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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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Reporter and in vitro differentiation analyses of PPARγ activation by TPP (top) and ITP (bottom). (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), TPP (0.1–40 μM), or ITP (0.1–10 μg/mL; 0.3–60 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), TPP (0.1–20 μM), or ITP (0.1–10 μg/mL; 0.3–30 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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f4: Reporter and in vitro differentiation analyses of PPARγ activation by TPP (top) and ITP (bottom). (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), TPP (0.1–40 μM), or ITP (0.1–10 μg/mL; 0.3–60 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), TPP (0.1–20 μM), or ITP (0.1–10 μg/mL; 0.3–30 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 the organophosphate components of FM550. As with FM550, we examined the ability of TPP and ITP to activate PPARγ-driven reporter expression and induce adipogenesis. In the PPARγ/RXRα Cos7 cell reporter assay, TPP significantly induced PPARγ-driven reporter activity at concentrations ≥ 10 μM, with an EC50 of 8 μM and maximal activity of 7.5 ± 0.7-fold (Figure 4A), and ITP significantly induced PPARγ-driven reporter activity at concentrations ≥ 10 μg/mL (30 μM), with an EC50 of 8 μM and maximal activity of 5.1 ± 0.6-fold (Figure 4A). TPP and ITP are less potent and efficacious than rosiglitazone (1 μM; 11.3 ± 0.6-fold; EC50 of 0.02 μM; see Supplemental Material, Figure S2A). In the BMS2 adipogenesis assay, TPP significantly induced lipid accumulation at concentrations ≥ 5 μM with a maximal lipid accumulation of 614 ± 60 RFUs (Figure 4B), and ITP significantly induced lipid accumulation at concentrations ≥ 1 μg/mL (3 μM), with a maximal lipid accumulation of 796 ± 60 RFUs (Figure 4B); these lipid accumulations were lower than the lipid accumulation induced by a maximally efficacious concentration of rosiglitazone (1 μM; 1043 ± 45 RFUs; see Supplemental Material, Figure S2B). That TPP and ITP induced terminal adipocyte differentiation was confirmed by the observation that both compounds induced the expression of perilipin (Figure 4C). The results indicate that TPP and ITP are PPARγ ligands that can induce adipocyte differentiation.


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 TPP (top) and ITP (bottom). (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), TPP (0.1–40 μM), or ITP (0.1–10 μg/mL; 0.3–60 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), TPP (0.1–20 μM), or ITP (0.1–10 μg/mL; 0.3–30 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

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getmorefigures.php?uid=PMC4216168&req=5

f4: Reporter and in vitro differentiation analyses of PPARγ activation by TPP (top) and ITP (bottom). (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), TPP (0.1–40 μM), or ITP (0.1–10 μg/mL; 0.3–60 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), TPP (0.1–20 μM), or ITP (0.1–10 μg/mL; 0.3–30 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 the organophosphate components of FM550. As with FM550, we examined the ability of TPP and ITP to activate PPARγ-driven reporter expression and induce adipogenesis. In the PPARγ/RXRα Cos7 cell reporter assay, TPP significantly induced PPARγ-driven reporter activity at concentrations ≥ 10 μM, with an EC50 of 8 μM and maximal activity of 7.5 ± 0.7-fold (Figure 4A), and ITP significantly induced PPARγ-driven reporter activity at concentrations ≥ 10 μg/mL (30 μM), with an EC50 of 8 μM and maximal activity of 5.1 ± 0.6-fold (Figure 4A). TPP and ITP are less potent and efficacious than rosiglitazone (1 μM; 11.3 ± 0.6-fold; EC50 of 0.02 μM; see Supplemental Material, Figure S2A). In the BMS2 adipogenesis assay, TPP significantly induced lipid accumulation at concentrations ≥ 5 μM with a maximal lipid accumulation of 614 ± 60 RFUs (Figure 4B), and ITP significantly induced lipid accumulation at concentrations ≥ 1 μg/mL (3 μM), with a maximal lipid accumulation of 796 ± 60 RFUs (Figure 4B); these lipid accumulations were lower than the lipid accumulation induced by a maximally efficacious concentration of rosiglitazone (1 μM; 1043 ± 45 RFUs; see Supplemental Material, Figure S2B). That TPP and ITP induced terminal adipocyte differentiation was confirmed by the observation that both compounds induced the expression of perilipin (Figure 4C). The results indicate that TPP and ITP are PPARγ ligands that can induce adipocyte differentiation.

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