Impacts of Gestational FireMaster 550 Exposure on the Neonatal Cortex Are Sex Specific and Largely Attributable to the Organophosphate Esters

Abstract

Introduction: Flame retardants (FRs) are common bodily and environmental pollutants, creating concern about their potential toxicity. We and others have found that the commercial mixture FireMaster® 550 (FM 550) or its individual brominated (BFR) and organophosphate ester (OPFR) components are potential developmental neurotoxicants. Using Wistar rats, we previously reported that developmental exposure to FM 550 or its component classes produced sex- and compound-specific effects on adult socioemotional behaviors. The underlying mechanisms driving the behavioral phenotypes are unknown.

Methods: To further mechanistic understanding, here we conducted transcriptomics in parallel with a novel lipidomics approach using cortical tissues from newborn siblings of the rats in the published behavioral study. Inclusion of lipid composition is significant because it is rarely examined in developmental neurotoxicity studies. Pups were gestationally exposed via oral dosing to the dam to FM 550 or the BFR or OPFR components at environmentally relevant doses.

Results: The neonatal cortex was highly sexually dimorphic in lipid and transcriptome composition, and males were more significantly impacted by FR exposure. Multiple adverse modes of action for the BFRs and OPFRs on neurodevelopment were identified, with the OPFRs being more disruptive than the BFRs via multiple mechanisms including dysregulation of mitochondrial function and disruption of cholinergic and glutamatergic systems. Disrupted mitochondrial function by environmental factors has been linked to a higher risk of autism spectrum disorders and neurodegenerative disorders. Impacted lipid classes included ceramides, sphingomyelins, and triacylglycerides. Robust ceramide upregulation in the OPFR females could suggest a heightened risk of brain metabolic disease.

Conclusions: This study reveals multiple mechanisms by which the components of a common FR mixture are developmentally neurotoxic and that the OPFRs may be the compounds of greatest concern.

Keywords: Brain; Choline; DNT; Developmental neurotoxicity; Environmental toxicology; Flame retardants; ITPs IPP; Lipidomics; TPHP; TPP; Transcriptomics.

Figure 1.

Illustration of how cortical tissue for RNAseq and lipidomic analysis was collected in the PND1 offspring gestationally exposed to FM 550, BFR or OPFR. A) For RNAseq analysis, whole PND1 heads were cryosectioned at 20 μm and the forebrain slices collected using The Developing Rat Nervous System (Paxinos and Ashwell 2018) atlas as a guide. Tissue collection began on plate 212 as indicated by clear separation of cortex and olfactory bulb and stopped on plate 216, recognized by the distinct position of the corpus collosum, fornix and lateral ventricle. B) For lipidomic tissue collection, micropunches (1.25mm width by 1-1.15 mm deep) were then used to isolate the medial portion of the cortex from the same PND1 mounted heads. The isolated lipidomics tissue approximately contained cortical material from plates 216 – 221.

Figure 2.

Differentially expressed genes (DEGs) separated by sex, exposure group and direction of expression change. Males had the highest number of DEGs regardless of exposure group, with only OPFR females having greater than 100 DEGs in both directions. For this reason, data from all the male exposure groups, but only the OPFR female exposure group, underwent pathways analysis.

Figure 3.

Shared DEGs by direction across exposure group in males. Across all three exposure groups, 35% of upregulated (703) and 30.1% of downregulated (629) genes were identified as shared DEGs. The FM550 and BFR groups had 8.4% upregulated and 8.5% downregulated genes in common, while the FM550 and OPFR groups had 13.7% upregulated and 6.8% downregulated genes in common.

Figure 4.

Summary of the ten most significant upregulated and downregulated GO Biological Processes in the exposure groups examined. Five upregulated processes were shared across all groups (hatched bars). Within the OPFR male and females 6 upregulated processes were shared by both sexes (connected with dashed lines). Four downregulated process were shared across all groups examined (hatched bars). OPFR females had no downregulated processes.

Figure 5.

Upregulated KEGG pathways. A) List of 22 upregulated KEGG pathways shared by OPFR, BFR and FM550 males. B) List of the 12 upregulated KEGG pathways shared between the OPFR males and females. The five neuronal degenerative pathways both sexes had in common are indicated by *. C) List of the 18 genes shared across the 5 neuronal degenerative pathways enriched in the OPFR exposed groups. All are involved in cellular respiration.

Figure 6.

Statistically significant lipids observed in all exposure versus control comparaisons. A) Venn diagram of statistically significant lipids from each exposure comparison and their overlap. B) Circular dendrogram of all 87 statistically significant lipids generated with an ECFP6 fingerprint, Tanimoto distance, and average linkage for the corresponding exposure comparisons. Log₂ fold change is overlaid for all three comparisons to visualize trends in significant dysregulation. All identified lipids without statistically significance are shown in grey whereas statistically significant species that were either upregulated or downregulated are displayed in red and blue. Lipid class abbreviations include: FA = fatty acid, ANA = anandamide, SM = sphingomyelin, Cer = ceramide, PE O- = ether-linked phosphatidylethanolamine, PE P- = vinyl ether-linked phosphatidylethanolamine, LPE = lysophosphatidylethanolamine, LPC = lysophosphatidylcholine, PC O- = ether-linked phosphatidylcholine, PC P- = vinyl ether-linked phosphatidylcholine, PC = phosphatidylcholine, PE = phosphatidylethanolamine, PS = phosphatidylserine, TG = triglyceride, DG = diglyceride.

Figure 7.

Sex specific statistically significant lipids observed in exposure versus control comparisons. Circular dendrogram illustrating Female only (F) and Male only (M) comparisons. Log₂ fold change is overlaid for all six comparisons to visualize trends in significant dysregulation. All identified but insignificant lipids are shown in grey whereas statistically significant species that were either upregulated or downregulated are displayed in red and blue. Lipid class abbreviations include: FA = fatty acid, ANA = anandamide, SM = sphingomyelin, Cer = ceramide, AC = acylcarnitine, PE O- = ether-linked phosphatidylethanolamine, PE P- = vinyl ether-linked phosphatidylethanolamine, LPE = lysophosphatidylethanolamine, LPC = lysophosphatidylcholine, PC = phosphatidylcholine, PC O- = ether-linked phosphatidylcholine, PC P- = vinyl ether-linked phosphatidylcholine, PS = phosphatidylserine, TG = triglyceride, DG = diglyceride

Figure 8.

Example of how enriched genes factor into the identified upregulated KEGG pathways. The 18 commonly expressed genes within the neurodegenerative disease pathways identified were used to render KEGG pathways using Pathview (http://bioinformatics.sdstate.edu/go/). Depicted in red are FR-affected genes in the oxidative phosphorylation (A) and Alzheimer’s disease (B) pathways.

Figure 9.

Representative renderings of 4 downregulated KEGG pathways in the exposed groups. DEGs within each are depicted in red. A) Endocytosis was the only downregulated pathway shared across all male exposure groups and the OPFR females. B) Axon guidance was the only significant downregulated pathway in both OPFR males and females. C & D) In males, OPFR downregulated KEGG pathways included glutamatergic and cholinergic synapses.