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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 fire-related injury and property damage.

Penta-BDE; Octa-BDE; PBB

Two highly brominated flame retardants

TeDB-DiPhOBz and BDE-209 continues to be produced and marketed in some regions around the world.

Both are highly brominated, generally non-volatile, and have low bio-availability or potential to bio-accumulate (log Kow >10).

O Br

Br

Br

Br

Br

Br

Br Br

Br

Br

Br

Br BrO

Br

O

Br

Br

Br

Br

Br

Br

Br

Br

Br Br

TeDB-DiPhOBz (SAYTEX 120)

BDE-209 (DecaBDE)

Research Hypothesis: There might be toxicity shift of SAYTEX120&BDE-209 following exposed to sunlight.

E-waste landfill

Environ. Sci. Technol. 2013, 47, 1373

Cell Viability (Cytotoxicity)

&mRNA

expression

Avian ToxChip Polymerase Chain Reaction Array [6]Gene functions include:Phase I and II metabolismThyroid hormone pathwayLipid/cholesterol metabolismOxidative stressImmune responseCell death

Instrumental analysis (By-products identification)

Chicken embryonic hepatocytes (CEH) assay

(Toxicological effect)

Two highly brominated flame retardants (dissolved into hexane (30% THF)

Sunlight irradiation

Schematic flow diagram of the experiment

SAYTEX 120 was hardly dissolved into other solvents (hexane, methanol, DCM etc)

except for THF.

Based on what kind of standards we have in our lab.

Cytotoxic effects; gene expression.

CEH assay-Cytotoxic effect

TeDB-DiPhOBz, SI-TeDB-DiPhOBz 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

“SI” means “sunlight irradiation”.

Cell Viability (Cytotoxicity)

&mRNA

expression

Avian ToxChip Polymerase Chain Reaction Array [6]Gene functions include:Phase I and II metabolismThyroid hormone pathwayLipid/cholesterol metabolismOxidative stressImmune responseCell death

CEH assay-mRNA expression (PCR array)

TeDB-DiPhOBz and BDE-209 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.

CYP1A4: Aryl hydrocarbon receptor (AhR)-responsive gene

Cell Viability (Cytotoxicity)

&mRNA

expression

Avian ToxChip Polymerase Chain Reaction Array [6]Gene functions include:Phase I and II metabolismThyroid hormone pathwayLipid/cholesterol metabolismOxidative stressImmune responseCell death

Effects of Irradiation in Different Solvents

0

20

40

60

DMSO

BDE-209 in Methanol

BDE-209 in THF/HexaneBDE-209 in Hexane

0.001 0.0

1 0.11 10 10

0

Concentration (μM)

%T

CD

D m

ax.

At concentrations ranging from 0.001 to 50 µM, the maximal responses caused by SI-BDE-209 in 30% THF/n-hexane, 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 .

These results clearly demonstrated that the observed AHR-mediated reporter gene activity was altered by photodegradation by-products of BDE-209 and not solvent reaction products. TeDB-DiPhOBz was not included in the solvent assessment

because of its poor solubility in methanol or n-hexane.

Does THF contribute anything to the observed toxicity?

Instrumental analysis 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 non-detectable concentrations.

Br8- to Br11-PB-DiPhOBz homolog groups of congeners were the major debrominated products in the SI-TeDB-DiPhOBz solution, with the Br10-PB-DiPhOBz homolog group showing the greatest APPI(-)-MS-TOF response.

Target ions: 407/409 m/z (BDE-197, -201, 202); 485/487 m/z (BDE-207, -208, 209); 79 and 81 m/z (other 41 PBDEs congeners).

Target ions: [M-Br+O]- or [M+O]-.

Next steps?

• Quantification of PBDD/Fs in BDE-209&TeDB-DiPhOBz 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

Methods [1,5]

Results

ReferencesAcknowledgementsFunding for this project was provided by the Chemical ManagementPlan (to R.J.L, S.W.K. and D.C.) and the National Science andEngineering Research Council (NSERC; to R.J.L). We thank S.G.Chu with assistance with the LC-ESI-ToF-MS analysis.

IntroductionDue to their ability to reduce flammability and hinder fire ignition in theproducts that contain them, brominated flame retardants (BFRs),including 2,2’,3,3’,4,4’,5,5’,6,6’-decaBDE (BDE-209), have beenwidely used in various commercial products such as furniture, textiles,plastics, paints, and electronic appliances.

Tetradecabromo-1,4-diphenoxybenzene (TeDB-DiPhOBz) is ahalogenated polyphenyl ether (HPE) that is a replacement for BDE-209, and is the main constituent of commercial technical formulations.A 1973 US Patent (US3760003A) to the Dow Chemical Companydetailed the production of HPEs that included TeDB-DiPhOBz. In1986, Albermarle Corp. acquired Dow’s bromine business. AlthoughAlbermarle has claimed that production of their major TeDB-DiPhOBz-based BFR, SAYTEX-120, was phased-out and discontinuedcommercially in January 2011, we know that SAYTEX 120-relatedmixtures 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 non-volatile, and due to their log octanol-water partition coefficients of >10,have low bioavailability or potential to bioaccumulate. When exposedto UV-A, -B, -C, or natural sunlight, we recently showed that TeDB-DiPhOBz can undergo rapid photolysis and degrade via stepwise,reductive debromination [1]. When exposed to natural sunlight, half-lives of TeDB-DiPhOBz ranged from 4.9 to 7.4 min. Similarly, whensunlight irradiated, BDE-209 can be photolytically degraded [1,2], witha half-life in n-hexane (1% THF) of 5.3 min. BDE-209 has also beenmetabolically degraded to less brominated, more bioaccumulative,and potentially more toxic compounds in diet-exposed Americankestrels or via silastic implants with European starlings [3,4]. To ourknowledge, there are publications on biological effects in any biota asa result of exposure to TeDB-DiPhOBz or its degradation by-products.

Study Objectives [5]:Using an in vitro CEH assay and an Avian ToxChip polymerasechain reaction (PCR) array [6], examine mRNA expressionalterations following exposure to TeDB-DiPhOBz and BDE-209 ortheir photo-transformed by-products, generated in situ as aresult of natural sunlight irradiation.

Chicken Embryonic Hepatocyte (CEH) screening assay [6]

Cell Viability (Cytotoxicity)

&mRNA

expression

Avian ToxChip Polymerase Chain Reaction Array [6]Gene functions include:Phase I and II metabolismThyroid hormone pathwayLipid/cholesterol metabolismOxidative stressImmune responseCell death

Determination of TeDB-DiPhOBz and debrominated by-products of sunlight irradiated (SI) TeDB-DiPhOBz wascarried out using an Agilent 1200 liquid chromatographic(LC) system, coupled with an Agilent 6250A quadrupole-time-of-flight mass spectrometer (Q-TOF)-MS.

Determination of BDE-209 and the debrominated by-products of sunlight irradiated (SI) BDE-209 was carriedout using an Agilent gas chromatograph (GC) 6890coupled with a 5973 quadrupole mass spectrometer (MS)detector.

Methods

TeDB-DiPhOBz and BDE-209, in solid powder form, were kindlysupplied by Wellington Laboratories (Guelph, ON, Canada).

TeDB-DiPhOBz and BDE-209 powder was dissolved in 30 % THF/n-hexane solution (and also BDE-209 in methanol or n-hexanes toassess solvent effects) to achieve a final, nominal concentration of300 µM. The same volume of 30% THF/n-hexane was used as thesolvent control for the present study.

The sunlight irradiation was conducted from December 24, 2013 toJanuary 14, 2014 in Ottawa (21 days), and the location coordinateswere 45o40'06''N and 75o74'22''W.

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

Figure 1. GC-MS (ECNI) total ion mass chromatogram of 47 PBDE congeners in a standardmixture (black) and in the sunlight irradiated (SI-) BDE-209 (50 μM) after a 21-day sunlightirradiation in 30% THF/n-hexane (red).

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.

[1] D. Chen, R.J. Letcher, L.T. Gauthier, S.G. Chu, Environ. Sci. Technol. 47 (2013) 1373.[2] H.M. Stapleton, N.G. Dodder, Environ. Toxicol. Chem. 27 (2008) 306.[3] R.J. Letcher, S.C. Marteinson, K.J. Fernie, Environ. Int. 63 (2014) 182.[4] E. van den Steen, A. Covaci, V.L. Jaspers, T. Dauwe, S. Voorspoels, M. Eens, R. Pinxten, Environ. Pollut. 148 (2007) 648.[5] 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).[6] E. Porter, D. Crump, C. Egloff, S. Chiu, S.W. Kennedy, Environ. Toxicol. Chem. 33 (2014) 573.

Conclusions

Knowledge Transfer and Exchange• 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 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.

• 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)

• 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).

• 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).

Thank you! Poster number: RP147