Abstract

Organophosphate esters have become widely used as flame retardants since the phase out of polybrominated diphenyl ethers. Previously, we demonstrated that some organophosphate esters, such as tert-butylphenyl diphenyl phosphate (BPDP), were more detrimental to endochondral ossification in murine limb bud cultures than one of the major polybrominated diphenyl ethers that they replaced, 2,2′,4,4′-tetrabromodiphenyl ether. Here, we used a transcriptomic approach to elucidate the mechanism of action of BPDP in the developing limb. Limb buds collected from gestation day 13 CD1 mouse embryos were cultured for 3 or 24 h in the presence of vehicle, 1 μM, or 10 μM BPDP. RNA sequencing analyses revealed that exposure to 1 µM BPDP for 24 h increased the expression of 5 transcripts, including Ihh, and decreased 14 others, including Gli1, Ptch1, Ptch2, and other targets of Hedgehog (Hh) signaling. Pathway analysis predicted the inhibition of Hh signaling. Attenuation of Hh signaling activity began earlier and reached a greater magnitude after exposure to 10 µM BPDP. Because this pathway is part of the regulatory network governing endochondral ossification, we used a known Hh agonist, purmorphamine, to determine the contribution of Hh signaling inhibition to the negative impact of BPDP on endochondral ossification. Cotreatment of limbs with purmorphamine rescued the detrimental morphological changes in the cartilage template induced by BPDP exposure though it did not restore the expression of key transcription factors, Runx2 and Sp7, to control levels. These data highlight Hh signaling as a developmentally important pathway vulnerable to environmental chemical exposures.

中文翻译：

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接触叔丁基苯基二苯基磷酸酯（一种有机磷酸酯阻燃剂和增塑剂）会改变鼠肢芽培养物中的刺猬信号。

摘要

自从多溴联苯醚逐步淘汰以来，有机磷酸酯已被广泛用作阻燃剂。以前，我们证明了一些有机磷酸酯，例如叔丁基苯基二苯基磷酸酯 (BPDP)，比它们取代的主要多溴二苯醚之一对小鼠肢芽培养物中的软骨内骨化更有害，2,2',4， 4'-四溴二苯醚。在这里，我们使用转录组学方法来阐明 BPDP 在发育中的肢体中的作用机制。从妊娠第 13 天 CD1 小鼠胚胎收集的肢芽在存在载体、1 μM 或 10 μM BPDP 的情况下培养 3 或 24 小时。RNA 测序分析显示，暴露于 1 µM BPDP 24 小时会增加 5 个转录本的表达，包括Ihh，并减少了其他 14 个，包括Gli1、Ptch1、Ptch2和 Hedgehog (Hh) 信号传导的其他目标。通路分析预测了 Hh 信号的抑制。Hh 信号活动的衰减开始得更早，并在暴露于 10 µM BPDP 后达到更大的幅度。由于该途径是控制软骨内骨化的调节网络的一部分，我们使用已知的 Hh 激动剂嘌呤吗啡胺来确定 Hh 信号抑制对 BPDP 对软骨内骨化的负面影响的贡献。用嘌呤吗啉共同处理四肢挽救了 BPDP 暴露诱导的软骨模板的有害形态变化，尽管它没有恢复关键转录因子Runx2的表达和Sp7，以控制电平。这些数据强调 Hh 信号是一种易受环境化学物质暴露影响的重要发育途径。