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 Page 1