Uncovering the common factors of chemical exposure and behavior: Evaluating behavioral effects across a testing battery using factor analysis.

Although specific environmental chemical exposures, including flame retardants, are known risk factors for neurodevelopmental disorders (NDDs), direct experimental evidence linking specific chemicals to NDDs is limited. Studies focusing on the mechanisms by which the social processing systems are vulnerable to chemical exposure are underrepresented in the literature, even though social impairments are defining characteristics of many NDDs. We have repeatedly demonstrated that exposure to Firemaster 550 (FM 550), a prevalent flame retardant mixture used in foam-based furniture and infant products, can adversely impact a variety of behavioral endpoints. Our recent work in prairie voles (Microtus ochrogaster), a prosocial animal model, demonstrated that perinatal exposure to FM 550 sex specifically impacts socioemotional behavior. Here, we utilized a factor analysis approach on a battery of behavioral data from our prior study to extract underlying factors that potentially explain patterns within the FM 550 behavior data. This approach identified which aspects of the behavioral battery are most robust and informative, an outcome critical for future study designs. Pearson's correlation identified behavioral endpoints associated with distance and stranger interactions that were highly correlated across 5 behavioral tests. Using these behavioral endpoints, exploratory factor analysis (EFA) and confirmatory factor analysis (CFA) extracted 2 factors that could explain the data: Activity (distance traveled endpoints) and Sociability (time spent with a novel conspecific). Exposure to FM 550 significantly decreased Activity and decreased Sociability. This factor analysis approach to behavioral data offers the advantages of modeling numerous measured variables and simplifying the data set by presenting the data in terms of common, overarching factors in terms of behavioral function.

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.

Individual and Combined Effects of Paternal Deprivation and Developmental Exposure to Firemaster 550 on Socio-Emotional Behavior in Prairie Voles.

The prevalence of neurodevelopmental disorders (NDDs) is rapidly rising, suggesting a confluence of environmental factors that are likely contributing, including developmental exposure to environmental contaminants. Unfortunately, chemical exposures and social stressors frequently occur simultaneously in many communities, yet very few studies have sought to establish the combined effects on neurodevelopment or behavior. Social deficits are common to many NDDs, and we and others have shown that exposure to the chemical flame retardant mixture, Firemaster 550 (FM 550), or paternal deprivation impairs social behavior and neural function. Here, we used a spontaneously prosocial animal model, the prairie vole (Microtus ochrogaster), to explore the effects of perinatal chemical (FM 550) exposure alone or in combination with an early life stressor (paternal absence) on prosocial behavior. Dams were exposed to vehicle (sesame oil) or 1000 µg FM 550 orally via food treats from conception through weaning and the paternal absence groups were generated by removing the sires the day after birth. Adult offspring of both sexes were then subjected to open-field, sociability, and a partner preference test. Paternal deprivation (PD)-related effects included increased anxiety, decreased sociability, and impaired pair-bonding in both sexes. FM 550 effects include heightened anxiety and partner preference in females but reduced partner preference in males. The combination of FM 550 exposure and PD did not exacerbate any behaviors in either sex except for distance traveled by females in the partner preference test and, to a lesser extent, time spent with, and the number of visits to the non-social stimulus by males in the sociability test. FM 550 ameliorated the impacts of parental deprivation on partner preference behaviors in both sexes. This study is significant because it provides evidence that chemical and social stressors can have unique behavioral effects that differ by sex but may not produce worse outcomes in combination.

Effects of developmental exposure to FireMaster® 550 (FM 550) on microglia density, reactivity and morphology in a prosocial animal model.

Microglia are known to shape brain sex differences critical for social and reproductive behaviors. Chemical exposures can disrupt brain sexual differentiation but there is limited data regarding how they may impact microglia distribution and function. We focused on the prevalent flame retardant mixture Firemaster 550 (FM 550) which is used in foam-based furniture and infant products including strollers and nursing pillows because it disrupts sexually dimorphic behaviors. We hypothesized early life FM 550 exposure would disrupt microglial distribution and reactivity in brain regions known to be highly sexually dimorphic or associated with social disorders in humans. We used prairie voles (Microtus ochrogaster) because they display spontaneous prosocial behaviors not seen in rats or mice and are thus a powerful model for studying chemical exposure-related impacts on social behaviors and their underlying neural systems. We have previously demonstrated that perinatal FM 550 exposure sex-specifically impacts socioemotional behaviors in prairie voles. We first established that, unlike in rats, the postnatal colonization of the prairie vole brain is not sexually dimorphic. Vole dams were then exposed to FM 550 (0, 500, 1000, 2000 µg/day) via subcutaneous injections through gestation, and pups were directly exposed beginning the day after birth until weaning. Adult offspring's brains were assessed for number and type (ramified, intermediate, ameboid) of microglia in the medial prefrontal cortex (mPFC), cerebellum (lobules VI-VII) and amygdala. Effects were sex- and dose-specific in the regions of interests. Overall, FM 550 exposure resulted in reduced numbers of microglia in most regions examined, with the 1000 µg FM 550 exposed males particularly affected. To further quantify differences in microglia morphology in the 1000 µg FM 550 group, Sholl and skeleton analysis were carried out on individual microglia. Microglia from control females had a more ramified phenotype compared to control males while 1000 µg FM 550-exposed males had decreased branching and ramification compared to same-sex controls. Future studies will examine the impact on the exposure to FM 550 on microglia during development given the critical role of these cells in shaping neural circuits.

Endocrine disrupting chemicals (EDCs) and placental function: Impact on fetal brain development.

Pregnancy is a critical time of vulnerability for the development of the fetal brain. Exposure to environmental pollutants at any point in pregnancy can negatively impact many aspects of fetal development, especially the organization and differentiation of the brain. The placenta performs a variety of functions that can help protect the fetus and sustain brain development. However, disruption of any of these functions can have negative impacts on both the pregnancy outcome and fetal neurodevelopment. This review presents current understanding of how environmental exposures, specifically to endocrine disrupting chemicals (EDCs), interfere with placental function and, in turn, neurodevelopment. Some of the key differences in placental development between animal models are presented, as well as how placental functions such as serving as a xenobiotic barrier and exchange organ, immune interface, regulator of growth and fetal oxygenation, and a neuroendocrine organ, could be vulnerable to environmental exposure. This review illustrates the importance of the placenta as a modulator of fetal brain development and suggests critical unexplored areas and possible vulnerabilities to environmental exposure.

Sex-specific Disruption of the Prairie Vole Hypothalamus by Developmental Exposure to a Flame Retardant Mixture.

Prevalence of neurodevelopmental disorders (NDDs) with social deficits is conspicuously rising, particularly in boys. Flame retardants (FRs) have long been associated with increased risk, and prior work by us and others in multiple species has shown that developmental exposure to the common FR mixture Firemaster 550 (FM 550) sex-specifically alters socioemotional behaviors including anxiety and pair bond formation. In rats, FRs have also been shown to impair aspects of osmoregulation. Because vasopressin (AVP) plays a role in both socioemotional behavior and osmotic balance we hypothesized that AVP and its related nonapeptide oxytocin (OT) would be vulnerable to developmental FM 550 exposure. We used the prairie vole (Microtus ochrogaste) to test this because it is spontaneously prosocial. Using siblings of prairie voles used in a prior study that assessed behavioral deficits resulting from developmental FM 550 exposure across 3 doses, here we tested the hypothesis that FM 550 sex-specifically alters AVP and OT neuronal populations in critical nuclei, such as the paraventricular nucleus (PVN), that coordinate those behaviors, as well as related dopaminergic (determined by tyrosine hydroxylase (TH) immunolabeling) populations. Exposed females had fewer AVP neurons in the anterior PVN and more A13 TH neurons in the zona incerta than controls. By contrast, in FM 550 males, A13 TH neuron numbers in the zona incerta were decreased but only in 1 dose group. These results expand on previous work showing evidence of endocrine disruption of OT/AVP pathways, including to subpopulations of PVN AVP neurons that coordinate osmoregulatory functions in the periphery.

Perfluorobutane sulfonate exposure disrupted human placental cytotrophoblast cell proliferation and invasion involving in dysregulating preeclampsia related genes.

We reported that maternal PFBS, an emerging pollutant, exposure is positively associated with preeclampsia which can result from aberrant trophoblasts invasion and subsequent placental ischemia. In this study, we investigated the effects of PFBS on trophoblasts proliferation/invasion and signaling pathways. We exposed a human trophoblast line, HTR8/SVneo, to PFBS. Cell viability, proliferation, and cell cycle were evaluated by the MTS assay, Ki-67 staining, and flow cytometry, respectively. We assessed cell migration and invasion with live-cell imaging-based migration assay and matrigel invasion assay, respectively. Signaling pathways were examined by Western blot, RNA-seq, and qPCR. PFBS exposure interrupted cell proliferation and invasion in a dose-dependent manner. PFBS (100 µM) did not cause cell death but instead significant cell proliferation without cell cycle disruption. PFBS (10 and 100 µM) decreased cell migration and invasion, while PFBS (0.1 µM) significantly increased cell invasion but not migration. Further, RNA-seq analysis identified dysregulated HIF-1α target genes that are relevant to cell proliferation/invasion and preeclampsia, while Western Blot data showed the activation of HIF-1α, but not Notch, ERK1/2, (PI3K)AKT, and P38 pathways. PBFS exposure altered trophoblast cell proliferation/invasion which might be mediated by preeclampsia-related genes, suggesting a possible association between prenatal PFBS exposure and adverse placentation.

Sex-specific effects of perinatal FireMaster® 550 (FM 550) exposure on socioemotional behavior in prairie voles.

The rapidly rising incidence of neurodevelopmental disorders with social deficits is raising concern that developmental exposure to environmental contaminants may be contributory. Firemaster 550 (FM 550) is one of the most prevalent flame-retardant (FR) mixtures used in foam-based furniture and baby products and contains both brominated and organophosphate components. We and others have published evidence of developmental neurotoxicity and sex specific effects of FM 550 on anxiety-like and exploratory behaviors. Using a prosocial animal model, we investigated the impact of perinatal FM 550 exposure on a range of socioemotional behaviors including anxiety, attachment, and memory. Virtually unknown to toxicologists, but widely used in the behavioral neurosciences, the prairie vole (Microtus ochrogaster) is a uniquely valuable model organism for examining environmental factors on sociality because this species is spontaneously prosocial, biparental, and displays attachment behaviors including pair bonding. Dams were exposed to 0, 500, 1000, or 2000 µg of FM 550 via subcutaneous (sc) injections throughout gestation, and pups were directly exposed beginning the day after birth until weaning. Adult offspring of both sexes were then subjected to multiple tasks including open field, novel object recognition, and partner preference. Effects were dose responsive and sex-specific, with females more greatly affected. Exposure-related outcomes in females included elevated anxiety, decreased social interaction, decreased exploratory motivation, and aversion to novelty. Exposed males also had social deficits, with males in all three dose groups failing to show a partner preference. Our studies demonstrate the utility of the prairie vole for investigating the impact of chemical exposures on social behavior and support the hypothesis that developmental FR exposure impacts the social brain. Future studies will probe the possible mechanisms by which these effects arise.

Roles of Progesterone Receptor Membrane Component 1 in Oxidative Stress-Induced Aging in Chorion Cells.

INTRODUCTION: Oxidative stress-mediated fetal membrane cell aging is activated prematurely in preterm premature rupture of membranes (PPROMs). The mechanism of this phenomenon is largely understudied. Progesterone receptor membrane component 1 (PGRMC1) has been recognized as a potential protective component for maintaining fetal membrane integrity and healthy pregnancies. We aimed to investigate the effects of oxidative stress (represented by hydrogen peroxide [H2O2]) on fetal membrane and chorion cell senescence, p38 mitogen-activated protein kinase (MAPK) phosphorylation, and sirtuin 3 (SIRT3) and to examine the roles of PGRMC1 in these effects. METHODS: Following serum starvation for 24 hours, full-thickness fetal membrane explants and primary chorion cells were treated with H2O2 at 100, 300, and 500 µM for 24 hours. Cells were fixed for cell senescence-associated ß-galactosidase assay. Cell lysates were harvested for quantitive reverse transcription polymerase chain reaction to quantify SIRT3 messenger RNA. Cell lysates were harvested for Western blot to semi-quantify SIRT3 protein and p38 MAPK phosphorylation levels, respectively. To examine the role of PGRMC1, primary chorion cells underwent the same treatment mentioned above following PGRMC1 knockdown using validated PGRMC1-specific small-interfering RNA. RESULTS: Hydrogen peroxide significantly induced cell senescence and p38 MAPK phosphorylation, and it significantly decreased SIRT3 expression in full-thickness fetal membrane explants and chorion cells. These effects were enhanced by PGRMC1 knockdown. DISCUSSION: This study further demonstrated that oxidative stress-induced cell aging is one of the mechanisms of PPROM and PGRMC1 acts as a protective element for maintaining fetal membrane integrity by inhibiting oxidative stress-induced chorion cell aging.

Infection-induced thrombin production: a potential novel mechanism for preterm premature rupture of membranes (PPROM).

BACKGROUND: Preterm premature rupture of membranes is a leading contributor to maternal and neonatal morbidity and death. Epidemiologic and experimental studies have demonstrated that thrombin causes fetal membrane weakening and subsequently preterm premature rupture of membranes. Although blood is suspected to be the likely source of thrombin in fetal membranes and amniotic fluid of patients with preterm premature rupture of membranes, this has not been proved. Ureaplasma parvum is emerging as a pathogen involved in prematurity, which includes preterm premature rupture of membranes; however, until now, prothrombin production that has been induced directly by bacteria in fetal membranes has not been described. OBJECTIVE: This study was designed to investigate whether Ureaplasma parvum exposure can induce prothrombin production in fetal membranes cells. STUDY DESIGN: Primary fetal membrane cells (amnion epithelial, chorion trophoblast, and decidua stromal) or full-thickness fetal membrane tissue explants from elective, term, uncomplicated cesarean deliveries were harvested. Cells or tissue explants were infected with live Ureaplasma parvum (1×105, 1×106 or 1×107 colony-forming units per milliliter) or lipopolysaccharide (Escherichia coli J5, L-5014; Sigma Chemical Company, St. Louis, MO; 100 ng/mL or 1000 ng/mL) for 24 hours. Tissue explants were fixed for immunohistochemistry staining of thrombin/prothrombin. Fetal membrane cells were fixed for confocal immunofluorescent staining of the biomarkers of fetal membrane cell types and thrombin/prothrombin. Protein and messenger RNA were harvested from the cells and tissue explants for Western blot or quantitative reverse transcription polymerase chain reaction to quantify thrombin/prothrombin protein or messenger RNA production, respectively. Data are presented as mean values ± standard errors of mean. Data were analyzed using 1-way analysis of variance with post hoc Dunnett's test. RESULTS: Prothrombin production and localization were confirmed by Western blot and immunostainings in all primary fetal membrane cells and tissue explants. Immunofluorescence observations revealed a perinuclear localization of prothrombin in amnion epithelial cells. Localization of prothrombin in chorion and decidua cells was perinuclear and cytoplasmic. Prothrombin messenger RNA and protein expression in fetal membranes were increased significantly by Ureaplasma parvum, but not lipopolysaccharide, treatments in a dose-dependent manner. Specifically, Ureaplasma parvum at a dose of 1×107 colony-forming units/mL significantly increased both prothrombin messenger RNA (fold changes in amnion: 4.1±1.9; chorion: 5.7±4.2; decidua: 10.0±5.4; fetal membrane: 9.2±3.0) and protein expression (fold changes in amnion: 138.0±44.0; chorion: 139.6±15.1; decidua: 56.9±29.1; fetal membrane: 133.1±40.0) compared with untreated control subjects. Ureaplasma parvum at a dose of 1×106 colony-forming units/mL significantly up-regulated prothrombin protein expression in chorion cells (fold change: 54.9±5.3) and prothrombin messenger RNA expression in decidua cells (fold change: 4.4±1.9). CONCLUSION: Our results demonstrate that prothrombin can be produced directly by fetal membrane amnion, chorion, and decidua cells. Further, prothrombin production can be stimulated by Ureaplasma parvum exposure in fetal membranes. These findings represent a potential novel underlying mechanism of Ureaplasma parvum-induced rupture of fetal membranes.