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

Abstract

There is a growing need to understand the potential neurotoxicity of organophosphate 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 flame retardant 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 for the developing brain. We have shown in rats and humans that flame retardants accumulate in placental tissue, and induce functional changes, including altered neurotransmitter production. Here, we sought to establish if OPFRs (triphenyl phosphate and a mixture of isopropylated triarylphosphate isomers) alter placental function and fetal forebrain development, with disruption of tryptophan metabolism as a primary pathway of interest. Wistar rat dams were orally exposed to OPFRs (0, 500, 1000, or 2000 μg/day) or a serotonin (5-HT) agonist 5-methoxytryptamine 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 metabolic pathways were upregulated. In addition, 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 tryptophan metabolism.

Keywords: developmental; developmental toxicity; developmental/teratology; endocrine disruptors; endocrine toxicology; flame retardants; metabolome; neurotoxicity; neurotoxicology; neurotransmitter; prenatal; reproductive and developmental toxicology.

Figure 1.

A, A representative hematoxylin and eosin stained placental section depicting location of the junctional and labyrinth zones. The dotted circle indicates the region where the micropunches were obtained. B, Representative images of a placenta mounted on the cryostat before and after two 1.25-mm micropunches were collected.

Figure 2.

A, Levels of individual chemicals in whole placenta separated by sex. Graph depicts mean ± SEM. B, Total burden of OPFRs in whole placenta from the 8 mg/kg OPFR group separated by sex. Lines connect littermates. Abbreviations: OPFR, organophosphate flame retardant; TPHP, triphenyl phosphate.

Figure 3.

The top 15 canonical pathways identified by Ingenuity Pathway Analysis (IPA) analysis using annotations for all tissue types (A) and only the annotations specific to the placenta/nervous system (B). The dotted boxes indicate pathways identified in both analyses. C, Genes enriched in the pathways identified using annotations from all tissue types, and the processes they coordinate, with pathways identified by both IPA analyses highlighted in gray.

Figure 4.

Effects of prenatal organophosphate flame retardant (OPFR) exposure on placental genes responsible for protection against oxidative stress and exposure to corticosterone. (B and C) Sod2, the enzyme responsible for the inactivation of superoxide anion (A), was downregulated in the 4 mg/kg OPFR, 8 mg/kg OPFR, and 5-MT groups when males and females were grouped for analysis, as well as when they were analyzed within sex. (E) Similarly, Hsd11b2, the enzyme responsible for the inactivation of corticosterone (D), was downregulated in the 4 mg/kg OPFR and 8 mg/kg OPFR groups when sexes were combined. F, Expression of Hsd11b2 was downregulated in the female 8 mg/kg OPFR group, whereas males showed only a suggestive decrease in expression at the 4 mg/kg OPFR dose. G, Relative abundance of corticosterone appeared to be elevated in both female- and male-associated placentas exposed to 8 mg/kg OPFR, with males showing a greater exposure-related increase than females. Graphs depict median with whiskers from minimum to maximum (*p ≤ .05, **p ≤ .01, ***p ≤ .001, ****p ≤ .0001).

Figure 5.

Effects of prenatal organophosphate flame retardant (OPFR) exposure on the relative abundance of genes involved in the transport, synthesis, and metabolism of serotonin (5-HT) and levels of tryptophan, 5-HT and 5-hydroxyindole acetic acid (5-HIAA). A and B, Slc6a4, the 5-HT transporter, was lower in the 2 mg/kg and 4 mg/kg OPFR dose when sexes were combined for analysis, and in male-associated placentas when separated by sex. In female-associated placentas levels were only lower in the 4 mg/kg OPFR dose group. (D and E) Expression of Tph1, enzyme that converts tryptophan to 5-hydroxytryptophan (5-HTP) (C), was significantly lower in the 5-methoxytryptamine (5-MT) group in both the combined analysis and in male-associated placentas when separated by sex. E, Tph1 also showed a significant sex difference in the unexposed controls, with male-associated placentas having greater expression than female-associated placentas (^cp ≤ .05). (H) Expression of Ddc, the enzyme that converts 5-HTP to 5-HT (G), was lower in the 4 mg/kg OPFR group in the combined analysis, whereas a suggestive decrease was observed in the 5-MT animals. I, When separated by sex, lower expression of Ddc was observed in the 4 mg/kg and 8 mg/kg females. L and M. Expression of Maoa, the enzyme that converts 5-HT to 5-HIAA (K), was unaffected. F, Relative abundance of tryptophan was higher in placentas exposed to 8 mg/kg OPFR compared with unexposed placentas, with male-associated placentas showing consistently higher levels than female-associated placentas. J and N, Quantitative analysis of 5-HT and 5-HIAA showed a significant increase in the concentration of these neuroactive metabolites in male and female placentas exposed to 8 mg/kg OPFR. Graphs depict median with whiskers from minimum to maximum (*p ≤ .05, **p ≤ .01, ***p ≤ .001).

Figure 6.

Effects of prenatal organophosphate flame retardant (OPFR) exposure on the expression of a gene that metabolizes kynurenine as well as the relative abundance of kynurenine and its metabolites. (B and C) Expression of Kmo, which converts kynurenine to 3-hydroxykynurenine (A), was not impacted by OPFR exposure but significantly lower in the 5-methoxytryptamine (5-MT) animals in the combined analysis as well as in male-associated placentas when separated by sex. D–F, Quantitative analysis of kynurenine, kynurenic acid, and xanthurenic acid showed a significant increase in the concentration of these neuroactive metabolites in male and female placentas exposed to 8 mg/kg OPFR. G–J, Relative abundance of N-formylkynurenine, 3-hydroxyanthranilic acid, picolinic acid, and glutamate were all higher in placentas exposed to 8 mg/kg OPFR compared with unexposed placentas, with male-associated placentas showing consistently higher levels than female–associate placentas. Graphs depict median with whiskers from minimum to maximum (*p ≤ .05, **p ≤ .01).

Figure 7.

Summary of observed organophosphate flame retardant (OPFR) effects on metabolites and genes involved in the 5-HT and kynurenine metabolic pathways. Metabolites elevated by exposure are bolded and colored according to the pathway of interest (red for kynurenine and green for 5-HT). Outcomes, changes in expression, and sex-specific effects, are also depicted. Created with BioRender.com.

Figure 8.

Principal component analysis scores plot (A), variable importance in projections (VIP) plots (B and C), and hierarchical clustering heatmap (D) from MetaboAnalyst. Abbreviations: CF, control female; CM, control male; HF, high-dose female; HM, high-dose male.

Figure 9.

Effects of gestational organophosphate flame retardant (OPFR) exposure on the extension of serotonin (5-HT) and thalamocortical immunolabeled axons in the GD 14 brain. A, Length of 5-HTergic projections was greater in the forebrains of males exposed to 4 mg/kg and 8 mg/kg OPFR, whereas only a suggestive increase was observed in the 5-MT group. A significant linear trend was observed for 5-HT fiber length in female offspring. (B) No significant effect of exposure or sex was found for Netg1a length or (C) the spread of 5-HT cell bodies across the emerging dorsal raphe. (D) Representative diagram and (E) image of thalamocortical axons and 5-HT immunolabeled fibers with the neuronal marker Hu, which was used to confirm all 5-HT-labeled cells were neuronal, and identify key landmarks. (F and G) The point at which length measurements were obtained is indicated by the white arrow with length visibly less in the male controls (F) than the 8 mg/kg OPFR males (G). Graphs depict median with whiskers from minimum to maximum (*p ≤ .05). Abbreviations: DR, dorsal raphe; DT, dorsal thalamus.