Tung Emily W Y, Ahmed Shaimaa, Peshdary Vian, Atlas Ella
Environmental Health Science and Research Bureau, Health Canada, Ottawa, Ontario, Canada.
PLoS One. 2017 Apr 24;12(4):e0175855. doi: 10.1371/journal.pone.0175855. eCollection 2017.
Firemaster® 550 (FM550) is a chemical mixture currently used as an additive flame retardant in commercial products, and is comprised of 2-ethylhexyl-2,3,4,5-tertrabromobenzoate (TBB), bis(2-ethylhexyl) tetrabromophthalate (TBPH), triphenyl phosphate (TPP), and isopropylated triphenyl phosphate (IPTP). Animal and in vitro studies suggest that FM550, TPP and IPTP may have adipogenic effects and may exert these effects through PPARγ activation. Using murine 3T3-L1 preadipocytes, we investigated the detailed expression of transcription factors and adipogenic markers in response to FM550 and its components. Further we investigated the mechanism of action of the peroxisome proliferator-activated receptor gamma (PPARγ) on downstream targets of the receptor by focussing on the mature adipocyte marker, adipocyte protein 2 (aP2). In addition, we set to elucidate the components responsible for the adipogenic effects seen in the FM550 mixture. We show that FM550 and its components TPP, IPTP, and TBPH, but not TBB induced lipid accumulation in a dose-dependent manner. Interestingly, despite displaying enhanced lipid accumulation, TBPH did not alter the mRNA or protein expression of terminal differentiation markers. In contrast, FM550, TPP, and IPTP treatment enhanced lipid accumulation, and mRNA and protein expression of terminal differentiation markers. To further delineate the mechanisms of action of FM550 and its components we focussed on aP2 promoter activity. For this purpose we used the enhancer region of the mouse aP2 promoter using a 584-bp reporter construct containing an active PPRE located 5.4 kb away from the transcription start site of aP2. Exposure to FM550, IPTP, and TPP significantly increased PPARγ mediated aP2 enhancer activity. Furthermore, we show that TPP- and IPTP-dependent upregulation of aP2 was significantly inhibited by the selective PPARγ antagonist GW9662. In addition, chromatin immunoprecipitation experiments showed that IPTP and TPP treatment led to the recruitment of PPARγ to the regulatory region of aP2.
Firemaster® 550（FM550）是一种化学混合物，目前在商业产品中用作添加型阻燃剂，它由2 - 乙基己基 - 2,3,4,5 - 四溴苯甲酸酯（TBB）、双（2 - 乙基己基）四溴邻苯二甲酸酯（TBPH）、磷酸三苯酯（TPP）和异丙基化磷酸三苯酯（IPTP）组成。动物和体外研究表明，FM550、TPP和IPTP可能具有促脂肪生成作用，并且可能通过激活过氧化物酶体增殖物激活受体γ（PPARγ）发挥这些作用。我们使用小鼠3T3 - L1前脂肪细胞，研究了转录因子和成脂标志物对FM550及其成分的详细表达情况。此外，我们通过聚焦于成熟脂肪细胞标志物脂肪细胞蛋白2（aP2），研究了过氧化物酶体增殖物激活受体γ（PPARγ）对该受体下游靶点的作用机制。另外，我们着手阐明FM550混合物中产生促脂肪生成作用的成分。我们发现，FM550及其成分TPP、IPTP和TBPH（但不包括TBB）以剂量依赖的方式诱导脂质积累。有趣的是，尽管TBPH显示出脂质积累增加，但其并未改变终末分化标志物的mRNA或蛋白质表达。相比之下，FM550、TPP和IPTP处理增强了脂质积累以及终末分化标志物的mRNA和蛋白质表达。为了进一步阐明FM550及其成分的作用机制，我们聚焦于aP2启动子活性。为此，我们使用了小鼠aP2启动子的增强子区域，采用一个584 bp的报告基因构建体，该构建体包含一个位于aP2转录起始位点5.4 kb处的活性过氧化物酶体增殖物反应元件（PPRE）。暴露于FM550、IPTP和TPP显著增加了PPARγ介导的aP2增强子活性。此外，我们发现选择性PPARγ拮抗剂GW9662显著抑制了TPP和IPTP依赖的aP2上调。另外，染色质免疫沉淀实验表明，IPTP和TPP处理导致PPARγ募集至aP2的调控区域。
