Supplemental Information Characterization of Adipogenic Activity of House Dust Extracts and Semi-Volatile Indoor Contaminants in 3T3-L1 Cells Christopher D. Kassotis, Kate Hoffman, and Heather M. Stapleton* Nicholas School of the Environment, Duke University, Durham, NC 27708 *Corresponding author: heather.stapleton@duke.edu Contents Figure S1, Rosiglitazone Positive Control Response Figure S2, Parabens Induce No Apparent Adipogenic Activity Figure S3, Strobins Inhibit Cell Viability Figure S4, Triglyceride Accumulation and PPARγ Potency Correlation Table S1, Chemical Purity and Ordering Information Table S3, List of Putative Adipogenic Pathways for Tested Chemicals Page S2 Page S3 Page S4 Page S5 Page S6 Page S8 S1
Figure S1: Rosiglitazone Positive Control Response. Zenbio 3T3-L1 cells were differentiated as described in Methods and assessed for adipocyte differentiation (Nile Red staining of lipid accumulation) and cell proliferation (Hoechst staining) after ten days of differentiation while exposed to a rosiglitazone positive control from 0.1 nm to 1.0 μm in concentration. Percent raw triglyceride accumulation per well relative to maximal rosiglitazone response (A), increase (cell proliferation) or decrease (potential cytotoxicity) in DNA content relative to vehicle control (B), and percent normalized triglyceride accumulation per cell relative to maximal rosiglitazone response (normalized to DNA content) (C), fold induction of triglyceride accumulation responses over vehicle control for rosiglitazone (D). Data presented as mean ± SEM from three independent experiments. * indicates lowest concentration with significant increase in triglyceride over vehicle control or cell proliferation/cytotoxicity relative to vehicle control, p<0.05, as per linear mixed model in SAS 9.4. S2
Figure S2: Parabens Induce No Apparent Adipogenic Activity. Zenbio 3T3-L1 cells were differentiated as described in Methods and assessed for adipocyte differentiation (Nile Red staining of lipid accumulation) and cell proliferation (Hoechst staining) after ten days of differentiation while exposed to various parabens from 0.1 nm to 10 μm in concentration. Percent raw triglyceride accumulation per well relative to maximal response for rosiglitazone (A), increase (cell proliferation) or decrease (potential cytotoxicity) in DNA content relative to vehicle control (B), and percent normalized triglyceride accumulation per cell relative to maximal rosiglitazone response (normalized to DNA content) (C). Data presented as mean ± SEM from three independent experiments. * indicates lowest concentration with significant increase in triglyceride over vehicle control or cell proliferation/cytotoxicity relative to vehicle control, p<0.05, as per linear mixed model in SAS 9.4. S3
Figure S3: Strobins Inhibit Cell Viability. Zenbio 3T3-L1 cells were differentiated for ten days as described in Methods and assessed for adipocyte differentiation and cell proliferation after exposure to pyraclostrobin, azoxystrobin, trifloxystrobin, or fluoxastrobin. Following these assays, cell viability was assessed using the CellTiter Glo assay (Promega) as described in the methods, using the cell s metabolic potential (ATP production) as a marker for metabolically viable cells. Data presented as mean ± SEM from three independent experiments. * indicates lowest concentration with significant decrease in cell viability relative to vehicle control, p<0.05, as per linear mixed model in SAS 9.4. S4
Figure S4: Triglyceride Accumulation and PPARγ Activity Correlations. Spearman s correlations of: potencies for Zenbio 3T3-L1 cell triglyceride accumulation via EC 20 (concentration of a chemical that exhibits 20% of its maximal activity; see Table 1) and using the GeneBlazer PPARγ reporter gene assay (human embryonic kidney cells; HEK293H) activation potencies (EC 20 values) published from our lab previously (A), and efficacies for each of these compounds in each assay based on maximal response relative to the rosiglitazone positive control (B). See Fang et al. 2015, Activation of Human Peroxisome Proliferator Activated Nuclear Receptors (PPARγ1) by Semi-Volatile Compounds (SVOCs) and Chemical Mixtures in Indoor Dust. Chemicals included in analysis: triphenyl phosphate, tris (4-butyl-phenyl phosphate), isopropylated triaryl phosphate mixture, Firemaster 550 mixture, tri-(2-butoxyethyl) phosphate, 2,4,6-tribromophenol, tetrachlorobisphenol A, tetrabromobisphenol A, triclosan, tetrabromodiphenyl ether, 6-hydroxy-tetrabromodiphenyl ether, di-isobutyl phthalate, dibutyl phthalate, benzyl butyl phthalate, bis (2-ethylhexyl) phthalate, and rosiglitazone. S5
Table S1. Chemical Purity and Ordering Information. Chemical Acronym CAS # Supplier Catalog # Purity Polybrominated Diphenyl Ethers (PBDEs) 2,2,4,4 -tetrabromodiphenyl ether BDE-47 5436-43-1 Accustandard BDE-047N 100.0% 6-hydroxide-2,2,4,4 -tetrabromodiphenyl ether 6-OH BDE- N/A Accustandard HBDE- 98.6% 47 4005N 2,2',4,4',5-pentabromodiphenyl ether BDE-99 32534-81-9 Accustandard BDE-099N 99.2% Decabromodiphenyl ether BDE-209 1163-19-5 Sigma 194425 98.0% Brominated Flame Retardants (BFRs) 2-ethyl hexyl-2,3,4,5-tetrabromobenzoate TBB 183658-27-7 Accustandard FRS-041N 97.7% Bis (2-ethyl hexyl)-2,3,4,5-tetrabromophthalate TBPH 26040-51-7 SCBT sc-391703 >97.0% Tris (2,3-dibromopropyl) isocyanurate TDBPIC 52434-90-9 Sigma 269999 99.0% Organophosphate Flame Retardants (OPFRs) Tris (1-chloro-isopropyl) phosphate (mix of isomers) TCPP 13674-84-5 Sigma 32952 97.5% Tris (2,4-dichloro-isopropyl) phosphate TDCPP 13674-87-8 Sigma 32951 95.7% triphenyl phosphate TPP 115-86-6 Sigma 442829 99.9% tris (4-butyl-phenyl) phosphate TBPP 78-33-1 Aldrich S356336 N/A Tri-iso-butyl-phosphate TiBP 126-71-6 Millipore 818606 99.7% Tri-n-butyl-phosphate TnBP 126-73-8 Sigma 240494 99.6% Tri-(2-butoxyethyl)-phosphate TBOEP 78-51-3 Sigma 130591 96.1% Tert-buty-phenyl, diphenyl phosphate TBPDP 56803-37-3 TRC B693910 98.0% Firemaster 550 (mixture) FM550 - Chemtura N/A N/A isopropylated triaryl phosphates (mixture) ITP - Chemtura N/A N/A Phthalates Bis(2-ethylhexyl) phthalate DEHP 117-81-7 Sigma 36735 99.7% dibutyl phthalate DBP 84-74-2 Sigma 524980 99.3% di-isobutyl phthalate DiBP 84-69-5 ChemService N11728 99.5% S6
benzyl butyl phthalate BBP 85-68-7 Sigma 442503 99.9% di-ethyl phthalate DEP 84-66-2 Sigma 524972 99.9% Bis (2-ethylhexyl) terephthalate DEHT 6422-86-2 Sigma 525189 98.0% Pesticides Chlorpyrifos 2921-88-2 ChemService 45395 99.5% Permethrin (mix of isomers) 52645-53-1 Sigma 45614 98.1% Cypermethrin (mix of isomers) 52315-07-8 Sigma 36128 94.3% Chlorfenapyr 122453-73-0 Sigma 16785 99.6% Fipronil 120068-37-3 Sigma 16785 99.6% Pyraclostrobin 175013-18-0 Sigma 33696 99.9% Azoxystrobin 131860-33-8 SCBT sc-364349 99.0% Fluoxastrobin 361377-29-9 SCBT sc-235159 99.9% Trifloxystrobin 141517-21-7 SCBT sc-229576 99.0% Perfluorinated Chemicals (PFCs) 1H, 1H,2H,2H-Perfluorodecyl acrylate 8:2 FTAcr 27905-45-9 Synquest 2324-3-41 99.0% 2-Perfluorooctyl ethanol 8:2 FTOH 678-39-7 Sigma 532789 99.5% 2-Perfluorohexyl ethanol 6:2 FTOH 647-42-7 Sigma 370533 99.4% Parabens Butyl paraben 94-26-8 USP RS 1084000 99.9% Ethyl paraben 120-47-8 Sigma PHR1011 100.0% Methyl paraben 99-76-3 Sigma PHR1012 99.5% Propyl paraben 94-13-3 Sigma P53357 99.3% Phenols 2,4,6-tribromophenol 2,4,6 TBP 118-79-6 Sigma 137715 99.5% Triclosan TCS 3380-34-5 Sigma 72779 99.7% Identifying information for all chemicals tested herein, including acronyms, chemical abstract service numbers (CAS), ordering information (catalog number and company), and purities. TRC = Toronto Research Chemicals; USP RS = USP Reference Standards; SCBT = Santa Cruz Biotechnology S7
Table S2. List of Putative Adipogenic Pathways for Tested Chemicals Chemical Acronym CAS # Adipogenic Activity PPARγ Activity Other Adipogenic Pathways Polybrominated Diphenyl Ethers (PBDEs) 2,2,4,4 -tetrabromodiphenyl ether BDE-47 5436-43-1 + + TR 6-hydroxide-2,2,4,4 -tetrabromodiphenyl ether 6-OH N/A - - Unlisted BDE-47 2,2',4,4',5-pentabromodiphenyl ether BDE-99 32534-81- - TR, AR 9 Decabromodiphenyl ether BDE-209 1163-19-5 - - Brominated Flame Retardants (BFRs) 2-ethyl hexyl-2,3,4,5-tetrabromobenzoate TBB 183658- - Unlisted 27-7 Bis (2-ethyl hexyl)-2,3,4,5-tetrabromophthalate TBPH 26040-51- +++ GR 7 Tris (2,3-dibromopropyl) isocyanurate TDBPIC 52434-90- + Unlisted 9 Organophosphate Flame Retardants (OPFRs) Tris (1-chloro-isopropyl) phosphate (mix of isomers) TCPP 13674-84- - - 5 Tris (2,4-dichloro-isopropyl) phosphate TDCPP 13674-87- 8 - x AR, RAR, RXR, FXR, LXR Triphenyl phosphate TPP 115-86-6 ++ ++ PXR, LDLR, LXR, FXR Tris (4-butyl-phenyl) phosphate TBPP 78-33-1 ++ + Unlisted Tri-iso-butyl-phosphate TiBP 126-71-6 - x PXR, LXR Tri-n-butyl-phosphate TnBP 126-73-8 + x PXR, LXR, FXR Tri-(2-butoxyethyl)-phosphate TBOEP 78-51-3 ++ + PXR, RAR, TR, FXR Tert-buty-phenyl, diphenyl phosphate TBPDP 56803-37- +++ +++ RAR, LDLR, PXR, LXR 3 Firemaster 550 (mixture) FM550 - +++ ++ Unlisted S8
Isopropylated triaryl phosphates (mixture) ITP - +++ ++ Unlisted Phthalates Bis(2-ethylhexyl) phthalate DEHP 117-81-7 ++ x PXR, GR Dibutyl phthalate DBP 84-74-2 +++ + TR, PXR, RXR, GR Di-isobutyl phthalate DiBP 84-69-5 + + RXR, RAR, LXR Benzyl butyl phthalate BBP 85-68-7 + ++ FXR, PXR Di-ethyl phthalate DEP 84-66-2 - - Bis (2-ethylhexyl) terephthalate DEHT 6422-86-2 + RXR, PXR Perfluoralkylated substances (PFAS) 1H, 1H,2H,2H-Perfluorodecyl acrylate 8:2 FTAcr 27905-45- ++ Unlisted 9 2-Perfluorooctyl ethanol 8:2 FTOH 678-39-7 - LXR, PXR 2-Perfluorohexyl ethanol 6:2 FTOH 647-42-7 - LXR, GR Parabens Butyl paraben 94-26-8 - x RXR, RAR, FXR, PXR Ethyl paraben 120-47-8 - - Methyl paraben 99-76-3 - RXR Propyl paraben 94-13-3 - x RXR, PXR, RAR, LXR Pesticides Chlorpyrifos 2921-88-2 + x LXR, PXR, RXR, LDLR Permethrin (mix of isomers) 52645-53- +++ x PXR, RAR, GR, LXR 1 Cypermethrin (mix of isomers) 52315-07- ++ x PXR, RAR, LXR 8 Chlorfenapyr 122453-73-0 - x RXR, FXR, AR, PXR, RAR, LXR Fipronil 120068-37-3 - LXR, PXR, TR, RAR, GR Pyraclostrobin 175013- +++ TR, FXR, AR, RAR, S9
18-0 LXR Azoxystrobin 131860-33-8 + TR, FXR, AR, CAR, PXR, RAR Fluoxastrobin 361377-29-9 ++ TR, FXR, AR, ROR, PXR, RAR Trifloxystrobin 141517-21-7 + TR, FXR, AR, CAR, PXR Phenols 2,4,6-tribromophenol 2,4,6 TBP 118-79-6 - + TR, FXR, RAR Triclosan TCS 3380-34-5 - - RXR, AR, LDLR, PXR, LXR Descriptive receptor pathway activity assessment for each tested chemical. Qualitative assessment of adipogenic activity (triglyceride accumulation) is provided for each chemical based on the testing presented herein. PPARγ activity is provided for chemicals that have been tested by our laboratory, Fang et al. 2015, and from Janesick et al. 2016. - = inactive, 0-8% activity; + = low activity, 9-25% activity; ++ = medium activity, 26-50%, +++ = high activity, 51% or greater. x = chemicals are reported to disrupt PPARγ (either agonism or antagonism) based on ToxCast assays, available at: https://actor.epa.gov/dashboard/. Other adipogenic pathways known to influence adipogenesis and/or lipogenesis as determined through assessment of ToxCast reporter gene assay results (https://actor.epa.gov/dashboard/). FXR = farnesoid X receptor, AR = androgen receptor, TR = thyroid receptor, RXR = retinoid X receptor, LXR = liver X receptor, GR = glucocorticoid receptor, LDLR = low-density lipoprotein receptor, PXR = pregnane X receptor, RAR = retinoic acid receptor. Unlisted chemicals are not currently listed in the ToxCast database. S10