Toxicology Centre
Effects of TBCO on the Reproductive Capacity of 2
Generations of Japanese Medaka (Oryzias latipes)
Br
Br
Br
Br
Steve Wiseman
University of Saskatchewan
Title or place of presentation
Date of presentation
Toxicology Centre
Multigenerational Effects
Pesticides
Vinclozolin
(anti-androgen)
(multigenerational)
(all generations exposed)
4 generations of males from
vinclozolin-treated females show
a higher level of testis apoptosis.
(transgenerational)
(no direct exposure)
Title or place of presentation
Date of presentation
Toxicology Centre
Generational Effects in Fish
BPA
EE2
 No effects in F0 and F1 generations
 Reduced fertilization in F2 generation
 Reduced embryo-survival in F3 generation
Title or place of presentation
Date of presentation
Toxicology Centre
Are there Multigenerational Effects of TBCO?
Saunders, D.M.V., M. Podaima, S. Wiseman, J.P. Giesy. 2015. Effects of the Brominated Flame Retardant TBCO on
Fecundity
Profiles of Transcripts of the HPGL-Axis in Japanese Medaka. Aquatic Toxicol. 160:180-187.Date of presentation
Title
or placeand
of presentation
Toxicology Centre
1,2,5,6-Tetrabromocyclooctane (TBCO)
Br
Br
Hexobromocyclododecane (HBCD)
Br
•
•
•
•
•
Br
Thermal insulation foams
Textile coatings
Meets EU criteria aquatic hazard
Not detected in the environment
EDC in vitro
• Disruption of sex- steroid
•
•
•
•
2010: 28,000 tons/yr
Being phased out (some exceptions)
Replacements being assessed.
receptor signaling
Disruption of steroidogenesis
Title or place of presentation
Date of presentation
Toxicology Centre
1. Effects of Dietary Exposure to TBCO on Reproductive
Capacity of F0 Generation Japanese Medaka
Saunders, D.M.V., M. Podaima, S. Wiseman, J.P. Giesy. 2015. Effects of the Brominated Flame Retardant TBCO on
Fecundity
Profiles of Transcripts of the HPGL-Axis in Japanese Medaka. Aquatic Toxicol. 160:180-187.Date of presentation
Title
or placeand
of presentation
Toxicology Centre
Egg Number
Cumulative Egg Production
500
450
400
350
300
250
200
150
100
50
0
Control
0
3
6
9
12
15
18
21
Time (Days)
Title or place of presentation
Date of presentation
Toxicology Centre
Egg Number
Cumulative Egg Production
500
450
400
350
300
250
200
150
100
50
0
Control
TBCO - 58 ug/g food
 Decreased fecundity
0
3
6
9
12
15
18
21
Time (Days)
Title or place of presentation
Date of presentation
Toxicology Centre
Egg Number
Cumulative Egg Production
500
450
400
350
300
250
200
150
100
50
0
Control
TBCO - 607 ug/g food
TBCO - 58 ug/g food
 Decreased fecundity caused
only by lowest dose of TBCO
0
3
6
9
12
15
18
21
Time (Days)
Title or place of presentation
Date of presentation
Toxicology Centre
Maternal Transfer of TBCO
Egg [TBCO]
ug/g
25
20
15
10
5
0
BDL
Ctrl
TBCO - 58
ug/g
TBCO - 607
ug/g
Dietary [TBCO]
Title or place of presentation
Date of presentation
Toxicology Centre
2. Effects of Maternally Transferred TBCO on Reproductive
Capacity of F1 Generation
Title or place of presentation
Date of presentation
Toxicology Centre
Question: Does maternally deposited TBCO affect reproductive capacity?
Control
Maternal
TBCO
Title or place of presentation
Date of presentation
Toxicology Centre
Question: Does maternally deposited TBCO affect reproductive capacity?
Control
Freshwater
Maternal
TBCO
Freshwater
Title or place of presentation
Date of presentation
Toxicology Centre
Question: Does maternally deposited TBCO affect reproductive capacity?
Control
Freshwater
Control
Diet
Maternal
TBCO
Freshwater
Control
Diet
Title or place of presentation
Ctrl / Ctrl
TBCO / Ctrl
Date of presentation
Toxicology Centre
Cumulative Egg Production (F1)
1200
Ctrl-Ctrl
Egg Number
1000
800
600
400
200
0
1
Title or place of presentation
3
5
7
9
11
13
Time (Days)
15
17
19
21
Date of presentation
Toxicology Centre
Cumulative Egg Production (F1)
1200
Ctrl-Ctrl
Egg Number
1000
TBCO-Ctrl
800
600
 Decreased fecundity caused by
exposure to maternal TBCO
400
200
0
1
Title or place of presentation
3
5
7
9
11
13
Time (Days)
15
17
19
21
Date of presentation
Toxicology Centre
Question: What is the effect of re-exposure to TBCO?
Control
Freshwater
Control
Diet
Maternal
TBCO
Freshwater
Control
Diet
Title or place of presentation
Ctrl / Ctrl
TBCO / Ctrl
Date of presentation
Toxicology Centre
Question: What is the effect of re-exposure to TBCO at sexual maturity.
Control
Maternal
TBCO
Control
Diet
Ctrl / Ctrl
Dietary
TBCO
Ctrl / TBCO
Freshwater
Freshwater
Control
Diet
Dietary
TBCO
Title or place of presentation
TBCO / Ctrl
TBCO / TBCO
Date of presentation
Toxicology Centre
Cumulative Egg Production (F1)
1200
Ctrl-Ctrl
Egg Number
1000
TBCO-Ctrl
800
600
400
200
0
1
Title or place of presentation
3
5
7
9
11
13
Time (Days)
15
17
19
21
Date of presentation
Toxicology Centre
Cumulative Egg Production (F1)
1200
Ctrl-Ctrl
Egg Number
1000
Ctrl-TBCO
800
TBCO-Ctrl
600
 Decreased fecundity caused by
exposure to TBCO
400
200
0
1
Title or place of presentation
3
5
7
9
11
13
Time (Days)
15
17
19
21
Date of presentation
Toxicology Centre
Cumulative Egg Production (F1)
1200
Ctrl-Ctrl
Ctrl-TBCO
TBCO-Ctrl
TBCO-TBCO
Egg Number
1000
800
600
 Re-exposure to TBCO during
reproduction ameliorates effect
of maternal TBCO
400
200
0
1
Title or place of presentation
3
5
7
9
11
13
Time (Days)
15
17
19
21
Date of presentation
Toxicology Centre
3. Mechanism of Effects
•
Epigenetic?
•
•
•
•
•
Methylome is erased and re-established during early development
Chemical stressors can alter the methylome
Changes in DNA methylation are mitotically heritable
Hypermethylation – decreased expression (transcriptional silencing)
Hypomethylation – increased expression (transcriptional activation)
Title or place of presentation
Date of presentation
Toxicology Centre
Embryo-Exposure
Freshwater
100 ug/L TBCO
Day 0
(1hpf)
(Hatch)
TBCO
Title or place of presentation
Conc. in Embryo (ug/g)
600
y = 54.451x - 30.699
R² = 0.9825
500
400
300
200
100
0
0
2
4
6
8
10
Days
Date of presentation
Toxicology Centre
Question: Does maternally deposited TBCO affect reproductive capacity?
Freshwater
TBCO
Title or place of presentation
Date of presentation
Toxicology Centre
Question: Does maternally deposited TBCO affect reproductive capacity?
Freshwater
Freshwater
TBCO
Freshwater
Title or place of presentation
Date of presentation
Toxicology Centre
Question: What is the effect of re-exposure to TBCO at sexual maturity.
Freshwater
TBCO
Control
Diet
Ctrl / Ctrl
Dietary
TBCO
Ctrl / TBCO
Freshwater
Freshwater
Control
Diet
Dietary
TBCO
Title or place of presentation
TBCO / Ctrl
TBCO / TBCO
Date of presentation
Toxicology Centre
Candidate Gene Approach – Female Fry
8
Vtg II
6
*
ER-β
2
4
1
2
0
0
Ctrl
25
3
CHG-H
20
TBCO
*
Ctrl
AR
4
3
15
TBCO
*
2
10
5
1
0
0
Ctrl
TBCO
Ctrl
TBCO
• Greater expression of genes for sex steroid signalling
Title or place of presentation
Date of presentation
Toxicology Centre
Candidate Gene Approach – Female Fry
StAR
1.5
1.5
1
Cyp19a (aromatase)
1
*
0.5
0
Ctrl
TBCO
*
0.5
0
Ctrl
TBCO
• Lesser expression of genes for sex steroid synthesis.
Title or place of presentation
Date of presentation
Toxicology Centre
Candidate Gene Approach – Female Liver
1.2
1.5
VTG II
0.8
*
*
1
0.4
0.5
0
0
1.5
*
*
ERa
1
0.5
CHG-H
*
*
• Lesser expression of genes for
sex steroid signalling.
0
Title or place of presentation
Date of presentation
Toxicology Centre
Candidate Gene Approach – Female Gonad
2.5
2
1.5
1
0.5
0
CYP19a
*
*
4
3
*
StAR
*
*
2
1
0
• Greater expression of genes for sex steroid synthesis.
Title or place of presentation
Date of presentation
Toxicology Centre
Conclusions
• Dietary exposure to TBCO lowers fecundity of F0 female medaka.
• Maternally deposited TBCO lowers fecundity of F1 medaka
• However, fecundity of F1 fish exposed to maternally deposited TBCO is
attenuated by re-exposure to dietary TBCO during reproduction.
• Predictive adaptive response?
• Compared to F0 fish, female F1 fish exposed to TBCO during ELS have
altered basal expression of key genes of the reproductive axis.
• Altered methylation/epigenetic effect?
• Effects on F2 ?
Title or place of presentation
Date of presentation
Toxicology Centre
• David Saunders (PhD Student)
• Jianxian Sun (PDF)
• Hui Peng (PDF)
• Song Tang (PDF)
• Jeremy Kuzma (BSc Student)
• Markus Hecker
• John Giesy
e-mail: steve.wiseman@usask.ca
Title or place of presentation
Date of presentation