Gestational exposure to FireMaster® 550 (FM 550) disrupts the placenta-brain axis in a socially monogamous rodent species, the prairie vole (Microtus ochrogaster).

Gestational flame retardant (FR) exposure has been linked to heightened risk of neurodevelopmental disorders, but the mechanisms remain largely unknown. Historically, toxicologists have relied on traditional, inbred rodent models, yet those do not always best model human vulnerability or biological systems, especially social systems. Here we used prairie voles (Microtus ochrogaster), a monogamous and bi-parental rodent, leveraged for decades to decipher the underpinnings of social behaviors, to examine the impact of fetal FR exposure on gene targets in the mid-gestational placenta and fetal brain. We previously established gestational exposure to the commercial mixture Firemaster 550 (FM 550) impairs sociality, particularly in males. FM 550 exposure disrupted placental monoamine production, particularly serotonin, and genes required for axon guidance and cellular respiration in the fetal brains. Effects were dose and sex specific. These data provide insights on the mechanisms by which FRs impair neurodevelopment and later in life social behaviors.

Maternal organophosphate flame retardant exposure alters the developing mesencephalic dopamine system in fetal rat.

Organophosphate flame retardants (OPFRs) have become the predominant substitution for legacy brominated flame retardants but there is concern about their potential developmental neurotoxicity (DNT). OPFRs readily dissociate from the fireproofed substrate to the environment, and they (or their metabolites) have been detected in diverse matrices including air, water, soil, and biota, including human urine and breastmilk. Given this ubiquitous contamination, it becomes increasingly important to understand the potential effects of OPFRs on the developing nervous system. We have previously shown that maternal exposure to OPFRs results in neuroendocrine disruption, alterations to developmental metabolism of serotonin (5-HT) and axonal extension in male fetal rats, and potentiates adult anxiety-like behaviors. The development of the serotonin and dopamine systems occur in parallel and interact, therefore, we first sought to enhance our prior 5-HT work by first examining the ascending 5-HT system on embryonic day 14 using whole mount clearing of fetal heads and 3-dimensional (3D) brain imaging. We also investigated the effects of maternal OPFR exposure on the development of the mesocortical dopamine system in the same animals through 2-dimensional and 3D analysis following immunohistochemistry for tyrosine hydroxylase (TH). Maternal OPFR exposure induced morphological changes to the putative ventral tegmental area and substantia nigra in both sexes and reduced the overall volume of this structure in males, whereas 5-HT nuclei were unchanged. Additionally, dopaminergic axogenesis was disrupted in OPFR exposed animals, as the dorsoventral spread of ventral telencephalic TH afferents were greater at embryonic day 14, while sparing 5-HT fibers. These results indicate maternal exposure to OPFRs alters the development trajectory of the embryonic dopaminergic system and adds to growing evidence of OPFR DNT.

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

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.

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

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.

Gene expression networks in the Drosophila Genetic Reference Panel.

A major challenge in modern biology is to understand how naturally occurring variation in DNA sequences affects complex organismal traits through networks of intermediate molecular phenotypes. This question is best addressed in a genetic mapping population in which all molecular polymorphisms are known and for which molecular endophenotypes and complex traits are assessed on the same genotypes. Here, we performed deep RNA sequencing of 200 Drosophila Genetic Reference Panel inbred lines with complete genome sequences and for which phenotypes of many quantitative traits have been evaluated. We mapped expression quantitative trait loci for annotated genes, novel transcribed regions, transposable elements, and microbial species. We identified host variants that affect expression of transposable elements, independent of their copy number, as well as microbiome composition. We constructed sex-specific expression quantitative trait locus regulatory networks. These networks are enriched for novel transcribed regions and target genes in heterochromatin and euchromatic regions of reduced recombination, as well as genes regulating transposable element expression. This study provides new insights regarding the role of natural genetic variation in regulating gene expression and generates testable hypotheses for future functional analyses.

A Drosophila model for toxicogenomics: Genetic variation in susceptibility to heavy metal exposure.

The genetic factors that give rise to variation in susceptibility to environmental toxins remain largely unexplored. Studies on genetic variation in susceptibility to environmental toxins are challenging in human populations, due to the variety of clinical symptoms and difficulty in determining which symptoms causally result from toxic exposure; uncontrolled environments, often with exposure to multiple toxicants; and difficulty in relating phenotypic effect size to toxic dose, especially when symptoms become manifest with a substantial time lag. Drosophila melanogaster is a powerful model that enables genome-wide studies for the identification of allelic variants that contribute to variation in susceptibility to environmental toxins, since the genetic background, environmental rearing conditions and toxic exposure can be precisely controlled. Here, we used extreme QTL mapping in an outbred population derived from the D. melanogaster Genetic Reference Panel to identify alleles associated with resistance to lead and/or cadmium, two ubiquitous environmental toxins that present serious health risks. We identified single nucleotide polymorphisms (SNPs) associated with variation in resistance to both heavy metals as well as SNPs associated with resistance specific to each of them. The effects of these SNPs were largely sex-specific. We applied mutational and RNAi analyses to 33 candidate genes and functionally validated 28 of them. We constructed networks of candidate genes as blueprints for orthologous networks of human genes. The latter not only provided functional contexts for known human targets of heavy metal toxicity, but also implicated novel candidate susceptibility genes. These studies validate Drosophila as a translational toxicogenomics gene discovery system.

Genetic and Genomic Response to Selection for Food Consumption in Drosophila melanogaster.

Food consumption is an essential component of animal fitness; however, excessive food intake in humans increases risk for many diseases. The roles of neuroendocrine feedback loops, food sensing modalities, and physiological state in regulating food intake are well understood, but not the genetic basis underlying variation in food consumption. Here, we applied ten generations of artificial selection for high and low food consumption in replicate populations of Drosophila melanogaster. The phenotypic response to selection was highly asymmetric, with significant responses only for increased food consumption and minimal correlated responses in body mass and composition. We assessed the molecular correlates of selection responses by DNA and RNA sequencing of the selection lines. The high and low selection lines had variants with significantly divergent allele frequencies within or near 2081 genes and 3526 differentially expressed genes in one or both sexes. A total of 519 genes were both genetically divergent and differentially expressed between the divergent selection lines. We performed functional analyses of the effects of RNAi suppression of gene expression and induced mutations for 27 of these candidate genes that have human orthologs and the strongest statistical support, and confirmed that 25 (93 %) affected the mean and/or variance of food consumption.

Obp56h Modulates Mating Behavior in Drosophila melanogaster.

Social interactions in insects are driven by conspecific chemical signals that are detected via olfactory and gustatory neurons. Odorant binding proteins (Obps) transport volatile odorants to chemosensory receptors, but their effects on behaviors remain poorly characterized. Here, we report that RNAi knockdown of Obp56h gene expression in Drosophila melanogaster enhances mating behavior by reducing courtship latency. The change in mating behavior that results from inhibition of Obp56h expression is accompanied by significant alterations in cuticular hydrocarbon (CHC) composition, including reduction in 5-tricosene (5-T), an inhibitory sex pheromone produced by males that increases copulation latency during courtship. Whole genome RNA sequencing confirms that expression of Obp56h is virtually abolished in Drosophila heads. Inhibition of Obp56h expression also affects expression of other chemoreception genes, including upregulation of lush in both sexes and Obp83ef in females, and reduction in expression of Obp19b and Or19b in males. In addition, several genes associated with lipid metabolism, which underlies the production of cuticular hydrocarbons, show altered transcript abundances. Our data show that modulation of mating behavior through reduction of Obp56h is accompanied by altered cuticular hydrocarbon profiles and implicate 5-T as a possible ligand for Obp56h.