INVESTIGATING THE MECHANISMS OF

TOXICITY OF CURRENT FLAME

RETARDANTS Boris Krivoshiev

Prof. Steven Husson

Prof. Ronny Blust

Introduction

• Previously used FRs toxic and have thus been tightly regulated

• Studies showed toxicity ranging from carcinogenesis to endocrine disruption

What do we know?

What is lacking?

Toxicological data for current FRs

Mechanistic insight into how these compounds exert their toxic phenotypes

What now?

• Establish potential toxicological and mechanistic profiles of current FRs

Aim

Monitoring in vitro responses Prokaryotic stress gene profiling

Eukaryotic stress gene profiling

Endocrine disruption

How?

Develop an unbiased mechanistic overview Differential proteomics

Prokaryotic Stress Gene Assay

Stress Response Stress Promoters

Oxidative stress KatG, Zwf, Soi28, Nfo Protein perturbation ClpB

DNA damage RecA, UmuDC, Ada

Membrane damage MicF, OsmY

Growth arrest UspA , SfiA

Class Chemical Name Abbreviation CAS Number

OPFR Tris (1-chloro-2-propyl) phosphate TCPP 13674-84-5

Triphenyl phosphate TPP 115-86-6

Tris(2-butoxyethyl) phosphate TBEP 78-51-3

Tris (1,3-dichloro-2-propyl) phosphate TDCPP 13674-87-8

Tri(2-chloroethyl)phosphate TCEP 115-96-8

Tris(butyl) phosphate TnBP 126-73-8

Triethyl phosphate TEP 78-40-0

9,10-Dihydro-9-oxa-10-phosphaphenanthrene DOPO 35948-25-5

NBFR Hexabromocyclododecane HBCD 3194-55-6

Tetrabromobisphenol A TBBPA 79-94-7

Bis(2-ethylhexyl) tetrabromophthalate TBPH 26040-51-7

Tris(2,3,-dibromopropyl) isocyanurate TBC 52434-90-9

TBBPA

Significance of inductions of stress genes by compound at highest concentration. Results are the average of three replicates. *p<0.05 **p<0.01 ***p<0.001 ****p<0.00001.

• Significant induction of a majority of stress genes by a majority of compounds

• No correlation between inductions and class of compound

Protein Damage

Class Abbreviation KatG Zwf Soi Nfo ClpB RecA UmuDC Ada MicF OsmY UspA SfiA

OPFR TCPP ** *** ** * ** **** ** * ** **TPP ** ** **** ** ** *TBEP *** * * ** ** **** ***TDCPP ** * ** *** ** * **TCEP ** ** ** ** ** ** *** *** **TnBP * * * ** ** ** **** *TEP ** **** * ** **DOPO **** *** **** ** *** *** *** ***

NBFR HBCD *** ** ** * *** *

TBBPA ** ** ** **** ***TBPH * * *** * ** **TBC *** *** ** ** ** *** * * ** *** **** *

Oxidative Stress DNA Damage Membrane Damage Growth Arrest

C lp B

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1

2

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T B B P A

T D C P P

T nB P* *

* ** * * *

R e c A

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-6 -4 -2 0 2 4

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1

2

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T C E P

T B B P A* *

* * * *

M ic F

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1 2 3 4

T P P

T B E P

T D C P P

T C E P

T nB P

T B B P A

* * *

* *

* ** * *

* *

• DNA damage, protein perturbation, and loss in membrane integrity

• OPFRs over-represented as BFRs hydrophilic therefore cannot be tested at same concentrations as OPFRs

• Majority of genes induced

• Genes of interest: ClpB, RecA, MicF

▫ Protein perturbation, DNA damage, & loss in membrane integrity

• TEP & DOPO induced the least amount of genes even at 5mM, likely to be the least toxic of all compounds tested ▫ Unfortunately, compounds are not used extensively

yet

• Manuscript in preparation

• Recent focus given its role in a variety of adverse effects (lower sperm count, poor fertility, neuronal toxicity, behavioural effects, etc)

• Previously used BFRs shown to affect T4, E2, and androgen networks

• Information on currently used FRs incomplete and contradictory

Estrogenicity

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%]

-6 -2

-5 0

0

5 0

1 0 0

1 5 0

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E 2

T P P

T B B P A

T D C P P

**

*

E2 TPP TDCPP TBBPA

EC50 (µM) 2.29x10-6 66.31 12.96 227.7

• TPP, TBBPA, TDCPP estrogenic • However, EC50 values of compounds are several orders of

magnitudes greater than E2 • EC50 values are much higher than likely to experience in

environment, biologically relevant?

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E 2

F as lo dex

T B E P

T nB P

T D C P P*

**

E2 Faslodex TDCPP TBEP TnBP

IC50 (µM) 2.12x10-6 4.57x10-2 53.79 454.6 638.9

• TBEP, TnBP, and TDCPP anti-estrogenic

• TPP, TBBPA, & TDCPP estrogenic

• TBEP, TnBP, & TDCPP anti-estrogenic

• Concentrations are much higher than likely to experience in environment

• Don’t know whether this is receptor or ligand-mediated

▫ Need to conduct steroidogenesis assay in H295R cells

• How can a compound be both estrogenic and anti-estrogenic?

▫ Interacting with E2?

Differential Proteomics

• Need to develop an understanding of the modes-of-action of these FRs to exert their toxic phenotypes

• Differential proteomics approach ▫ Result in understanding what the compounds do

in vitro, elucidate other toxicological effects that could not be investigated when monitoring endpoints, identification of effect biomarkers

• Will be conducted in HepG2 cells, thus giving insight into effects of metabolites in vitro

Construction of effected pathways

Identification of other potential toxicological effects

Identification of key proteins/other effector biomarkers

Thank you