Effects of Organophosphate Ester Flame Retardants on Endochondral Ossification in Ex Vivo Murine Limb Bud Cultures.

Abstract

Phasing out the usage of polybrominated diphenyl ether (PBDE) flame retardants (FRs) in consumer products led to their widespread replacement with organophosphate ester (OPE) FRs, despite scarce safety data. PBDE exposures were associated with the suppression of endochondral ossification but little is known about the effects of OPEs on bones. Here, we used a novel ex vivo murine limb bud culture system to compare the effects of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) with those of several OPEs. Gestation day 13 embryos were collected from transgenic CD1 mice expressing fluorescent markers for the major stages of endochondral ossification: COL2A1-ECFP (chondrogenesis), COL10A1-mCherry (early osteogenesis), and COL1A1-YFP (late osteogenesis). Limbs were excised and cultured for 6 days in the presence of vehicle, BDE-47, or an OPE FR: triphenyl phosphate (TPHP), tert-butylphenyl diphenyl phosphate (BPDP), tris(methylphenyl) phosphate (TMPP), or isopropylated triphenyl phosphate (IPPP). BDE-47 (50 μM) decreased the extent of chondrogenesis in the digits and COL1A1-YFP expression in the radius and ulna relative to control. In comparison, concentrations of ≥1 μM of all 4 OPEs limited chondrogenesis; osteogenesis (both COL10A1-mCherry and COL1A1-YFP fluorescence) was markedly inhibited at concentrations ≥3 μM. The expression of Sox9, the master regulator of chondrogenesis, was altered by BDE-47, TPHP, and BPDP. BDE-47 exposure had minimal impact on the expression of Runx2 and Sp7, which drive osteogenesis, whereas TPHP and BPDP both suppressed the expression of these transcription factors. These data suggest that OPE FRs may be more detrimental to bone formation than their brominated predecessors.