Supplemental Information. Characterization of Adipogenic Activity of House Dust Extracts and Semi-Volatile Indoor. Contaminants in 3T3-L1 Cells

Transcription

1 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 *Corresponding author: 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

2 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

3 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

4 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

5 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

6 Table S1. Chemical Purity and Ordering Information. Chemical Acronym CAS # Supplier Catalog # Purity Polybrominated Diphenyl Ethers (PBDEs) 2,2,4,4 -tetrabromodiphenyl ether BDE Accustandard BDE-047N 100.0% 6-hydroxide-2,2,4,4 -tetrabromodiphenyl ether 6-OH BDE- N/A Accustandard HBDE- 98.6% N 2,2',4,4',5-pentabromodiphenyl ether BDE Accustandard BDE-099N 99.2% Decabromodiphenyl ether BDE Sigma % Brominated Flame Retardants (BFRs) 2-ethyl hexyl-2,3,4,5-tetrabromobenzoate TBB Accustandard FRS-041N 97.7% Bis (2-ethyl hexyl)-2,3,4,5-tetrabromophthalate TBPH SCBT sc >97.0% Tris (2,3-dibromopropyl) isocyanurate TDBPIC Sigma % Organophosphate Flame Retardants (OPFRs) Tris (1-chloro-isopropyl) phosphate (mix of isomers) TCPP Sigma % Tris (2,4-dichloro-isopropyl) phosphate TDCPP Sigma % triphenyl phosphate TPP Sigma % tris (4-butyl-phenyl) phosphate TBPP Aldrich S N/A Tri-iso-butyl-phosphate TiBP Millipore % Tri-n-butyl-phosphate TnBP Sigma % Tri-(2-butoxyethyl)-phosphate TBOEP Sigma % Tert-buty-phenyl, diphenyl phosphate TBPDP TRC B % 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 Sigma % dibutyl phthalate DBP Sigma % di-isobutyl phthalate DiBP ChemService N % S6

7 benzyl butyl phthalate BBP Sigma % di-ethyl phthalate DEP Sigma % Bis (2-ethylhexyl) terephthalate DEHT Sigma % Pesticides Chlorpyrifos ChemService % Permethrin (mix of isomers) Sigma % Cypermethrin (mix of isomers) Sigma % Chlorfenapyr Sigma % Fipronil Sigma % Pyraclostrobin Sigma % Azoxystrobin SCBT sc % Fluoxastrobin SCBT sc % Trifloxystrobin SCBT sc % Perfluorinated Chemicals (PFCs) 1H, 1H,2H,2H-Perfluorodecyl acrylate 8:2 FTAcr Synquest % 2-Perfluorooctyl ethanol 8:2 FTOH Sigma % 2-Perfluorohexyl ethanol 6:2 FTOH Sigma % Parabens Butyl paraben USP RS % Ethyl paraben Sigma PHR % Methyl paraben Sigma PHR % Propyl paraben Sigma P % Phenols 2,4,6-tribromophenol 2,4,6 TBP Sigma % Triclosan TCS Sigma % 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

8 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 TR 6-hydroxide-2,2,4,4 -tetrabromodiphenyl ether 6-OH N/A - - Unlisted BDE-47 2,2',4,4',5-pentabromodiphenyl ether BDE TR, AR 9 Decabromodiphenyl ether BDE Brominated Flame Retardants (BFRs) 2-ethyl hexyl-2,3,4,5-tetrabromobenzoate TBB Unlisted 27-7 Bis (2-ethyl hexyl)-2,3,4,5-tetrabromophthalate TBPH GR 7 Tris (2,3-dibromopropyl) isocyanurate TDBPIC Unlisted 9 Organophosphate Flame Retardants (OPFRs) Tris (1-chloro-isopropyl) phosphate (mix of isomers) TCPP Tris (2,4-dichloro-isopropyl) phosphate TDCPP x AR, RAR, RXR, FXR, LXR Triphenyl phosphate TPP PXR, LDLR, LXR, FXR Tris (4-butyl-phenyl) phosphate TBPP Unlisted Tri-iso-butyl-phosphate TiBP x PXR, LXR Tri-n-butyl-phosphate TnBP x PXR, LXR, FXR Tri-(2-butoxyethyl)-phosphate TBOEP PXR, RAR, TR, FXR Tert-buty-phenyl, diphenyl phosphate TBPDP RAR, LDLR, PXR, LXR 3 Firemaster 550 (mixture) FM Unlisted S8

9 Isopropylated triaryl phosphates (mixture) ITP Unlisted Phthalates Bis(2-ethylhexyl) phthalate DEHP x PXR, GR Dibutyl phthalate DBP TR, PXR, RXR, GR Di-isobutyl phthalate DiBP RXR, RAR, LXR Benzyl butyl phthalate BBP FXR, PXR Di-ethyl phthalate DEP Bis (2-ethylhexyl) terephthalate DEHT RXR, PXR Perfluoralkylated substances (PFAS) 1H, 1H,2H,2H-Perfluorodecyl acrylate 8:2 FTAcr Unlisted 9 2-Perfluorooctyl ethanol 8:2 FTOH LXR, PXR 2-Perfluorohexyl ethanol 6:2 FTOH LXR, GR Parabens Butyl paraben x RXR, RAR, FXR, PXR Ethyl paraben Methyl paraben RXR Propyl paraben x RXR, PXR, RAR, LXR Pesticides Chlorpyrifos x LXR, PXR, RXR, LDLR Permethrin (mix of isomers) x PXR, RAR, GR, LXR 1 Cypermethrin (mix of isomers) x PXR, RAR, LXR 8 Chlorfenapyr x RXR, FXR, AR, PXR, RAR, LXR Fipronil LXR, PXR, TR, RAR, GR Pyraclostrobin TR, FXR, AR, RAR, S9

10 18-0 LXR Azoxystrobin TR, FXR, AR, CAR, PXR, RAR Fluoxastrobin TR, FXR, AR, ROR, PXR, RAR Trifloxystrobin TR, FXR, AR, CAR, PXR Phenols 2,4,6-tribromophenol 2,4,6 TBP TR, FXR, RAR Triclosan TCS 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 = 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: Other adipogenic pathways known to influence adipogenesis and/or lipogenesis as determined through assessment of ToxCast reporter gene assay results ( 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