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Ligand binding and activation of PPARγ by Firemaster® 550: effects on adipogenesis and osteogenesis in vitro.

Pillai HK, Fang M, Beglov D, Kozakov D, Vajda S, Stapleton HM, Webster TF, Schlezinger JJ - Environ. Health Perspect. (2014)

Bottom Line: Our findings suggest that FM550 components bind and activate PPARγ.TPP likely is a major contributor to these biological actions.Given that TPP is ubiquitous in house dust, further studies are warranted to investigate the health effects of FM550.

View Article: PubMed Central - PubMed

Affiliation: Department of Environmental Health, Boston University, Boston, Massachusetts, USA.

ABSTRACT

Background: The use of alternative flame retardants has increased since the phase out of pentabromodiphenyl ethers (pentaBDEs). One alternative, Firemaster® 550 (FM550), induces obesity in rats. Triphenyl phosphate (TPP), a component of FM550, has a structure similar to that of organotins, which are obesogenic in rodents.

Objectives: We tested the hypothesis that components of FM550 are biologically active peroxisome proliferator-activated receptor γ (PPARγ) ligands and estimated indoor exposure to TPP.

Methods: FM550 and its components were assessed for ligand binding to and activation of human PPARγ. Solvent mapping was used to model TPP in the PPARγ binding site. Adipocyte and osteoblast differentiation were assessed in bone marrow multipotent mesenchymal stromal cell models. We estimated exposure of children to TPP using a screening-level indoor exposure model and house dust concentrations determined previously.

Results: FM550 bound human PPARγ, and binding appeared to be driven primarily by TPP. Solvent mapping revealed that TPP interacted with binding hot spots within the PPARγ ligand binding domain. FM550 and its organophosphate components increased human PPARγ1 transcriptional activity in a Cos7 reporter assay and induced lipid accumulation and perilipin protein expression in BMS2 cells. FM550 and TPP diverted osteogenic differentiation toward adipogenesis in primary mouse bone marrow cultures. Our estimates suggest that dust ingestion is the major route of exposure of children to TPP.

Conclusions: Our findings suggest that FM550 components bind and activate PPARγ. In addition, in vitro exposure initiated adipocyte differentiation and antagonized osteogenesis. TPP likely is a major contributor to these biological actions. Given that TPP is ubiquitous in house dust, further studies are warranted to investigate the health effects of FM550.

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Related in: MedlinePlus

Reporter and in vitro differentiation analyses of PPARγ activation by FM550. (A) Cos-7 cells were transiently transfected with human PPARG1 and PPRE x3-TK-luc, with either pcDNA3 or PPARγ-DN vectors. Transfected cultures were treated with vehicle (Veh; DMSO, reported as 10–2 μM) or FM550 (0.1–20 μg/mL; 0.2–50 μM) and incubated for 24 hr; reporter activation was assessed by luciferase expression and normalized by eGFP fluorescence. (B–C) Confluent BMS2 cultures were treated with Veh (DMSO, reported as 10–2 μM) or FM550 (0.1–10 μg/mL; 0.2–20 μM), and lipid accumulation (B) and perilipin expression (C) were quantified after 7 days. (A,B) Data are presented as mean ± SE of 3–7 biological replicates. (C) Data are representative of 3–7 biological replicates.*p < 0.05, and **p < 0.01, by ANOVA and Dunnett’s multiple comparisons test, compared with Veh treatment.
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f1: Reporter and in vitro differentiation analyses of PPARγ activation by FM550. (A) Cos-7 cells were transiently transfected with human PPARG1 and PPRE x3-TK-luc, with either pcDNA3 or PPARγ-DN vectors. Transfected cultures were treated with vehicle (Veh; DMSO, reported as 10–2 μM) or FM550 (0.1–20 μg/mL; 0.2–50 μM) and incubated for 24 hr; reporter activation was assessed by luciferase expression and normalized by eGFP fluorescence. (B–C) Confluent BMS2 cultures were treated with Veh (DMSO, reported as 10–2 μM) or FM550 (0.1–10 μg/mL; 0.2–20 μM), and lipid accumulation (B) and perilipin expression (C) were quantified after 7 days. (A,B) Data are presented as mean ± SE of 3–7 biological replicates. (C) Data are representative of 3–7 biological replicates.*p < 0.05, and **p < 0.01, by ANOVA and Dunnett’s multiple comparisons test, compared with Veh treatment.

Mentions: Assessment of PPARγ activation by FM550. To test the hypothesis that FM550 can activate PPARγ transcriptional activity and directly induce adipogenesis, we began by investigating the ability of FM550 to activate PPARγ-driven reporter activity. Cos7 cells were transfected with human PPARG1 and RXRA expression vectors and a PPRE-driven reporter construct and treated with vehicle or FM550. FM550 significantly induced PPARγ-driven reporter activity at concentrations ≥ 10 μg/mL (20 μM), with an EC50 (concentration required to produce 50% of maximal effect) of 47 μM (Figure 1A). The maximal FM550-induced activity of 5.7 ± 0.3-fold was less than the activity induced by a maximally efficacious concentration of rosiglitazone (1 μM; 11.3 ± 0.9-fold; EC50 of 0.02 μM; see Supplemental Material, Figure S2A). The specificity of reporter activity was determined by co-transfecting Cos7 cells with a DN-PPARγ expression vector. The presence of DN-PPARγ significantly reduced FM550-induced reporter expression (Figure 1A).


Ligand binding and activation of PPARγ by Firemaster® 550: effects on adipogenesis and osteogenesis in vitro.

Pillai HK, Fang M, Beglov D, Kozakov D, Vajda S, Stapleton HM, Webster TF, Schlezinger JJ - Environ. Health Perspect. (2014)

Reporter and in vitro differentiation analyses of PPARγ activation by FM550. (A) Cos-7 cells were transiently transfected with human PPARG1 and PPRE x3-TK-luc, with either pcDNA3 or PPARγ-DN vectors. Transfected cultures were treated with vehicle (Veh; DMSO, reported as 10–2 μM) or FM550 (0.1–20 μg/mL; 0.2–50 μM) and incubated for 24 hr; reporter activation was assessed by luciferase expression and normalized by eGFP fluorescence. (B–C) Confluent BMS2 cultures were treated with Veh (DMSO, reported as 10–2 μM) or FM550 (0.1–10 μg/mL; 0.2–20 μM), and lipid accumulation (B) and perilipin expression (C) were quantified after 7 days. (A,B) Data are presented as mean ± SE of 3–7 biological replicates. (C) Data are representative of 3–7 biological replicates.*p < 0.05, and **p < 0.01, by ANOVA and Dunnett’s multiple comparisons test, compared with Veh treatment.
© Copyright Policy - public-domain
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4216168&req=5

f1: Reporter and in vitro differentiation analyses of PPARγ activation by FM550. (A) Cos-7 cells were transiently transfected with human PPARG1 and PPRE x3-TK-luc, with either pcDNA3 or PPARγ-DN vectors. Transfected cultures were treated with vehicle (Veh; DMSO, reported as 10–2 μM) or FM550 (0.1–20 μg/mL; 0.2–50 μM) and incubated for 24 hr; reporter activation was assessed by luciferase expression and normalized by eGFP fluorescence. (B–C) Confluent BMS2 cultures were treated with Veh (DMSO, reported as 10–2 μM) or FM550 (0.1–10 μg/mL; 0.2–20 μM), and lipid accumulation (B) and perilipin expression (C) were quantified after 7 days. (A,B) Data are presented as mean ± SE of 3–7 biological replicates. (C) Data are representative of 3–7 biological replicates.*p < 0.05, and **p < 0.01, by ANOVA and Dunnett’s multiple comparisons test, compared with Veh treatment.
Mentions: Assessment of PPARγ activation by FM550. To test the hypothesis that FM550 can activate PPARγ transcriptional activity and directly induce adipogenesis, we began by investigating the ability of FM550 to activate PPARγ-driven reporter activity. Cos7 cells were transfected with human PPARG1 and RXRA expression vectors and a PPRE-driven reporter construct and treated with vehicle or FM550. FM550 significantly induced PPARγ-driven reporter activity at concentrations ≥ 10 μg/mL (20 μM), with an EC50 (concentration required to produce 50% of maximal effect) of 47 μM (Figure 1A). The maximal FM550-induced activity of 5.7 ± 0.3-fold was less than the activity induced by a maximally efficacious concentration of rosiglitazone (1 μM; 11.3 ± 0.9-fold; EC50 of 0.02 μM; see Supplemental Material, Figure S2A). The specificity of reporter activity was determined by co-transfecting Cos7 cells with a DN-PPARγ expression vector. The presence of DN-PPARγ significantly reduced FM550-induced reporter expression (Figure 1A).

Bottom Line: Our findings suggest that FM550 components bind and activate PPARγ.TPP likely is a major contributor to these biological actions.Given that TPP is ubiquitous in house dust, further studies are warranted to investigate the health effects of FM550.

View Article: PubMed Central - PubMed

Affiliation: Department of Environmental Health, Boston University, Boston, Massachusetts, USA.

ABSTRACT

Background: The use of alternative flame retardants has increased since the phase out of pentabromodiphenyl ethers (pentaBDEs). One alternative, Firemaster® 550 (FM550), induces obesity in rats. Triphenyl phosphate (TPP), a component of FM550, has a structure similar to that of organotins, which are obesogenic in rodents.

Objectives: We tested the hypothesis that components of FM550 are biologically active peroxisome proliferator-activated receptor γ (PPARγ) ligands and estimated indoor exposure to TPP.

Methods: FM550 and its components were assessed for ligand binding to and activation of human PPARγ. Solvent mapping was used to model TPP in the PPARγ binding site. Adipocyte and osteoblast differentiation were assessed in bone marrow multipotent mesenchymal stromal cell models. We estimated exposure of children to TPP using a screening-level indoor exposure model and house dust concentrations determined previously.

Results: FM550 bound human PPARγ, and binding appeared to be driven primarily by TPP. Solvent mapping revealed that TPP interacted with binding hot spots within the PPARγ ligand binding domain. FM550 and its organophosphate components increased human PPARγ1 transcriptional activity in a Cos7 reporter assay and induced lipid accumulation and perilipin protein expression in BMS2 cells. FM550 and TPP diverted osteogenic differentiation toward adipogenesis in primary mouse bone marrow cultures. Our estimates suggest that dust ingestion is the major route of exposure of children to TPP.

Conclusions: Our findings suggest that FM550 components bind and activate PPARγ. In addition, in vitro exposure initiated adipocyte differentiation and antagonized osteogenesis. TPP likely is a major contributor to these biological actions. Given that TPP is ubiquitous in house dust, further studies are warranted to investigate the health effects of FM550.

Show MeSH
Related in: MedlinePlus