Cytotoxicity and mRNA Expression Alterations of Two Highly
Brominated Flame Retardants after Sunlight Irradiation
Guanyong Su,1,2 Robert J. Letcher,1,2 Doug Crump,1 Reza Farmahin,1,3
John P. Giesy,4,5,6 Sean W. Kennedy1,3
1 Environment Canada, National Wildlife Research Centre, Ottawa, ON, Canada;
2 Department of Chemistry, Carleton University, Ottawa, ON, Canada;
3 Department of Biology, University of Ottawa, Ottawa, ON, Canada;
4 University of Saskatchewan, Saskatoon, SK, Canada;
5 Michigan State University, East Lansing, USA;
6 City University of Hong Kong, Kowloon, Hong Kong, SAR, China
Poster number: RP147
Brominated flame retardants
(BFRs) have routinely been
added to consumer products
for several decades in a
successful effort to reduce firerelated injury and property
damage.
Penta-BDE; Octa-BDE;
PBB
Two highly brominated flame retardants
Br
Br
Br
O
Br
Br
Br Br
 TeDB-DiPhOBz and BDE-209 continues to be
produced and marketed in some regions around
the world.
TeDB-DiPhOBz (SAYTEX 120)
Br Br
Br
Br
 Both are highly brominated, generally nonvolatile, and have low bio-availability or potential
to bio-accumulate (log Kow >10).
Br
Br
Br
Br
Br
Br
O
Br Br
Br
Br
Br
O
Br
Br
BDE-209 (DecaBDE)
E-waste landfill
Environ. Sci. Technol. 2013, 47, 1373
Research Hypothesis:
There might be toxicity shift of SAYTEX120&BDE-209 following exposed to sunlight.
Schematic flow diagram
of the experiment
Sunlight
irradiation
Two highly brominated flame retardants
SAYTEX 120 was hardly dissolved
into other solvents (hexane, methanol, DCM etc) (dissolved into hexane (30% THF)
except for THF.
Chicken embryonic
hepatocytes (CEH) assay
(Toxicological effect)
Cytotoxic effects; gene expression.
Instrumental analysis
(By-products identification)
Based on what kind of standards
we have in our lab.
Cell Viability
(Cytotoxicity)
&
mRNA
expression
Avian ToxChip Polymerase Chain Reaction Array [6]
Gene functions include:
Phase I and II metabolism
Thyroid hormone pathway
Lipid/cholesterol metabolism
Oxidative stress
Immune response
Cell death
Cell Viability
(Cytotoxicity)
&
mRNA
expression
CEH assay-Cytotoxic effect
Avian ToxChip Polymerase Chain Reaction Array [6]
Gene functions include:
Phase I and II metabolism
Thyroid hormone pathway
Lipid/cholesterol metabolism
Oxidative stress
Immune response
Cell death
“SI” means “sunlight irradiation”.
 TeDB-DiPhOBz, SI-TeDBDiPhOBz and BDE-209
showed no significant
cytotoxic effect on CEH
at any examined
concentration relative to
the DMSO solvent control.
 SI-BDE-209 significantly
decreased cell viability at
a nominal concentration
of 50 µM (LC50: 26 ± 3.1
µM).
Parent Chemicals
Sunlight irradiated
by-products
Cell Viability
(Cytotoxicity)
&
mRNA
expression
CEH assay-mRNA expression (PCR array)
Avian ToxChip Polymerase Chain Reaction Array [6]
Gene functions include:
Phase I and II metabolism
Thyroid hormone pathway
Lipid/cholesterol metabolism
Oxidative stress
Immune response
Cell death
CYP1A4: Aryl hydrocarbon receptor (AhR)-responsive gene
 TeDB-DiPhOBz and BDE209 caused limited
mRNA expression
changes across 27 genes.
 However, 12 and 14 of
the 27 genes were
altered following exposed
to SI-TeDB-DiPhOBz and
SI-BDE-209.
 CYP1A4 showed an
extremely large mRNA
expression change
ranged from 560 to 5200.
Effects of Irradiation in Different Solvents
Does THF contribute anything to
the observed toxicity?
%TCDD max.
60
40
 At concentrations ranging
from 0.001 to 50 µM, the
maximal responses caused by
SI-BDE-209 in 30% THF/nhexane, n-hexane, and
methanol relative to a 300
nM TCDD positive control
were similar; 31 ± 4.8 %, 37
± 3.4 %, and 45 ± 6.5 %,
respectively .
BDE-209 in THF/Hexane
BDE-209 in Hexane
BDE-209 in Methanol
20
0
10
10
1
0 .1
1
0 .0
01
0 .0
DM
SO
 These results clearly
0
demonstrated that the
observed AHR-mediated
reporter gene activity was
altered by photodegradation
Concentration (μM)
by-products of BDE-209 and
TeDB-DiPhOBz was not included in the solvent assessment not solvent reaction products.
because of its poor solubility in methanol or n-hexane.
Instrumental analysis
Target ions: 407/409 m/z (BDE-197, -201, 202); 485/487 m/z (BDE207, -208, 209); 79 and 81 m/z (other 41 PBDEs congeners).
 GC-MS(ECNI)
 There was no detectable BDE-209.
 Eighteen PBDE congeners were
quantifiable.
 Down to the earliest retention times
of 5 to 10 min, there were numerous
unidentified peaks representing
compounds containing bromide
anions.
 LC-APPI(-)-MS-TOF
 TeDB-DiPhOBz was depleted to nondetectable concentrations.
 Br8- to Br11-PB-DiPhOBz homolog
groups of congeners were the major
debrominated products in the SI-TeDBDiPhOBz solution, with the Br10-PBDiPhOBz homolog group showing the
greatest APPI(-)-MS-TOF response.
Target ions: [M-Br+O]- or [M+O]-.
Next steps?
• Quantification of PBDD/Fs in BDE-209&TeDBDiPhOBz byproducts (Collaboration with Dr. John
Giesy in University of Saskatchewan)
• Expose the BDE-209&TeDB-DiPhOBz powder
under sunlight, and then investigate its
toxicological effects
Sunlight-Induced Photolytic Degradation of Highly Brominated Flame Retardants Cause
Cytotoxicity and mRNA Expression Alterations in Chicken Embryonic Hepatocytes
Guanyong Su,1,2 Robert J. Letcher,1,2 Doug Crump,1 Reza Farmahin,1,3 John P. Giesy,4,5,6 Sean W. Kennedy1,3
1 Ecotoxicology and Wildlife Health Division, Environment Canada, National Wildlife Research Centre, Carleton University, Ottawa, ON, K1A 0H3, Canada; 2 Department of Chemistry, Carleton
University, Ottawa, ON, K1S 5B6, Canada; 3 Centre for Advanced Research in Environmental Genomics, Department of Biology, University of Ottawa, Ottawa, Ontario, Canada; 4 Department of
Veterinary Biomedical Sciences and Toxicology Centre, University of Saskatchewan, Saskatoon, SK S7N 5B3, Canada; 5 Department of Zoology and Center for Integrative Toxicology, Michigan
State University, East Lansing, MI 48824, USA; 6 Department of Biology & Chemistry, State Key Laboratory in Marine Pollution, City University of Hong Kong, Kowloon, Hong Kong, SAR, China
Results
Introduction
Due to their ability to reduce flammability and hinder fire ignition in the
products that contain them, brominated flame retardants (BFRs),
including 2,2’,3,3’,4,4’,5,5’,6,6’-decaBDE (BDE-209), have been
widely used in various commercial products such as furniture, textiles,
plastics, paints, and electronic appliances.
Tetradecabromo-1,4-diphenoxybenzene
(TeDB-DiPhOBz)
is
a
halogenated polyphenyl ether (HPE) that is a replacement for BDE209, and is the main constituent of commercial technical formulations.
A 1973 US Patent (US3760003A) to the Dow Chemical Company
detailed the production of HPEs that included TeDB-DiPhOBz. In
1986, Albermarle Corp. acquired Dow’s bromine business. Although
Albermarle has claimed that production of their major TeDB-DiPhOBzbased BFR, SAYTEX-120, was phased-out and discontinued
commercially in January 2011, we know that SAYTEX 120-related
mixtures are produced and marketed in some global regions,
especially Asia (Energy Chemical, MHP Chemical, TCI Chemicals).
TeDB-DiPhOBz and BDE-209 are highly brominated, generally nonvolatile, and due to their log octanol-water partition coefficients of >10,
have low bioavailability or potential to bioaccumulate. When exposed
to UV-A, -B, -C, or natural sunlight, we recently showed that TeDBDiPhOBz can undergo rapid photolysis and degrade via stepwise,
reductive debromination [1]. When exposed to natural sunlight, halflives of TeDB-DiPhOBz ranged from 4.9 to 7.4 min. Similarly, when
sunlight irradiated, BDE-209 can be photolytically degraded [1,2], with
a half-life in n-hexane (1% THF) of 5.3 min. BDE-209 has also been
metabolically degraded to less brominated, more bioaccumulative,
and potentially more toxic compounds in diet-exposed American
kestrels or via silastic implants with European starlings [3,4]. To our
knowledge, there are publications on biological effects in any biota as
a result of exposure to TeDB-DiPhOBz or its degradation by-products.
Study Objectives [5]:
Using an in vitro CEH assay and an Avian ToxChip polymerase
chain reaction (PCR) array [6], examine mRNA expression
alterations following exposure to TeDB-DiPhOBz and BDE-209 or
their photo-transformed by-products, generated in situ as a
result of natural sunlight irradiation.
Figure 2.
Transcriptional
profiles of 27 target
genes on the Avian
ToxChip PCR array
following CEH
exposure to
non-irradiated (NI-)
and sunlight
irradiated (SI-)
TeDB-DiPhOBz or
BDE-209.
Figure 1. GC-MS (ECNI) total ion mass chromatogram of 47 PBDE congeners in a standard
mixture (black) and in the sunlight irradiated (SI-) BDE-209 (50 μM) after a 21-day sunlight
irradiation in 30% THF/n-hexane (red).
Methods [1,5]
Determination of TeDB-DiPhOBz and debrominated byproducts of sunlight irradiated (SI) TeDB-DiPhOBz was
carried out using an Agilent 1200 liquid chromatographic
(LC) system, coupled with an Agilent 6250A quadrupoletime-of-flight mass spectrometer (Q-TOF)-MS.
Conclusions
• After sunlight irradiation, both TeDB-DiPhOBz and BDE-209 were depleted to non-detectable
concentrations. Br8- to Br11-PB-DiPhOBz homolog groups of congeners were the major
debrominated products in the SI-TeDB-DiPhOBz solution, and 18 PBDE congeners were
debrominated products in the SI-BDE-209 solution (Fig. 1)
Determination of BDE-209 and the debrominated byproducts of sunlight irradiated (SI) BDE-209 was carried
out using an Agilent gas chromatograph (GC) 6890
coupled with a 5973 quadrupole mass spectrometer (MS)
detector.
•
None of the tested concentrations of NI-TeDB-DiPhOBz or NI-BDE-209 caused significant (one
way ANOVA; Dunnett’s p<0.05) cytotoxic effects to the CEH relative to the DMSO solvent control.
Extensive cytotoxic effects were observed following exposure to 50 µM SI-BDE-209 (and thus
lower conc. were assessed for possible gene regulation).
Chicken Embryonic Hepatocyte (CEH) screening assay [6]
Cell Viability
(Cytotoxicity)
&
mRNA
expression
Methods
•
The Avian ToxChip PCR array showed that 12 and 14 of the 27 genes were altered after
exposure to 25 µM SI-TeDB-DiPhOBz or 10 µM SI-BDE-209, respectively (Fig. 2). As shown for
BDE-209, since mRNA alterations were found regardless of irradiation in THF and non-THF
solvents (THF, methanol or n-hexane).
Knowledge Transfer and Exchange
Avian ToxChip Polymerase Chain Reaction Array [6]
TeDB-DiPhOBz and BDE-209, in solid powder form, were kindly
supplied by Wellington Laboratories (Guelph, ON, Canada).
TeDB-DiPhOBz and BDE-209 powder was dissolved in 30 % THF/nhexane solution (and also BDE-209 in methanol or n-hexanes to
assess solvent effects) to achieve a final, nominal concentration of
300 µM. The same volume of 30% THF/n-hexane was used as the
solvent control for the present study.
The sunlight irradiation was conducted from December 24, 2013 to
January 14, 2014 in Ottawa (21 days), and the location coordinates
were 45o40'06''N and 75o74'22''W.
Gene functions include:
Phase I and II metabolism
Thyroid hormone pathway
Lipid/cholesterol metabolism
Oxidative stress
Immune response
Cell death
• Knowledge gap addressed: this is the first report on the in vitro cytotoxicity and gene
expression effects of TeDB-DiPhOBz on any cell system or level of biological organization.
• Science implication: very large induction of CYP1A4 mRNA (Fig. 2) following CEH
Thank you!
Acknowledgements
Funding for this project was provided by the Chemical Management
Plan (to R.J.L, S.W.K. and D.C.) and the National Science and
Engineering Research Council (NSERC; to R.J.L). We thank S.G.
Chu with assistance with the LC-ESI-ToF-MS analysis.
exposure to by-products of SI-TeDB-DiPhOBz and SI-BDE-209, strongly suggests more
environmental focus on by-products rather than TeDB-DiPhOBz and BDE-209.
CMP3 risk assessment: TeDB-DiPhOBz is a priority substance in the OFR grouping, and
these results contribute the exposure and in vitro effects within our avian AOP framework.
•
References
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[5]
[6]
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E. van den Steen, A. Covaci, V.L. Jaspers, T. Dauwe, S. Voorspoels, M. Eens, R. Pinxten, Environ. Pollut. 148 (2007) 648.
G. Su, R.J. Letcher, D. Crump, R. Farmahin, J.P. Giesy, S.W. Kennedy, Environ. Sci. Technol. (2014) “Just Accepted” on-line (Sept. 16th).
E. Porter, D. Crump, C. Egloff, S. Chiu, S.W. Kennedy, Environ. Toxicol. Chem. 33 (2014) 573.
Poster number: RP147