDE-209 was used to predict the potential effects on

occupational exposure workers. Occupational exposure to

higher levels of BDE-209 can occur in workers manufactur-

ing or handling flame-retarded products. The highest BDE-

209 concentration was 3100 ng g21 lipid in electronics dis-

mantling workers (Bi et al., 2007). The high-dosage group

(1500 mg kg21) had a level of BDE-209 that was at least a

207 times higher level of BDE-209 than current maternal

cord serum levels and a 24 times higher level of BDE-209

than workers’ serum levels. From the reproduction toxicity

point of view, the chance of humans being exposed to such

a high dose of BDE-209 is very small. Findings in mice

prenatally exposed to high levels of BDE-209, however,

provide valuable information on male reproductive effects

of PBDEs and related chemicals in humans. The evidence

found concerning the adverse effects of PBDE congeners

both in our animal studies and those of others, suggests that

further studies using environmentally relevant doses are im-

portant for hazard identification.

Oxidative stress is one of the major contributors to DNA

damage in the male germ line. Moreover, DNA damage is a

major contributor to infertility, miscarriage and birth

defects in the offspring (reviewed by Aitken and De Iuliis,

2010). It has been proposed that the source of the oxidative

stress responsible for sperm DNA damage has been

involves a loss of antioxidant protection, infection, chemi-

cal toxicants exposure, and intrinsic radical generation by

spermatozoa. BDE-209-induced sperm oxidative damage

could therefore be associated with ROS generation. At pres-

ent, there is very little information available on the impact

of BDE-209 on ROS generation and DNA damage in sper-

matozoa. The mechanisms responsible for the association

between BDE-209 exposure and ROS generation are

unknown and validation of this concept is required.

This is the first study to document the lowest-observed-

adverse-effect level (LOAEL) for sperm DNA damage and

TABLE III. Pathological changes in testis tissue of male mice prenatally exposed to decabrominated diphenyl ether(BDE-209)

Pathological changes

Control (a/b) Treatment of BDE-209 (a/b)

0 10 mg kg21 500 mg kg21 1500 mg kg21

Vacuolization in interstitial cells 0/5 2/5 11 3/5 1 3/5 11

2/5 1 2/5 1

Vacuolization in seminiferous tubules 0/5 0/5 0/5 2/5 111

2/5 1

a/b: number of mice with positive pathological change/total number of mice in each group.

1: slight; 11: moderate; 111: severe.

TABLE IV. A summary of adverse effects on various endpoints related to in male offspring prenatally exposed todecabrominated diphenyl ether (BDE-209)

Endpoints

Doses

10 mg kg21 500 mg kg21 1500 mg kg21

Anogenital distance (AGD) --- --- ;*

Abnormal sperms head --- --- :*

Sperm chromatin DNA damage levels (X aT) :* :* :*

DNA fragmentation index (DFI) :* :* :*

H2O2 generation :* --- :*

Histopathology of testis 11 1 111

---: no significant effects.* : P\0.05 as compared to control group.

1: slight; 11: moderate; 111: severe.

387DEVELOPMENTAL EXPOSURE TO DECABROMINATED DIPHENYL ETHER (BDE-209)

Environmental Toxicology DOI 10.1002/tox

excessive H2O2 production resulting from in utero exposure

to doses as low as 10 mg kg21 day21. In conclusion, our

study has found that lower concentrations of BDE-209

might cause sperm chromatin DNA damage via sperm oxi-

dative stress in mouse offspring, based on the evidence of

association between sperm H2O2 production and increased

fraction of chromatin defect. We believe that these results

make an important contribution to the issue of environmen-

tal pollution, and provide critical information for health-

risk assessments.

The authors thank Dr. Wei-Jen Yao from National Cheng

Keng University Medical Center for his technical assistance in the

serum hormone assay. They also thank Dr. Chee-Yin Chai and

Ms. Wan-Tzu Chen from Kaohsiung Medical University for shar-

ing their experience in histopathology.

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