Novel Brominated Flame Retardants Affect Fecundity and
Transcript Profiles of the HPGL-axis in Japanese Medaka
SETAC Prairie Northern Presentation
David M.V. Saunders*, Steve Wiseman, Michelle Podaima, Garry Coddling, John P. Giesy
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Brominated Flame Retardants (BFRs)
•BFRs, additives in consumer products OH
Br
Br
Br
HO
O
HO
Br
Br
Br
H3C
CH3
Br
BrBr
Br
Br
Br
Br
Br
Br
Br
OH
PBDE
TBBPA
HBCD
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Global Phase-Out of Legacy BFRs
• PBDEs meet PBT criteria – Members of the Nasty Nine (UN)
1970s - Ban on Use of Polychlorinated Biphenyls
- Commercial production of PBDEs began
1990s - Peak PBDE production
- Global demand for PBDEs doubled
2004 - EU banned use of Penta BDE and Octa BDE
- Voluntary phase-out in North America
2008 - EU banned use of Deca
Today -North American phase-out of DecaBDE by end 2013
-HBCD being phased out in NA, 2014-2015
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Novel Brominated Flame Retardants (NBFRs)
• Three NBFRs:
•
•
•
2-ethylhexyl tetrabromobenzoate (TBB)
Bis(2-ethylhexyl) tetrabromophthalate (TBPH)
Tetrabromocyclooctane (TBCO)
• TBPH :A structural analogue to a known toxicant, di(2-ethylhexyl) phthalate (DEHP)
•
Known EDC with non-genotoxic hepatocarcinogenic effects
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TBPH, TBB & TBCO
Uses:
• TBPH & TBB: components of the technical mixtures Firemaster 550 (35% TBB, 15%
TBPH), Firemaster BZ-54 (70% TBB, 30% TBPH), and DP-45 (TBPH only)
• TBPH and TBB are used as a replacement for PentaBDE mixtures in polyurethane foams,
PVC, cable insulation,
• TBCO is a component of Saytex BC-48 - textiles and plastics
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TBPH & TBB in the Environment
•Discovered in environmental matrices - air, sewer sludge, foam, dust, baby products (Stapleton et al. 2011)
•Integrated Atmospheric Deposition Network (IADN)
2008 – 2010 - detected in gas and particle-phase at six locations near N.A Great Lakes
Greatest concentration near urban centres – Chicago, Cleveland
• Global Atmospheric Passive Sampling (GAPS) Network
•TBPH, TBB present in >60% all sampled sites
America, Europe, Africa, Asia, Australia
•Recent Canadian Study (Gentes, 2012): industrialized section of St. Lawrence
Greatest detection frequency – 89% of all samples (ring-billed gulls)
Greatest biotic concentrations – max. 17.6 ng/g ww
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Toxic Profiles of TBPH and TBB
• Brominated analogues of the phthalate, DEHP and its metabolite MEHP
•
Controlled substance under CEPA, Schedule 1
• Initial screening level investigations (YES/YAS assays) have highlighted potential receptor
mediated endocrine disrupting effects (Saunders, 2013)
TBB
100
90
80
70
60
50
40
30
20
10
0
150
30
15
3
0.3
0.03
0.003
HT
*
TBPH
100
90
80
70
60
50
40
30
20
10
0
TBB
TBPH
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1500
300
150
30
15
3
0.3
0.03
HF
CTRL
YAS
* * *
CTRL
5.E…
5.E…
5.E…
5.E…
5.E…
5.E…
5.E…
CT…
HT
*
Signal Intensity
* *
*
100
90
80
70
60
50
40
30
20
10
0
Signal Intensity
(YAS) TBB: Weak anti-androgenic effects
TBPH: Significant anti-androgenic effects
YES
* * *
*
CTRL
HF
5E-10
5.E-08
5.E-06
5.E-04
5.E-03
5.E-02
5.E-01
•
(YES) TBB: Weak anti-estrogenic effects
TBPH: No anti-estrogenic effects
Signal Intensity
•
Effects of TBPH and TBB
Signal Intensity
•
100
90
80
70
60
50
40
30
20
10
0
Toxic Profiles of TBPH and TBB
• Exposure alters sex hormone concentrations
•
[17β-estradiol] - porcine primary testicular cells and H295R steroidogenesis cellular assay
TBPH: approx. 5-fold
TBB: approx. 2-fold
(Mankidy, 2013)
TBPH: approx. 5-fold
TBB: approx. 3-fold
(Saunders, 2013)
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Toxic Profiles of TBPH and TBB
•
Exposure alters expression of steroidogenic genes - porcine primary testicular cells
TBPH up-regulated: CYP11A
3β-HSD
CYP19A
TBB up-regulated: CYP21A
down-regulated: CYP17A
•
TBPH/TBB alter hormone concentrations and gene expression in screening level in vitro systems
•
Require investigations of in vivo systems & integrative measures of endocrine disruption
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In vivo investigation of endocrine disruption
• OECD 21-day fecundity assay (test 229)
• Exposure parameters
•
•
•
•
•
Japanese Medaka – dietary exposure
3 chemical groups
5 dosing groups
4 tanks per dosing group
8 male – 8 female per tank
• Experimental parameters
•
•
•
TBPH/TBB mixture
High (1500:1500 μg/g food)
Low (150:150 μg/g food)
• Endpoint measurements
1.
2.
Cumulative Fecundity
Expression profiles of genes across the hypothalamic-pituitary-gonadal-liver axis
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Cumulative Fecundity – TBPH/TBB
• Integrated measure of endocrine disruption: high dose significantly disrupted fecundity
Egg production relative to solvent control: 68% and 94%, high and low doses
600
500
Control
TBPH/TBB high
TBPH/TBB low
400
*
Egg Number
•
300
200
100
0
0
5
10
15
20
25
Time (days)
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Trends in Daily Fecundity
50
30
A
A
Control
TBPH/TBB high
25
30
Profile analysis:
High dose: Sig. Dif. Profile
Low dose: No Dif.
20
10
Egg Number
Egg Number
40
20
15
10
0
5
0
5
10
15
20
25
0
5
10
Time (days)
15
20
25
Time (days)
26
45
B
B
Control
TBPH/TBB low
40
24
22
20
Within-group analysis:
High dose: Sig. Dif
Grouped for stat analysis
30
25
Egg Productino
Egg Number
35
18
*
16
*
14
*
12
20
10
15
8
10
6
0
5
10
15
Time (days)
20
25
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
Groups
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4.5
Hypothalamic-Pituitary-Gonadal-Liver Axis
• Changes to cumulative fecundity and daily egg deposition
•
Mechanisms and/or profiles of effect?
(Villeneuve,2007)
•
Chose 35 genes across the HPGL axis to determine changes in transcript abundances
• Genes incorporated: signal initiation (brain), steroidogenesis (gonad), vitellogenesis (liver)
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Changes to Gene Expression Profiles Along the
HPGL-Axis
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Focus on Initiating Signals – Gonadotropin Releasing
Hormones
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Focus on Initiating Signals – Gonadotropin Releasing
Hormone Receptors
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Focus on Propagation of Signals – Gonadotropin
Sub-Units
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Down-Regulation of GnRH Transcripts in Brain
T
T
E2
E2
Hypothalamus
GnRHs
• Hypothalamus integrates several signals including E2, T, progesterone,
and neurotransmitters (serotonin, dopamine)
Negative feedback mechanism
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Down-Regulation of GnRHRs Transcripts in Brain
T
T
E2
E2
Hypothalamus
GnRHs
GnRH
GnRH
GnRH
GnRH
Pituitary
•
Gonadotropin releasing hormone receptors I/II/III
• GnRHs interact with receptors – propagation
of signal
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Down-Regulation of Gonadotropin Transcripts in
Brain
GnRH
GnRH
GnRH
GnRH
Pituitary
GTHα
LH-β
FSH-β
•
Down-regulation of genes encoding sub-units of gonadotropins
• Down-regulation of gonadotropin hormones
• Key regulators of steroidogenesis
• Down-regulation of steroidogenesis
FSH
LH
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Conclusion - Toxic Profiles of TBPH/TBB
1.
TBPH/TBB significantly inhibited cumulative fecundity of Medaka
a)
2.
TBPH/TBB significantly inhibited daily egg production of Medaka
a)
3.
Integrative measure of endocrine disruption – population level effects
Within-group analysis – pattern of initial toxic insult - compensatory
response
TBPH/TBB altered expression profiles of key genes across the HPGL axis
a)
Global down-regulation of key genes involved in initiation of endocrine
signaling - subsequent inhibition of steroidogenesis
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Acknowledgements
Dr. Steve Wiseman
Miss. Michelle Podaima
Mrs. Eric Higley
Dr. John P. Giesy
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Questions
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