Skeletal effects following developmental flame-retardant exposure are specific to sex and chemical class in the adult Wistar rat.

Introduction: Accumulating evidence reveals that endocrine disrupting chemicals (EDCs) can disrupt aspects of metabolic programming, suggesting that skeletal development may be at risk, a possibility that is rarely examined. The commercial flame retardant (FR) mixture, Firemaster 550 (FM 550), has repeatedly been shown to negatively influence metabolic programming, raising concerns that skeletal integrity may consequently be impaired. We have previously shown that gestational and lactational exposure to 1,000 µg FM 550 negatively affected sex-specific skeletal traits in male, but not female, rats assessed at 6 months of age. Whether this outcome is primarily driven by the brominated (BFR) or organophosphate ester (OPFR) portions of the mixture or the effects persist to older ages is unknown. Materials and methods: To address this, in the present study, dams were orally exposed throughout gestation and lactation to either 1,000 µg BFR, 1,000 µg OPFR, or 2,000 µg FM 550. Offspring (n = 8/sex/exposure) were weaned at PND 21 and assessed for femoral cortical and trabecular bone parameters at 8 months of age by high-resolution X-ray micro-computed tomography (micro-CT). Serum levels of serotonin, osteocalcin, alkaline phosphatase, and calcium were quantified. Results: FM 550 affected both sexes, but the females were more appreciably impacted by the OPFRs, while the males were more vulnerable to the BFRs. Conclusion: Although sex specificity was expected due to the sexual dimorphic nature of skeletal physiology, the mechanisms accounting for the male- and female-specific phenotypes remain to be determined. Future work aims to clarify these unresolved issues.

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.

Reproductive and developmental toxicity following exposure to organophosphate ester flame retardants and plasticizers, triphenyl phosphate and isopropylated phenyl phosphate, in Sprague Dawley rats.

Two organophosphate esters used as flame retardants and plasticizers, triphenyl phosphate (TPHP) and isopropylated phenyl phosphate (IPP), have been detected in environmental samples around the world. Human exposure primarily occurs via oral ingestion with reported higher concentrations in children. Currently, there are no data to evaluate potential risk from exposure to either TPHP or IPP during fetal development. These short-term perinatal studies in rats provide preliminary toxicity data for TPHP and IPP, including information on transfer to fetus/offspring and across the pup blood-brain barrier. In separate experiments, TPHP or IPP were administered via dosed feed at concentrations 0, 1000, 3000, 10 000, 15 000, or 30 000 ppm to time-mated Hsd:Sprague Dawley SD rats from gestation day (GD) 6 through postnatal day (PND) 28; offspring were provided dosed feed at the same concentration as their dam (PND 28-PND 56). TPHP- and IPP-related toxicity resulted in removal of both 30 000 ppm groups on GD 12 and 15 000 ppm IPP group after parturition. Body weight and organ weights were impacted with exposure in remaining dams. Reproductive performance was perturbed at ≥10 000 ppm TPHP and all IPP exposure groups. In offspring, both TPHP- and IPP-related toxicity was noted in pups at ≥10 000 ppm as well as reduction in bodyweights, delays in pubertal endpoints, and/or reduced cholinesterase enzyme activity starting at 1000 ppm TPHP or IPP. Preliminary internal dose assessment indicated gestational and lactational transfer following exposure to TPHP or IPP. These findings demonstrate that offspring development is sensitive to 1000 ppm TPHP or IPP exposure.

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.

Roadbumps at the Crossroads of Integrating Behavioral and In Vitro Approaches for Neurotoxicity Assessment.

With the appreciation that behavior represents the integration and complexity of the nervous system, neurobehavioral phenotyping and assessment has seen a renaissance over the last couple of decades, resulting in a robust database on rodent performance within various testing paradigms, possible associations with human disorders, and therapeutic interventions. The interchange of data across behavior and other test modalities and multiple model systems has advanced our understanding of fundamental biology and mechanisms associated with normal functions and alterations in the nervous system. While there is a demonstrated value and power of neurobehavioral assessments for examining alterations due to genetic manipulations, maternal factors, early development environment, the applied use of behavior to assess environmental neurotoxicity continues to come under question as to whether behavior represents a sensitive endpoint for assessment. Why is rodent behavior a sensitive tool to the neuroscientist and yet, not when used in pre-clinical or chemical neurotoxicity studies? Applying new paradigms and evidence on the biological basis of behavior to neurobehavioral testing requires expertise and refinement of how such experiments are conducted to minimize variability and maximize information. This review presents relevant issues of methods used to conduct such test, sources of variability, experimental design, data analysis, interpretation, and reporting. It presents beneficial and critical limitations as they translate to the in vivo environment and considers the need to integrate across disciplines for the best value. It proposes that a refinement of behavioral assessments and understanding of subtle pronounced differences will facilitate the integration of data obtained across multiple approaches and to address issues of translation.

Perinatal exposure to FireMaster® 550 (FM550), brominated or organophosphate flame retardants produces sex and compound specific effects on adult Wistar rat socioemotional behavior.

Firemaster 550 (FM550) is a flame retardant (FR) mixture that has become one of the most commonly used FRs in household items such as foam-based furniture and baby products. Because this mixture readily leaches from products, contamination of the environment and human tissues is widespread. Prior work by us and others has reported sex-specific behavioral deficits in rodents and zebrafish following early life exposure. In an effort to understand the mechanisms by which these behavioral effects occur, here we explored the effects of its constituents on behavioral outcomes previously shown to be altered by developmental FM550 exposure. The FM550 commercial mixture is composed of two brominated compounds (BFR) and two organophosphate compounds (OPFRs) at almost equivalent proportions. Both the BFR and the OPFR components are differentially metabolized and structurally distinct, but similar to known neurotoxicants. Here we examined adult Wistar rat offspring socioemotional behaviors following perinatal exposure (oral, to the dam) to vehicle, 2000 µg/day FM550, 1000 µg/day BFR or 1000 µg/day OPFR from gestation day 0 to weaning. Beginning on postnatal day 65 offspring from all groups were subjected to a series of behavioral tasks including open field, elevated plus maze, marble burying, social interaction tests, and running wheel. Effects were exposure-, sex- and task-specific, with BFR exposure resulting in the most consistent behavioral deficits. Overall, exposed females showed more deficits compared to males across all dose groups and tasks. These findings help elucidate how different classes of flame retardants, independently and as a mixture, contribute to sex-specific behavioral effects of exposure.

Perinatal bisphenol A (BPA) exposure alters brain oxytocin receptor (OTR) expression in a sex- and region- specific manner: A CLARITY-BPA consortium follow-up study.

Bisphenol A (BPA) is a well-characterized endocrine disrupting chemical (EDC) used in plastics, epoxy resins and other products. Neurodevelopmental effects of BPA exposure are a major concern with multiple rodent and human studies showing that early life BPA exposure may impact the developing brain and sexually dimorphic behaviors. The CLARITY-BPA (Consortium Linking Academic and Regulatory Insights on BPA Toxicity) program was established to assess multiple endpoints, including neural, across a wide dose range. Studies from our lab as part of (and prior to) CLARITY-BPA have shown that BPA disrupts estrogen receptor expression in the developing brain, and some evidence of oxytocin (OT) and oxytocin receptor (OTR) disruption in the hypothalamus and amygdala. While BPA disruption of steroid hormone function is well documented, less is known about its capacity to alter nonapeptide signals. In this CLARITY-BPA follow up study, we used remaining juvenile rat tissues to test the hypothesis that developmental BPA exposure affects OTR expression across the brain. Perinatal BPA exposure (2.5, 25, or 2500 µg/kg body weight (bw)/day) spanned gestation and lactation with dams gavaged from gestational day 6 until birth and then the offspring gavaged directly through weaning. Ethinyl estradiol (0.5 µg/kg bw/day) was used as a reference estrogen. Animals of both sexes were sacrificed as juveniles and OTR expression assessed by receptor binding. Our results demonstrate prenatal exposure to BPA can eliminate sex differences in OTR expression in three hypothalamic regions, and that male OTR expression may be more susceptible. Our data also identify a sub-region of the BNST with sexually dimorphic OTR expression not previously reported in juvenile rats that is also susceptible to BPA.

Social Context, Stress, Neuropsychiatric Disorders, and the Vasopressin 1b Receptor.

The arginine vasopressin 1b receptor (Avpr1b) is involved in the modulation of a variety of behaviors and is an important part of the mammalian hormonal stress axis. The Avpr1b is prominent in hippocampal CA2 pyramidal cells and in the anterior pituitary corticotrophs. Decades of research on this receptor has demonstrated its importance to the modulation of social recognition memory, social forms of aggression, and modulation of the hypothalamic-pituitary-adrenal axis, particularly under conditions of acute stress. Further, work in humans suggests that the Avpr1b may play a role in human neuropsychiatric disorders and its modulation may have therapeutic potential. This paper reviews what is known about the role of the Avpr1b in the context of social behaviors, the stress axis, and human neuropsychiatric disorders. Further, possible mechanisms for how Avpr1b activation within the hippocampus vs. Avpr1b activation within anterior pituitary may interact with one another to affect behavioral output are proposed.

Oxytocin and behavior: Lessons from knockout mice.

It is well established that the nonapeptide oxytocin (Oxt) is important for the neural modulation of behaviors in many mammalian species. Since its discovery in 1906 and synthesis in the early 1950s, elegant pharmacological work has helped identify specific neural substrates on which Oxt exerts its effects. More recently, mice with targeted genetic disruptions of the Oxt system-i.e., both the peptide and its receptor (the Oxtr)-have further defined Oxt's actions and laid some important scientific groundwork for studies in other species. In this article, we highlight the scientific contributions that various mouse knockouts of the Oxt system have made to our understanding of Oxt's modulation of behavior. We specifically focus on how the use of these mice has shed light on our understanding of social recognition memory, maternal behavior, aggression, and several nonsocial behaviors. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 190-201, 2017.

Genotypic differences in intruder-evoked immediate early gene activation in male, but not female, vasopressin 1b receptor knockout mice.

BACKGROUND: The neuropeptide arginine vasopressin (Avp) modulates social behaviors via its two centrally expressed receptors, the Avp 1a receptor and the Avp 1b receptor (Avpr1b). Recent work suggests that, at least in mice, Avp signaling through Avpr1b within the CA2 region of the hippocampus is critical for normal aggressive behaviors and social recognition memory. However, this brain area is just one part of a larger neural circuit that is likely to be impacted in Avpr1b knockout (-/-) mice. To identify other brain areas that are affected by altered Avpr1b signaling, genotypic differences in immediate early gene activation, i.e. c-FOS and early growth response factor 1 (EGR-1), were quantified using immunocytochemistry following a single exposure to an intruder. RESULTS: In females, no genotypic differences in intruder-evoked c-FOS or EGR-1 immunoreactivity were observed in any of the brain areas measured. In males, while there were no intruder-evoked genotypic differences in c-FOS immunoreactivity, genotypic differences were observed in EGR-1 immunoreactivity within the ventral bed nucleus of the stria terminalis and the anterior hypothalamus; with Avpr1b -/- males having less EGR-1 immunoreactivity in these regions than controls. CONCLUSIONS: These data are the first to identify specific brain areas that may be a part of a neural circuit that includes Avpr1b-expressing cells in the CA2 region of the hippocampus. It is thought that this circuit, when working properly, plays a role in how an animal evaluates its social context.