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Stacy Mann Presentation
- 1. Defining the Developmental Toxicity of Commonly Used Flame Retardant Chemicals Using Zebrafish Oregon State University Dept. of Molecular & Environmental Toxicology Tanguay Laboratory Stacy Mann University of California, Davis
- 2. Resist or inhibit the spread of fire Increase in the use of flammable plastics and electronics Fire safety standards Exposure via inhalation, ingestion, dermal contact Used in consumer products Chemicals used: Aryl Phosphate Esters: IPP-1, IPP-2, IPP-3, TEHP, TBEP Chlorinated Phosphate Esters: TCEP, TDCPP Other Brominated FRs: TBBPA, TBBPA-DBPE
- 3. Human health, hazard, and disease Easy to manipulate genetically and pharmacologically Zebrafish and mammalian brains share anatomical and functional features ~80% genetic homology with humans
- 4. Noyes et al. Mortality & Teratogenicity Morphology (LEL) Chemical Name Chemical Structure 24 hpf 120 hpf 24 hpf 120 hpf IPP-1* ✓ ✓ ✓ ✓ IPP-2* ✓ ✓ ✓ IPP-3* ✓ ✓ ✓ ✓ TBBPA ✓ ✓ ✓ ✓ TBBPA-DBPE TBEP ✓ ✓ TCPP ✓ TDCPP ✓ ✓ ✓ ✓ TEHP ✓ ✓ ✓ *chemical is a complex mixture of numerous positional isomers & phenol groups may be mono-, di-, or tri-isopropylated
- 5. Expose embryos to chemical at different points in development to measure susceptibility to toxicity 6 hr10 min 1 day 5 days Time Point Development 6 hpf Before organogenesis 24 hpf Major organs exist Heartbeat Gene expression Organogenesis 48 hpf Circulatory system formed Behavioral development (swimming) Early touch reflex and motility Blood circulates Somites 72 hpf Metabolism Internal organs easily recognizable Morphogenesis complete 96 hpf Elongated body length 120 hpf All organs done forming
- 6. Tested 5 concentrations: 15 μM, 10 μM, 5 μM, 1 μM, 0.05 μM Control: 0 μM Time Points: 6 hpf, 24 hpf, 48 hpf, 72 hpf, 96 hpf N=32 animals/concentration 2 replicate plates (n=16) 22 end points 2 behavioral assays Embryonic Photomotor Response (EPR) Larval Photomotor Response (LPR) uM 15 10 5 1 0.05 0 15 10 5 1 0.05 0
- 7. Fertilization @ 0 hours Chorion Removal @ 4 hpf Load 96 well plates Parafilm & Wrap in Foil 24 hpf End Points End Point Abbreviation Mortality MO24 Delayed Progression DP24 Chemical added @ 6 24 48 72 96 24 hpf End Points End Point Abbreviation Mortality MO24 Delayed Progression DP24 Spontaneous Movement SM24 Notochord NC24
- 10. Concentration: 5 uM Control Chemical added at: 6 hpf 24 hpf 48 hpf 72 hpf 96 hpf
- 12. Concentration: 15 uM Control Chemical added at: 6 hpf 24 hpf 48 hpf 72 hpf 96 hpf
- 13. Noyes et al. My Data Chemical Mortality & Teratogenicity At 5 days Morphology At 5 days 6 hpf 24 hpf 48 hpf 72 hpf 96 hpf IPP-1 + + + + + + + IPP-2 + + + + − − + IPP-3 + + + + − + + TBBPA + + + + + + + TBPPA- DBPE − − − − − − − TBEP + + − − − − − TCPP − + − − − − − TCDPP + + + + − − − TEHP + + − − − − −
- 14. Developing zebrafish are sensitive to many FR chemicals There are clear differences in the developmental toxicity of these flame retardant chemicals The developmental time of chemical exposure influences the toxicity of some FRs Condition Chemical Developmental toxicity IS impacted by the time of chemical addition IPP-1 IPP-3 TBBPA TDCPP Developmental toxicity is NOT impacted by the time of chemical addition IPP-2 TBEP TBBPA-DBPE TCPP TEHP
- 15. Test additional flame retardant chemicals for stage specific toxicity Determine the amount of chemical taken up by the zebrafish Determine the mechanism that causes the axial defects following TBBPA exposure
- 16. Dr. Robert Tanguay Dr. Lisa Truong Gloria Garcia Greg Gonnerman Jane La Du Michael Simonich Carrie Barton Mitra Geier Dr. Craig Marcus Dr. Andrew Buermeyer
Editor's Notes
- Flame retardants are chemicals or other manufactured materials… Exposure to flame retardants occurs mainly through inhalation or ingestion of dust along with food and water contaminated with flame retardants. And exposure from dermal contact with contaminated soil and dust. Flame retardants are widely used in consumer products, such as furniture, textiles, electronics, and insulation. Also in children’s products including car seats and changing pads Children may be more likely to be exposed to flame retardants due to the amount of time spent on the floor and the higher level of hand-to-mouth contact. Some have been banned or voluntarily phased out by manufacturers due to health concerns, however they may still persist in the environment.
- Zebrafish is an important biological sensor and model for screening chemicals for human health hazard and disease. Are small, prolific spawners that are easy to manipulate genetically and pharmacologically. …Including well-conserved neuronal morphology and neurotransmitter systems
- Broad concentrations chosen based off of Pam’s data. Columns for concentrations to account for variability during behavioral assays. 2 plates for statistical significance due to variability from one fish to another.
- Dechorionate because chorion has limited permeability and protects the embryo from exposure to some chemicals
- TBBPA is the most widely used Brominated FR It is a brominated analog of BPA that is used primarily as a reactive flame retardant in printed circuit boards and also as an additive flame retardant in polymers. Pam added the chemical at 6 hpf Wanted to see if adding the chemical at different points in the development effected the toxicity of the chemical adding it later decreases the severity of the chemical Last column- Any effect: all the effects added together per fish 2nd to last: anything except mort More sublethal effects at 24 hr, more dead at 6 hr 6 hr so many signaling effects happening, killing them 24 hrs, less sensitive pathways, not killing them but showing adverse effects
- Pam tested from 6 hpf -120 hpf and this chemical did not show hits for morphology, teratogenicity, or mortality. Could have not been a hit due to chemical stability? Metabolism? Wanted to see if you add the chemical later in development would make a difference in the toxicity Results show that adding it later does not produce significant effects, so we are more confident that this chemical is simply not toxic to zebrafish
- The blue columns show whether there was at hit at any of the concentrations tested by Pam. + is a hit, - is not a hit. Grey shows my data and whether there was statistically significant data for that time point at any concentration I tested.
- Although zebrafish development shows to be sensitive to different FR chemicals, the toxicity of these chemicals are not necessarily coupled with when the chemical appears in their development The developmental time of chemical exposure influences the toxicity of some FRs