Effects of Prenatal Exposure to a Mixture of Organophosphate Flame Retardants on Placental Gene Expression and Serotonergic Innervation in the Developing Rat Brain

There is a growing need to understand the potential neurotoxicity of organophosphate ester flame retardants (OPFRs) and plasticizers because use and, consequently, human exposure, is rapidly expanding. We have previously shown in rats that developmental exposure to the commercial FR mixture Firemaster® 550 (FM 550), which contains OPFRs, results in sex-specific behavioral effects, and identified the placenta as a potential target of toxicity. The placenta is a critical coordinator of fetal growth and neurodevelopment, and a source of neurotransmitters (NTs) for the developing brain. We have shown in rats and humans that FRs accumulate in placental tissue, and induce functional changes, including altered neurotransmitter (NT) production. Here we sought to establish if OPFRs (triphenyl phosphate, TPHP, and a mixture of isopropylated triarylphosphate isomers, ITPs) alter placental function and fetal forebrain development, with disruption of tryptophan (Trp) metabolism as a primary pathway of interest. Wistar rat dams were orally exposed to OPFRs (0, 500, 1,000, or 2,000 μg/day) or a serotonin (5-HT) agonist (5-MT) for 14 days during gestation and placenta and fetal forebrain tissues collected for analysis by transcriptomics and metabolomics. Relative abundance of genes responsible for the transport and synthesis of placental 5-HT were disrupted, and multiple neuroactive metabolites in the 5-HT and kynurenine (Kyn) metabolic pathways were upregulated. Additionally, 5-HTergic projections were significantly longer in the fetal forebrains of exposed males. These findings suggest that OPFRs have the potential to impact the 5-HTergic system in the fetal forebrain by disrupting placental Trp metabolism. As an initial hypothesis-generating approach, placental punches from the fetal side (5 per sex per group, from vehicle and 8mg/kg OPFRs) underwent RNA sequencing (RNA-seq), a total of 10 control and 10 with high dose treatment. RNA extraction was performed with the Qiagen RNEasy Miniprep kit according to the manufacturer protocol (Qiagen, Cat. 74134). RNA quality was determined using an Agilent 2100 Bioanalyzer and all samples, for both tissue types, had an RNA integrity number (RIN) = 10. Sequencing libraries were prepared as described previously (Puzianowska-Kuznicka et al. 2006), using NEBNext Ultra Directional RNA Library Prep kit and NEBNext Poly(A) mRNA Magnetic Isolation Module (catalogs E7420 and E7490; New England Biolabs), to be compatible with Illumina sequencing. Isolation, heat fragmentation, and priming was performed according to manufacturer’s instructions. cDNA synthesis was followed by purification and size selection. Finally, library clean-up was performed used AMPure XP beads (Beckman Coulter Genomics, Cat. A63881) and quality was assessed using the Agilent 2100 Bioanalyzer. Sequencing was performed using a 125-bp single-end protocol on a single lane of an Illumina HiSeq2500 sequencer. Approximately, 33 million uniquely mapped reads were generated per placental library.