A Combined Transcriptomic and Proteomic Approach to Elucidate Adverse Effects
of the Brominated Flame Retardant TBCO on Early-Life Stages of Japanese Medaka
Jianxian Sun1, Song Tang2, Hui Peng1, David M.V. Saunders1, Jon Doering1, Markus Hecker1,2, Paul Jones1,2, John P. Giesy1,3, and Steve Wiseman1
Centre, University of Saskatchewan, Saskatoon, SK, Canada; 2 School of Environment and Sustainability, 117 Science Place, Saskatoon, SK, Canada
3. Department of Veterinary Biomedical Sciences, University of Saskatchewan, Saskatoon, SK, Canada
Results
Background
Knowledge of toxic effects of TBCO is very limited. Studies to date
have demonstrated endocrine disruptive effects of TBCO in vitro and in
vivo.2,3
50
600
A
40
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500
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20
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y = 4.6x - 1.9
r² = 0.97
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y = 54.5x - 30.8
r² = 0.98
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y = 5.7x + 650.1
R² = 0.0043
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Days (dpf)
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Fig 1. Up take and accumulation of TBCO in eggs of Japanese medaka.
(A) 10 μg/L, (B) (100 μg/L), and (C) 1 mg/L. Data are mean ± st. dev of 7
replicate exposures.
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Objectives
Identify potential molecular mechanisms of toxicity of TBCO by:
1. Quantifying transcriptome and proteome-wide responses of
early-life stages of Japanese medaka to TBCO.
2. Linking transcriptome and proteome-wide responses to
adverse apical effects.
***
Changes in gene expression determined by use of transcriptomics and
proteomics were predictive of adverse effects of TBCO on the following
apical endpoints:
high
 Cardiac function: Abundances of proteins related to cardiac
muscle development and contraction were significantly lesser in fish
exposed to TBCO. This indicates that development of the
cardiovascular system is a targets for TBCO in medaka embryos.
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Fig 2. Effect of TBCO on hatching success of Japanese medaka.
(A) Time to hatch. (B) Percentage of embryos hatched at day 13.
Data are mean ± st. dev of 7 replicate exposures. *** p < 0.001.
A
Pvalb4
B
Pvalb1
tpt1
Gene reads
VTG1
lancl2
Proteome
high
VTG6
VTG3
Transcriptome
medium
Protein spectra count
Fig 3. Heat map illustrating effects of TBCO on
abundance of proteins and transcripts in Japanese
medaka fry. Among the 20,425 transcripts identified,
240 were differentially expressed (142 of greater
abundance, and 98 of lesser abundance). Of the 1,256
proteins identified, 252 proteins were differentially
expressed (98 of greater abundance, and 154 of lesser
abundance) in medium dose, while 254 differentially
expressed proteins were identified in high dose (139 of
greater abundance, and 115 of lesser abundance).
Fig 4. Distance-based redundancy analysis of measured
responses of the (A) transcriptome and (B) proteome of
early-life stages of Japanese medaka exposed to TBCO.
Proximity of points in the biplot is an approximation of the
similarity among samples with respect to their response.
Significant differences among groupings were observed
(p=0.001). Variability of each axis is significant (Axis1
p=0.001; Axis2 p=0.001). Ovals represent the 95%
confidence ellipse around the group centroids.
Results 3. Linking Molecular Responses to Apical Effects
A
A
Proteomics: Proteins from four pools (n=4)
of fifty fry exposed to control, medium or high
concentration of TBCO were analyzed by use
of a LTQ Orbitrap Velos hybrid instrument.
Raw MS files were analyzed by MaxQuant
based on the Ensembl protein database for
Japanese medaka.
Gene Ontology (GO) Enrichment Analysis: Pathway enrichment was
based on significantly altered transcripts and proteins and was
investigated by use of ClueGO,4 based on zebrafish orthologs.
Created by Peter Downing – Educational Media Access and Production © 2011
-1 )
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HeartBeat(min
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Fig 6. Effect of TBCO on heart rate in Japanese medaka embryos.
(A) Results of the proteome analysis suggested that TBCO decreases
expression of genes required for cardiac development and performance.
Proteins of lesser abundance are shown in green and proteins of greater
abundance are shown in red. Size of circles is representative of the
significance of enrichment in a pathway (p value), and intensity of the
color is proportional to the extent of down or up regulation.
(B) In a separate experiment, embryos were exposed to TBCO starting at
2 hpf and the count of heart beats per minute were determined at 5 dpf.
Data are mean ± st. dev of 12-19 trials. *** p < 0.001.
(C and D) Heart contractions in Japanese medaka embryos at 5 dpf.
Development of Adverse Outcome Pathways (AOPs)
Results might be useful in support of the development of AOPs for the
disruption of cardiac system development and visual performance.
References
1. Alaee, M.; Arias, P.; Sjodin, A.; Bergman, A., An overview of commercially used
brominated flame retardants, their applications, their use patterns in different
countries/regions and possible modes of release. Environ. Int. 2003, 29, (6), 683689.
2. Saunders, D. M.; Higley, E. B.; Hecker, M.; Mankidy, R.; Giesy, J. P., In vitro
endocrine disruption and TCDD-like effects of three novel brominated flame
retardants: TBPH, TBB, & TBCO. Toxicology Letters 2013, 223, (2), 252-259.
3. Saunders, D. M.; Podaima, M.; Wiseman, S.; Giesy, J. P., Effects of the
brominated flame retardant TBCO on fecundity and profiles of transcripts of the
HPGL-axis in Japanese medaka. Aquatic Toxicology 2015, 160, 180-187.
4. Shannon, P.; Markiel, A.; Ozier, O.; Baliga, N. S.; Wang, J. T.; Ramage, D.; Amin,
N.; Schwikowski, B.; Ideker, T., Cytoscape: a software environment for integrated
models of biomolecular interaction networks. Genome Res. 2003, 13, (11), 24982504.
5. Viczian, A. S.; Zuber, M. E., A simple behavioral assay for testing visual function
in Xenopus laevis. JoVE (Journal of Visualized Experiments) 2014, (88), e51726e51726.
Acknowledgements
E
F
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Ongoing and Future Research
Fig 5. Regression analysis of relationship
between abundance of proteins and
transcripts in freshly hatched medaka
larvae from control group. Black dash line
indicates 95% confidence level. Presence of
proteins with high abundance in proteome but
low in transcriptome, such as VTG, is a clear
evidence of maternal transfer (dots in red).
Exposure: Fertilized eggs were exposed to waterborne TBCO from 2
hpf until hatch. Fish were exposed to control (0.1% v/v acetone), low
(10 μg/L), medium (100 μg/L), and high (1000 μg/L) concentrations of
TBCO. All exposures were replicated 7 times.
6
0
B 1
Maternally transferred proteins and great variation of rates of turnover
of proteins and mRNAs are complicating factors that can influence
gene expression profiling studies with early-life stages of fishes.
Simultaneous application of open-format transcriptomics and
proteomics can overcome this limitation.
R=0.26, p=9e-5
Methodology
Transcriptomics: RNA from three pools (n=3)
of five fry exposed to control or medium
concentration of TBCO was sequenced using
2×150 bp paired-end reads on an Illumina
MiSeq platform. RNASeq was performed
using TopHat2 and Cufflinks by aligning reads
to the Japanese medaka transcriptome
available in Ensembl.
 Visual function: Abundances of transcripts of genes that regulate
development of the eye, photo-transduction, and sensory
perception of light were lesser in fish exposed to TBCO. Visual
performance was impaired in fish exposed to TBCO.
Results 2. Transcriptomics and Proteomics
Proteome
medium
TBCO
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Days (dpf)
Advances in high-throughput open format “omics” technologies that
facilitate investigation of transcriptome-wide and proteome-wide
responses to chemical stressors can be used to identify mechanisms of
toxicity of chemical stressors and to predict averse effects on apical
endpoints.
HBCD
Hatching rate
The novel BFR, 1,2,5,6-tetrabromocyclooctane (TBCO) is an
alternative to HBCD. Although concentrations of TBCO in some
environmental matrices are less than the limit of detection, amounts
are expected to increase if usage increases significantly. Thus,
information on potential toxicities of TBCO to ecological receptors,
such as fish, very important.
Effects of TBCO were quantified by identifying responses of both the
transcriptome and proteome of early-life stages of Japanese medaka.
Results 1. Uptake and Effects of TBCO on Hatching Success
Con. in embryo (μg/g)
In response to concerns about persistence, bioaccumulation, and
toxicity (PBT) of brominated flame retardants (BFRs)1, production and
use of PentaBDE and OctaBDE technical mixtures have been banned,
and HBCD and DecaBDE will be phased-out in European countries by
2015 and from North America in the near future.
Conclusions
Hatching Rate
1 Toxicology
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0
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Low
Med
ctrl
low
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Fig 7. Effects of TBCO on visual function of Japanese medaka fry at 7 dph.
(A) RNAseq analysis suggested that TBCO decreases expression of genes for eye
development and performance (sensory perception of light and phototransduction).
Size of circles is representative of the significance of enrichment in a pathway (p
value), and intensity of the color is proportional to the extent of down or up regulation.
(B-F) Effects on visual performance were determined by use of a background color
preference assay.5 (B) Percentage of fry that swam to the white side of the exposure
vessel within 30 seconds; (C) average time required to swim to the white side of the
exposure vessel. Swimming behavior of fry exposed to (D) control, (E) low and (F)
medium concentrations of TBCO are given. Data are mean ± st. dev of 4 replicate
exposures with 20 fry per trial. * p < 0.05.
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J.P. Giesy and M. Hecker are supported by the Canada Research Chairs
Program and Discovery Grants form the Natural Sciences and Engineering
Research Council (NSERC) of Canada.
An equipment grant from Western Economic Diversification
Aquatic Toxicology Research Facility at the University of Saskatchewan.
Contact
Jianxian Sun, PhD
Post-Doctoral Fellow
Email: sunsjx@gmail.com