David
1,*
Saunders ,
Rishikesh
1
Mankidy ,
Eric
1
Higley ,
John P.
1,2,3
Giesy
1. Toxicology Centre, University of Saskatchewan, Saskatoon, SK, Canada 2. Department of Veterinary Biomedical Sciences, University of Saskatchewan, Saskatoon, SK, Canada
3. Department of Biology and Chemistry, City University of Hong Kong, Kowloon, Hong Kong, China
Introduction
H295R Assay: Determination of NBFR induced changes in estradiol concentrations compared to
DMSO controls.
The H295R assay expresses the endogenous components that comprise the intracellular
biochemical pathway of steroidogenesis. Data derived from the assay represent a holistic
measurement of final hormonal concentrations
Results
There are over 75 current use brominated flame retardants (BFRs)
Total international production volumes continue to increase
In 2005: 8% increase from 310 000 tonnes in 2000
Yeast androgen screen: Determination of receptor mediated induction or inhibition of androgenic
activity of three NBFRs
Penta- Octa- mixtures banned internationally
Full phase out of deca-BDE in North America (2013)
70
TBB
International regulations banning PBDEs, and the potential toxicities of current BFRs
have led to increased production of novel brominated flame retardants (NBFRs)
Three NBFRs with potentials for several sub-lethal endpoints:
30
20
40
Br
6
2.5
5
2
1.5
10
10
0
3
1
2
0.5
1
0
0
DMSO
5.E-02
5.E-03
5.E-04
0
HF
4
30
20
2-ethylhexyl tetrabromobenzoate (TBB) – Firemaster 550, Firemaster BZ-54
Bis-(2-ethylhexyl) tetrabromophthalate (TBPH) – FM 550, FM BZ-54, DP-45
1, 2, 5, 6-tetrabromocyclooctane (TBCO) – BC-48
CH3
Inhibition (%)
Inhibition (%)
50
40
3
TBPH
60
50
TBPH
7
Fold Change
60
Fold Change
Several major use BFRs, the PBDEs, have been listed as persistent organic pollutants
(POPs)
CH3
TBB
3.5
5.E-01 5.E-02 5.E-03 5.E-04 5.E-06 5.E-08 5.E-10
HF
1500
300
150
30
15
3
0.3
5.E-06
DMSO
30
15
3
1.5
4.5
0.03
TBCO
4
Br
90
TBCO
3.5
CH3
O
Br
Br
O
O
-
H3C
Br
O
Br
Br
Br
TBB
O
O
H3C
70
Inhibition (%)
H3C
Br
Br
TBPH
Fold Change
80
Br
Br
60
50
2.5
2
1.5
40
1
30
0.5
20
TBCO
3
0
10
DMSO
Detection of TBB & TBPH in the environment and biota:
HF
300
150
30
15
3
0.3
0.03
0.003
Fig. 1. Antiandrogenic activity of TBB, TBPH, and TBCO at several concentrations (mg/L). The antiandrogenic activity is presented as
inhibition (%) of b-galactosidase production (mean ± SE) compared to activated control cells (5 nM DHT). HF represents the
antiandrogenic activity of the positive control hydroxyflutamide (10 -5 nM). All dose responses are significantly different than activated
controls (p<0.05).
Global Atmospheric Passive Sampling (GAPS) Network:
Both compounds >60% all sampled sites
1.5
0.3
Fig. 3. The effects of TBB, TBPH, and TBCO exposure on relative estradiol hormone concentrations measured in the H295R cell assay.
Several concentrations (mg/L) of the the NBFRs were tested and data are presented as fold change (mean ± SE) compared to DMSO
controls. All dose responses are significantly different than activated controls (p<0.05).
Conclusions
Yeast estrogen screen: Determination of receptor mediated induction or inhibition of estrogenic
activity of three NBFRs
Few toxicological data exist for these three NBFRs:
TBPH is a structural analogue of a known EDC (DEHP)
80
TBB
80.00
(a)Indicates compounds might have cellular effects similar to known EDCs
60
Inhibition (%)
Inhibition (%)
TBPH
(1) The three NBFRs (TBB, TBPH, and TBCO) show significant antagonistic effects in
bothAR and ER receptor controlled systems.
70
70.00
60.00
To address the gap in toxicological data for these three NBFRs we have tested for
endocrine disrupting abilities, and TCDD-like effects using the YES/YAS, H295R, and
H4IIE bioassay systems
3
0
Integrated Atmospheric Depositio Network (IADN)
Detected at six locations near Great Lakes (2008-2010)
Calculated doubling time 1.1 years
Gentes et al. 2012 detected the compounds in Ring-billed gulls (St. Lawrence River)
Detected in 89% of birds - greatest detection frequency to date
15
50.00
40.00
30.00
50
(b)Activator compounds (E2 and DHT) introduced to system prior to NBFRs
indicating NBFRs displaced compounds from receptors
40
30
20
20.00
(2) TBPH and TBCO produced minimal changes in testosterone concentrations in the
H295R cell line (data not shown)
(a) It is likely to observe minimal changes in hormone concentrations by
inherent variability of biological systems
10
10.00
0
Methods
0.00
HT
5.E-01 5.E-02 5.E-03 5.E-04 5.E-06 5.E-08 5.E-10
80.00
TBCO
70.00
YES/YAS Assays
Inhibition (%)
60.00
Permisson to use the recombinant YES/YAS cells was kindly provided by John. P Sumpter (Brunel University).
Yeast cell maintenance and growth was conducted as previously described (Sohoni, Sumpter. 1998). Yeast cells
were plated at a volume of 200 mL and a density of 1.0 absorbance unit by dilution of the growth stock. DMSO
was used as a carrier solvent and did not exceed 1% v/v. Yeast cells were dosed with 2 mL of the NBFR solutions
and incubated for 48 hr on an orbital shaker at 32 ◦C. To determine the inhibitory properties of the NBFRs, dosed
wells were previously exposed to activating compounds, 3nM E2 (YES) and 5 nM DHT (YAS). Inhibitory controls
were exposed to receptor specific antagonistic compounds, hydroxy tamoxifen [10-6 nM] (YES) and hydroxy
flutamide [10-5 nM] (YAS). Total b-galactosidase production was assayed using absorbances at 570 nm and 690
nm, where Total Activity = 570nm – 690nm.
H295R Assay
The H295R human cell line was grown as described previously (Higley et al., 2010). All exposures were
conducted in 24-well culture plates with a cell concentration of 300,000 cells/mL. Cells were exposed to the
NBFRs for 48 h. DMSO was used as a carrier solvent and did not exceed 0.1% v/v. Test plates included four
chemical concentrations, a solvent control (DMSO), as well as forskolin (10 mM) and prochloraz (3 mM) controls
in quadruplicate. At the end of each experiment, culture medium was collected and stored at -80◦C. Hormonal
analysis was conducted using ELISA following a liquid-liquid extraction of the media (Chang et al., 2009).
(3) TBPH, TBB, and TBCO resulted in significant increases in estradiol concentrations at
all exposures
50.00
(a) TBPH, the brominated analogue of DEHP, demonstrates similar endocrine
disrupting effects
40.00
30.00
20.00
(b)Potential for toxic endpoints similar to DEHP
Interactions with the peroxisome proliferator-activated receptor (PPARa)
Potential for induction of hepatic lesions, potential hepatic tumours
10.00
0.00
HT
30
15
3
0.3
0.03
0.003
Fig. 2. Antiestrogenic activity of TBB, TBPH, and TBCO at several concentrations (mg/L). The antiestrogenic activity is presented as
inhibition (%) of b-galactosidase production (mean ± SE) compared to activated control cells (3 nM E2). HT represents the
antiestrogenic activity of the positive control hydroxytamoifen(10-6 nM). All dose responses are significantly different than activated
controls (p<0.05).
Acknowledgements
Stipend support provided by the NSERC CREATE program
Facilities support by the Toxicology Graduate Program
Part of this research is supported by the Canada Research Chair Program (J.P. Giesy)
NSERC Discovery Grant (Project # 326415-07; J.P. Giesy)
References
Higley et al. 2010. Environ. Sci. Pollut. Res.
Chang et al. 2009. Journal of Hazardous
Sohoni et al. 1998. Journal of Endocrinology
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