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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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Determination of PPARγ ligand binding affinities of FM550 and its components. FM550 (0.01–70 μg/mL; 0.02–160 μM), TPP (0.01–1,400 μM), TBB (0.009–90 μM), TBPH (0.12–1,200 μM), and ITP (0.01–28 μg/mL; 0.02–80 μM) were tested in the PolarScreen™ PPARγ-competitor assay. IC50 values and dissociation constants were calculated as described in “Materials and Methods.” Data are presented as mean ± SE of three technical replicates and are representative of two independent experiments.**p < 0.01, by ANOVA and Dunnett’s multiple comparisons test, compared with the lowest concentration.
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f2: Determination of PPARγ ligand binding affinities of FM550 and its components. FM550 (0.01–70 μg/mL; 0.02–160 μM), TPP (0.01–1,400 μM), TBB (0.009–90 μM), TBPH (0.12–1,200 μM), and ITP (0.01–28 μg/mL; 0.02–80 μM) were tested in the PolarScreen™ PPARγ-competitor assay. IC50 values and dissociation constants were calculated as described in “Materials and Methods.” Data are presented as mean ± SE of three technical replicates and are representative of two independent experiments.**p < 0.01, by ANOVA and Dunnett’s multiple comparisons test, compared with the lowest concentration.

Mentions: Computational and in vitro analyses of PPARγ binding by components of FM550. To test the hypothesis that components of FM550 are PPARγ ligands, we assessed the ability of FM550 and its components to bind with the PPARγ LBD (Figure 2). We found that FM550 could competitively bind with the PPARγ LBD in a dose-dependent manner (IC50 = 400 μM; Kd = 210 μM). The brominated components of FM550, TBB, and TBPH did not demonstrate any binding over the concentration range tested. In contrast, TPP was found to be a ligand of PPARγ (IC50 = 38 μM; Kd = 20 μM). ITP showed an ability similar to that of TPP to compete for PPARγ binding (IC50 = 60 μM; Kd = 32 μM). In comparison, rosiglitazone competed for PPARγ binding with an IC50 of 0.23 μM and a Kd of 0.12 μM (see Supplemental Material, Figure S2C). Because ITP contains approximately 40% TPP (measured in the laboratory of H.M.S.), TPP was likely a significant contributor to PPARγ binding by the ITP mixture; however, the other isopropylated phosphate isomers in this mixture may also effectively bind to PPARγ-LBD.


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)

Determination of PPARγ ligand binding affinities of FM550 and its components. FM550 (0.01–70 μg/mL; 0.02–160 μM), TPP (0.01–1,400 μM), TBB (0.009–90 μM), TBPH (0.12–1,200 μM), and ITP (0.01–28 μg/mL; 0.02–80 μM) were tested in the PolarScreen™ PPARγ-competitor assay. IC50 values and dissociation constants were calculated as described in “Materials and Methods.” Data are presented as mean ± SE of three technical replicates and are representative of two independent experiments.**p < 0.01, by ANOVA and Dunnett’s multiple comparisons test, compared with the lowest concentration.
© Copyright Policy - public-domain
Related In: Results  -  Collection

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

f2: Determination of PPARγ ligand binding affinities of FM550 and its components. FM550 (0.01–70 μg/mL; 0.02–160 μM), TPP (0.01–1,400 μM), TBB (0.009–90 μM), TBPH (0.12–1,200 μM), and ITP (0.01–28 μg/mL; 0.02–80 μM) were tested in the PolarScreen™ PPARγ-competitor assay. IC50 values and dissociation constants were calculated as described in “Materials and Methods.” Data are presented as mean ± SE of three technical replicates and are representative of two independent experiments.**p < 0.01, by ANOVA and Dunnett’s multiple comparisons test, compared with the lowest concentration.
Mentions: Computational and in vitro analyses of PPARγ binding by components of FM550. To test the hypothesis that components of FM550 are PPARγ ligands, we assessed the ability of FM550 and its components to bind with the PPARγ LBD (Figure 2). We found that FM550 could competitively bind with the PPARγ LBD in a dose-dependent manner (IC50 = 400 μM; Kd = 210 μM). The brominated components of FM550, TBB, and TBPH did not demonstrate any binding over the concentration range tested. In contrast, TPP was found to be a ligand of PPARγ (IC50 = 38 μM; Kd = 20 μM). ITP showed an ability similar to that of TPP to compete for PPARγ binding (IC50 = 60 μM; Kd = 32 μM). In comparison, rosiglitazone competed for PPARγ binding with an IC50 of 0.23 μM and a Kd of 0.12 μM (see Supplemental Material, Figure S2C). Because ITP contains approximately 40% TPP (measured in the laboratory of H.M.S.), TPP was likely a significant contributor to PPARγ binding by the ITP mixture; however, the other isopropylated phosphate isomers in this mixture may also effectively bind to PPARγ-LBD.

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