Regrettable Substitutes and the Brain: What Animal Models and Human Studies Tell Us about the Neurodevelopmental Effects of Bisphenol, Per- and Polyfluoroalkyl Substances, and Phthalate Replacements.

In recent decades, emerging evidence has identified endocrine and neurologic health concerns related to exposure to endocrine-disrupting chemicals (EDCs), including bisphenol A (BPA), certain per- and polyfluoroalkyl compounds (PFASs), and phthalates. This has resulted in consumer pressure to remove these chemicals from the market, especially in food-contact materials and personal care products, driving their replacement with structurally or functionally similar substitutes. However, these "new-generation" chemicals may be just as or more harmful than their predecessors and some have not received adequate testing. This review discusses the research on early-life exposures to new-generation bisphenols, PFASs, and phthalates and their links to neurodevelopmental and behavioral alterations in zebrafish, rodents, and humans. As a whole, the evidence suggests that BPA alternatives, especially BPAF, and newer PFASs, such as GenX, can have significant effects on neurodevelopment. The need for further research, especially regarding phthalate replacements and bio-based alternatives, is briefly discussed.

The medial preoptic area and acute cocaine's stimulant effects in rats: Potential influences of estradiol and biological sex.

The medial preoptic area (mPOA) in the hypothalamus is an important integrator of neuroendocrine signaling and a key regulator of both natural and drug-induced reward. Although the mPOA modulates sex differences in other behaviors, whether it also modulates sex differences in cocaine response remains unclear. To help us better understand the mPOA's role in sex differences associated with cocaine response, we examined cocaine-induced changes in locomotion and neural activity in the mPOA of male and female rats. In addition, neural activity in the striatum, a brain area known to be involved in cocaine response, was examined for comparison purposes. Fos, the protein product of the immediate early gene c-fos, was used as the marker of neural activity. Locomotion chambers were used to measure behavior, radioimmunoassays and vaginal lavages were used to determine hormonal status, and immunohistochemical assays were used to quantify Fos. To account for the effects of gonadal hormones, rats were left gonadally intact and categorized as either 'low-estradiol' or 'high-estradiol' based on their hormonal status on test day. Results indicate that high-estradiol females experienced greater cocaine-induced mPOA Fos-immunoreactivity (Fos-ir) and displayed greater cocaine-induced locomotion than low estradiol females. Conversely, high-estradiol males experienced less cocaine-induced mPOA Fos-ir and displayed less cocaine-induced locomotion than low-estradiol males. Cocaine-induced Fos-ir in the mPOA also correlated with cocaine-induced Fos-ir in areas of the striatum already associated with cocaine response. These findings further support the mPOA's role in the endocrine-mediated response to cocaine. It also identifies the mPOA as a contributor to sex differences in cocaine response and potential differences in vulnerability to developing cocaine use disorders.

Transgenerational effects of polychlorinated biphenyls: 2. Hypothalamic gene expression in rats†.

Polychlorinated biphenyls (PCBs) are endocrine-disrupting chemicals (EDCs) with well-established effects on reproduction and behavior in developmentally-exposed (F1) individuals. Because of evidence for transgenerational effects of EDCs on the neuroendocrine control of reproductive physiology, we tested the hypothesis that prenatal PCB exposure leads to unique hypothalamic gene-expression profiles in three generations. Pregnant Sprague-Dawley rats were treated on gestational days 16 and 18 with the PCB mixture Aroclor 1221 (A1221), vehicle (3% DMSO in sesame oil), or estradiol benzoate (EB, 50 µg/kg), the latter a positive control for estrogenic effects of A1221. Maternal- and paternal-lineage F2 and F3 generations were bred using untreated partners. The anteroventral periventricular nucleus (AVPV) and arcuate nucleus (ARC), involved in the hypothalamic control of reproduction, were dissected from F1 to F3 females and males, RNA extracted, and gene expression measured in a qPCR array. We detected unique gene-expression profiles in each generation, which were sex- and lineage-specific. In the AVPV, treatment significantly changed 10, 25, and 11 transcripts in F1, F2, and F3 generations, whereas 10, 1, and 12 transcripts were changed in these generations in the ARC. In the F1 AVPV and ARC, most affected transcripts were decreased by A1221. In the F2 AVPV, most effects of A1221 were observed in females of the maternal lineage, whereas only Pomc expression changed in the F2 ARC (by EB). The F3 AVPV and ARC were mainly affected by EB. It is notable that results in one generation do not predict results in another, and that lineage was a major determinant in results. Thus, transient prenatal exposure of F1 rats to A1221 or EB can alter hypothalamic gene expression across three generations in a sex- and lineage-dependent manner, leading to the conclusion that the legacy of PCBs continues for generations.

Daily GnRH agonist treatment delays the development of reproductive physiology and behavior in male rats.

The present study was designed to examine the effects of suppressing pubertal onset with leuprolide acetate, a gonadotropin releasing hormone (GnRH) agonist. Starting on postnatal day (PD) 25, male Long-Evans rats were injected daily with either leuprolide acetate (25 µg/kg dissolved in 0.9% sterile physiological saline; n = 13) or sterile physiological saline (1.0 ml/kg 0.9% NaCl; n = 14) for a total of 25 days. Males were monitored daily for signs of puberty (i.e., preputial separation). On the last day of leuprolide treatment (PD 50), half of each treatment group was injected with 10.0 µg of estradiol benzoate (EB) daily for three consecutive days (PD 50-52) and 1.0 mg of progesterone (P) on the 4th day (PD 53), whereas the other half of each treatment group received oil injections. Four hours after P injections, all subjects were given the opportunity to interact with a gonadally-intact male and a sexually receptive female rat (i.e., a partner-preference test with and without physical contact). Copulatory behavior and sexual motivation were measured. Hormone injections and mating tests were repeated weekly for a total of 3 consecutive weeks. Results showed that leuprolide delayed puberty as well as the development of copulatory behavior and the expression of sexual motivation. By the last test, the leuprolide-treated subjects showed signs of catching up, however, many continued to be delayed. Estradiol and progesterone mildly feminized male physiology (e.g., decreased testes weight and serum testosterone) and behavior (e.g., increased lordosis), but did not interact with leuprolide treatment.

Endocrine-disrupting chemicals: Effects on neuroendocrine systems and the neurobiology of social behavior.

A contribution to SBN/ICN special issue. Endocrine-disrupting chemicals (EDCs) are pervasive in the environment. They are found in plastics and plasticizers (bisphenol A (BPA) and phthalates), in industrial chemicals such as polychlorinated biphenyls (PCBs), and include some pesticides and fungicides such as vinclozolin. These chemicals act on hormone receptors and their downstream signaling pathways, and can interfere with hormone synthesis, metabolism, and actions. Because the developing brain is particularly sensitive to endogenous hormones, disruptions by EDCs can change neural circuits that form during periods of brain organization. Here, we review the evidence that EDCs affect developing hypothalamic neuroendocrine systems, and change behavioral outcomes in juvenile, adolescent, and adult life in exposed individuals, and even in their descendants. Our focus is on social, communicative and sociosexual behaviors, as how an individual behaves with a same- or opposite-sex conspecific determines that individual's ability to exist in a community, be selected as a mate, and reproduce successfully.

Maternal care modulates transgenerational effects of endocrine-disrupting chemicals on offspring pup vocalizations and adult behaviors.

Endocrine-disrupting chemicals (EDCs) can act upon a developing organism to change its endocrine health and behavior in adulthood. Beyond actions on the exposed individuals, transgenerational effects of several EDCs have been reported. This study assessed the combinatorial impact of EDC-altered maternal care and transgenerational inheritance on F3 male and female offspring. Pregnant rats were exposed to EDCs with different modes of action: the weakly estrogenic polychlorinated biphenyl (PCB) mixture Aroclor 1221, the anti-androgenic fungicide vinclozolin (VIN), or the vehicle (6% dimethylsulfoxide in sesame oil; VEH) during embryonic development. The F1 male and female offspring were bred through the paternal- or maternal-lineage with untreated partners to generate F2 offspring. This process was repeated through both maternal and paternal lineages to create the F3 generation. Maternal care of F2 dams towards their F3 offspring was altered in a lineage-dependent manner, particularly in PCB paternal-lineage animals. When F3 pups were recorded for ultrasonic vocalizations (USVs) following separation from the mother, the rate of neonatal USVs in F3 offspring were decreased in PCB paternal-lineage pups. In adulthood, anxiety-like behaviors of the F3 rats were tested, with only small effects of EDCs detected. These interactions of maternal behaviors and EDC effects across generations, especially via the paternal lineage, has implications for health and environmental responses in wildlife and humans.

Hypothyroidism induced by loss of the manganese efflux transporter SLC30A10 may be explained by reduced thyroxine production.

SLC30A10 and SLC39A14 are manganese efflux and influx transporters, respectively. Loss-of-function mutations in genes encoding either transporter induce hereditary manganese toxicity. Patients have elevated manganese in the blood and brain and develop neurotoxicity. Liver manganese is increased in patients lacking SLC30A10 but not SLC39A14. These organ-specific changes in manganese were recently recapitulated in knockout mice. Surprisingly, Slc30a10 knockouts also had elevated thyroid manganese and developed hypothyroidism. To determine the mechanisms of manganese-induced hypothyroidism and understand how SLC30A10 and SLC39A14 cooperatively mediate manganese detoxification, here we produced Slc39a14 single and Slc30a10/Slc39a14 double knockout mice and compared their phenotypes with that of Slc30a10 single knockouts. Compared with wild-type controls, Slc39a14 single and Slc30a10/Slc39a14 double knockouts had higher manganese levels in the blood and brain but not in the liver. In contrast, Slc30a10 single knockouts had elevated manganese levels in the liver as well as in the blood and brain. Furthermore, SLC30A10 and SLC39A14 localized to the canalicular and basolateral domains of polarized hepatic cells, respectively. Thus, transport activities of both SLC39A14 and SLC30A10 are required for hepatic manganese excretion. Compared with Slc30a10 single knockouts, Slc39a14 single and Slc30a10/Slc39a14 double knockouts had lower thyroid manganese levels and normal thyroid function. Moreover, intrathyroid thyroxine levels of Slc30a10 single knockouts were lower than those of controls. Thus, the hypothyroidism phenotype of Slc30a10 single knockouts is induced by elevated thyroid manganese, which blocks thyroxine production. These findings provide new insights into the mechanisms of manganese detoxification and manganese-induced thyroid dysfunction.

Deficiency in the manganese efflux transporter SLC30A10 induces severe hypothyroidism in mice.

Manganese is an essential metal that becomes toxic at elevated levels. Loss-of-function mutations in SLC30A10, a cell-surface-localized manganese efflux transporter, cause a heritable manganese metabolism disorder resulting in elevated manganese levels and parkinsonian-like movement deficits. The underlying disease mechanisms are unclear; therefore, treatment is challenging. To understand the consequences of loss of SLC30A10 function at the organism level, we generated Slc30a10 knock-out mice. During early development, knock-outs were indistinguishable from controls. Surprisingly, however, after weaning and compared with controls, knock-out mice failed to gain weight, were smaller, and died prematurely (by ∼6-8 weeks of age). At 6 weeks, manganese levels in the brain, blood, and liver of the knock-outs were ∼20-60-fold higher than controls. Unexpectedly, histological analyses revealed that the brain and liver of the knock-outs were largely unaffected, but their thyroid exhibited extensive alterations. Because hypothyroidism leads to growth defects and premature death in mice, we assayed for changes in thyroid and pituitary hormones. At 6 weeks and compared with controls, the knock-outs had markedly reduced thyroxine levels (∼50-80%) and profoundly increased thyroid-stimulating hormone levels (∼800-1000-fold), indicating that Slc30a10 knock-out mice develop hypothyroidism. Importantly, a low-manganese diet produced lower tissue manganese levels in the knock-outs and rescued the phenotype, suggesting that manganese toxicity was the underlying cause. Our unanticipated discovery highlights the importance of determining the role of thyroid dysfunction in the onset and progression of manganese-induced disease and identifies Slc30a10 knock-out mice as a new model for studying thyroid biology.

Epigenetic impacts of endocrine disruptors in the brain.

The acquisition of reproductive competence is organized and activated by steroid hormones acting upon the hypothalamus during critical windows of development. This review describes the potential role of epigenetic processes, particularly DNA methylation, in the regulation of sexual differentiation of the hypothalamus by hormones. We examine disruption of these processes by endocrine-disrupting chemicals (EDCs) in an age-, sex-, and region-specific manner, focusing on how perinatal EDCs act through epigenetic mechanisms to reprogram DNA methylation and sex steroid hormone receptor expression throughout life. These receptors are necessary for brain sexual differentiation and their altered expression may underlie disrupted reproductive physiology and behavior. Finally, we review the literature on histone modifications and non-coding RNA involvement in brain sexual differentiation and their perturbation by EDCs. By putting these data into a sex and developmental context we conclude that perinatal EDC exposure alters the developmental trajectory of reproductive neuroendocrine systems in a sex-specific manner.

Sex differences in effects of gestational polychlorinated biphenyl exposure on hypothalamic neuroimmune and neuromodulator systems in neonatal rats.

Polychlorinated biphenyls (PCBs) are ubiquitous in the environment and exposure to them is associated with immune, endocrine and neural dysfunction. Effects of PCBs on inflammation and immunity are best described in spleen and blood, with fewer studies on neural tissues. This is an important gap in knowledge, as molecules typically associated with neuroinflammation also serve neuromodulatory roles and interact with hormones in normal brain development. The current study used Sprague-Dawley rats to assess whether gestational PCB exposure altered hypothalamic gene expression and serum cytokine concentration in neonatal animals given an immune challenge. Dams were fed wafers containing a mixture of PCBs at an environmentally relevant dose and composition (20 µg/kg, 1:1:1 Aroclor 1242:1248:1254) or oil vehicle control throughout their pregnancy. One day old male and female offspring were treated with an inflammatory challenge (lipopolysaccharide, LPS, 50 µg/kg, sc) or saline vehicle control approximately 3.5 h prior to tissue collection. Across both basal and activated inflammatory states, PCB exposure caused greater expression of a subset of inflammatory genes in the hypothalamus and lower expression of genes involved in dopamine, serotonin, and opioid systems compared to oil controls. PCB exposure also altered reactions to inflammatory challenge: it reversed the normal decrease in Esr2 hypothalamic expression and induced an abnormal increase in IL-1b and IL-6 serum concentration in response to LPS. Many of these effects were sex specific. Given the potential long-term consequences of neuroimmune disruption, our findings demonstrate the need for further research.

Mate choice, sexual selection, and endocrine-disrupting chemicals.

Humans have disproportionately affected the habitat and survival of species through environmental contamination. Important among these anthropogenic influences is the proliferation of organic chemicals, some of which perturb hormone systems, the latter referred to as endocrine-disrupting chemicals (EDCs). EDCs are widespread in the environment and affect all levels of reproduction, including development of reproductive organs, hormone release and regulation through the life cycle, the development of secondary sexual characteristics, and the maturation and maintenance of adult physiology and behavior. However, what is not well-known is how the confluence of EDC actions on the manifestation of morphological and behavioral sexual traits influences mate choice, a process that requires the reciprocal evaluation of and/or acceptance of a sexual partner. Moreover, the outcomes of EDC-induced perturbations are likely to influence sexual selection; yet this has rarely been directly tested. Here, we provide background on the development and manifestation of sexual traits, reproductive competence, and the neurobiology of sexual behavior, and evidence for their perturbation by EDCs. Selection acts on individuals, with the consequences manifest in populations, and we discuss the implications for EDC contamination of these processes, and the future of species.

The timing and duration of estradiol treatment in a rat model of the perimenopause: Influences on social behavior and the neuromolecular phenotype.

This study tested the effects of timing and duration of estradiol (E2) treatment, factors that are clinically relevant to hormone replacement in perimenopausal women, on social behavior and expression of genes in brain regions that regulate these behaviors. Female rats were ovariectomized (OVX) at 1year of age, roughly equivalent to middle-age in women, and given E2 or vehicle for different durations (3 or 6months) and timing (immediately or after a 3-month delay) relative to OVX. Social and ultrasonic vocalization (USV) behaviors were assessed at the 3 and 6month timepoints, and the rats' brains were then used for gene expression profiling in hypothalamus (supraoptic nucleus, paraventricular nucleus), bed nucleus of the stria terminalis, medial amygdala, and prefrontal cortex using a 48-gene qPCR platform. At the 3-month post-OVX testing period, E2 treatment significantly decreased the number of frequency-modulated USVs emitted. No effects of hormone were found at the 6-month testing period. There were few effects of timing and duration of E2 in a test of social preference of a rat given a choice between her same-sex cagemate and a novel conspecific. For gene expression, effects of timing and duration of E2 were region-specific, with the majority of changes found for genes involved in regulating social behavior such as neuropeptides (Oxt, Oxtr &Avp), neurotransmitters (Drd1, Drd2, Htr2a, Grin2d &Gabbr1), and steroid hormone receptors (Esr2, Ar, Pgr). These data suggest that the mode of E2 treatment has specific effects on social behavior and expression of target genes involved in the regulation of these behaviors.

Transgenerational effects of polychlorinated biphenyls: 1. Development and physiology across 3 generations of rats.

BACKGROUND: Polychlorinated biphenyls (PCBs) are persistent organic environmental contaminants and known endocrine-disrupting chemicals (EDCs). Previous studies demonstrated that developmental exposure to the weakly estrogenic PCB mixture Aroclor 1221 (A1221) in Sprague-Dawley rats altered sexual development, adult reproductive physiology and body weight. The current study tested the hypothesis that prenatal A1221 exposure not only disrupts these endpoints within an exposed individual's (F1 generation) lifespan, but may also affect subsequent generations (F2-F3). METHODS: We treated pregnant female rats on embryonic days (E) 16 and E18 with A1221 (1 mg/kg), estradiol benzoate (50 µg/kg, positive estrogenic control), or vehicle (3% DMSO in sesame oil, negative control). Endpoints related to sexually dimorphic developmental trajectories of reproductive and developmental physiology were measured, and as adults, reproductive endocrine status was assessed, in the F1, F2, and F3 generations. RESULTS: Significant effects of transgenerational EDCs were found for body weight and serum hormones. The A1221 descendants had significantly higher body weight in the F2-maternal lineage throughout postnatal development, and in F3-maternal lineage animals after weaning. In females, generation- and lineage-specific effects of exposure were found for serum progesterone and estradiol. Specifically, serum progesterone concentrations were lower in F2-A1221 females, and higher in F3-A1221 females, compared to their respective F2- and F3-vehicle counterparts. Serum estradiol concentrations were higher in F3-A1221 than F3-vehicle females. Reproductive and adrenal organ weights, birth outcomes, sex ratio, and estrous cycles, were unaffected. It is notable that effects of A1221 were only sometimes mirrored by the estrogenic control, EB, indicating that the mechanism of action of A1221 was likely via non-estrogenic pathways. CONCLUSIONS: PCBs caused body weight and hormonal effects in rats that were not observed in the directly exposed F1 offspring, but emerged in F2 and F3 generations. Furthermore, most effects were in the maternal lineage; this may relate to the timing of exposure of the F1 fetuses at E16 and 18, when germline (the future F2 generation) epigenetic changes diverge in the sexes. These results showing transgenerational effects of EDCs have implications for humans, as we are now in the 3rd generation since the Chemical Revolution of the mid-twentieth century, and even banned chemicals such as PCBs have a persistent imprint on the health of our descendants.

Specific effects of prenatal DEHP exposure on neuroendocrine gene expression in the developing hypothalamus of male rats.

Endocrine disrupting chemicals may disrupt developing neuroendocrine systems, especially when the exposure occurs during a critical period. This study aimed to investigate whether prenatal exposure to di-(2-ethylhexyl) phthalate (DEHP), a major component of plasticizers used worldwide, disrupted the development of a network of genes important for neuroendocrine function in male rats. Pregnant rats were treated with corn oil (vehicle control), 2, 10 or 50 mg/kg DEHP by gavage from gestational day 14 to 19. The neuroendocrine gene expressions were quantified using a 48-gene Taqman qPCR array in the whole hypothalamus of neonatal rats (postnatal day 1) and in the anteroventral periventricular nucleus (AVPV), medial preoptic nucleus (MPN) and arcuate nucleus (ARC) of adult rats (postnatal day 70). Immunofluorescent signals of ERα and CYP19 were detected using the confocal microscopy in adult AVPV, MPN and ARC. The results showed that prenatal DEHP exposure perturbed somatic and reproductive development of offspring. Eleven genes were down-regulated in neonatal hypothalamus and showed non-monotonic dose-response relationships, that the 10 mg/kg DEHP dosage was associated with the greatest number of gene expression changes. Different from this, 14 genes were altered in adult AVPV, MPN and ARC and most of alterations were found in the 50 mg/kg DEHP group. Also, 50 mg/kg DEHP reduced ERα expression in the ARC, but no alterations were observed in CYP19 expression. These results indicated that prenatal DEHP exposure may perturb hypothalamic gene programming and the influences are permanent. The effects showed dependence on developmental stages and nuclei region.

The effects of long-term estradiol treatment on social behavior and gene expression in adult female rats.

This study tested the effects of long-term estradiol (E2) replacement on social behavior and gene expression in brain nuclei involved in the regulation of these social behaviors in adult female rats. We developed an ultrasonic vocalization (USV) test and a sociability test to examine communications, social interactions, and social preference, using young adult female cagemates. All rats were ovariectomized (OVX) and implanted with a Silastic capsule containing E2 or vehicle, and housed in same-treatment pairs for a 3-month period. Then, rats were behaviorally tested, euthanized, and 5 nuclei in the brain's social decision-making circuit were selected for neuromolecular profiling by a multiplex qPCR method. Our novel USV test proved to be a robust tool to measure numbers and types of calls emitted by cagemates that had been reintroduced after a 1-week separation. Results also showed that E2-treated OVX rats had profoundly decreased numbers of USV calls compared to vehicle-treated OVX rats. In a test of sociability, in which a female was allowed to choose between her cagemate or a same-treatment novel rat, we found few effects of E2 compared to vehicle, although interestingly, rats chose the cagemate over an unfamiliar conspecific. Gene expression results revealed that the supraoptic nucleus had the greatest number of gene changes caused by E2: Oxt, Oxtr and Avp were increased, and Drd2, Htr1a, Grin2b, and Gabbr1 were decreased, by E2. No genes were affected in the prefrontal cortex, and 1-4 genes were changed in paraventricular nucleus (Pgr), bed nucleus of the stria terminalis (Oxtr, Esr2, Dnmt3a), and medial amygdala (Oxtr, Ar, Foxp1, Tac3). Thus, E2 changes communicative interactions between adult female rats, together with selected expression of genes in the brain, especially in the supraoptic nucleus.

Age-related changes in sexual function and steroid-hormone receptors in the medial preoptic area of male rats.

Testosterone is the main circulating steroid hormone in males, and acts to facilitate sexual behavior via both reduction to dihydrotestosterone (DHT) and aromatization to estradiol. The mPOA is a key site involved in mediating actions of androgens and estrogens in the control of masculine sexual behavior, but the respective roles of these hormones is not fully understood. As males age they show impairments in sexual function, and a decreased facilitation of behavior by steroid hormones compared to younger animals. We hypothesized that an anatomical substrate for these behavioral changes is a decline in expression and/or activation of hormone receptor-sensitive cells in the mPOA. We tested this by quantifying and comparing numbers of AR- and ERα-containing cells, and Fos as a marker of activated neurons, in the mPOA of mature (4-5months) and aged (12-13months) male rats, assessed one hour after copulation to one ejaculation. Numbers of AR- and ERα cells did not change with age or after sex, but the percentage of AR- and ERα-cells that co-expressed Fos were significantly up-regulated by sex, independent of age. Age effects were found for the percentage of Fos cells that co-expressed ERα (up-regulated in the central mPOA) and the percentage of Fos cells co-expressing AR in the posterior mPOA. Interestingly, serum estradiol concentrations positively correlated with intromission latency in aged but not mature animals. These data show that the aging male brain continues to have high expression and activation of both AR and ERα in the mPOA with copulation, raising the possibility that differences in relationships between hormones, behavior, and neural activation may underlie some age-related impairments.

Anxiety-like behaviors in adulthood are altered in male but not female rats exposed to low dosages of polychlorinated biphenyls in utero.

Exposure to polychlorinated biphenyls (PCBs), a class of endocrine-disrupting chemicals, can result in altered reproductive behavior in adulthood, especially when exposure occurs during critical periods of brain sexual differentiation in the fetus. Whether PCBs alter other sexually dimorphic behaviors such as those involved in anxiety is poorly understood. To address this, pregnant rat dams were injected twice, on gestational days 16 and 18, with the weakly estrogenic PCB mixture Aroclor 1221 (A1221) at one of two low dosages (0.5mg/kg or 1.0mg/kg, hereafter 1.0 and 0.5), estradiol benzoate (EB; 50µg/kg) as a positive estrogenic control, or the vehicle (3% DMSO in sesame oil). We also conducted a comprehensive assessment of developmental milestones of the F1 male and female offspring. There were no effects of treatment on sex ratio at birth and age at eye opening. Puberty, assessed by vaginal opening in females and preputial separation in males, was not affected in females but was advanced in males treated with A1221 (1.0). Males and females treated with A1221 (both dosages) were heavier in early adulthood relative to controls. The earliest manifestation of this effect developed in males prior to puberty and in females slightly later, during puberty. Anxiety-like behaviors were tested using the light:dark box and elevated plus maze tests in adulthood. In females, anxiety behaviors were unaffected by treatment. Males treated with A1221 (1.0) showed reduced indices of anxiety and increased activity in the light:dark box but not the elevated plus maze. EB failed to replicate the phenotype produced by A1221 for any of the developmental and behavioral endpoints. Collectively, these results indicate that PCBs increase body weight in both sexes, but their effects on anxiety-like behaviors are specific to males. Furthermore, differences between the results of A1221 and EB suggest that the PCBs are likely acting through mechanisms distinct from their estrogenic activity.

Two-hit exposure to polychlorinated biphenyls at gestational and juvenile life stages: 1. Sexually dimorphic effects on social and anxiety-like behaviors.

Endocrine disrupting chemicals (EDCs) are widespread environmental contaminants that affect many neuroendocrine functions. The brain is particularly vulnerable to EDCs during critical periods of gestational development when gonadal hormones exert organizational effects on sexually dimorphic behaviors later in life. Peripubertal development is also a time of continued neural sensitivity to organizing effects of hormones, yet little is known about EDC actions at these times. We sought to determine effects of prenatal or juvenile exposures to a class of EDCs, polychlorinated biphenyls (PCBs) at human-relevant dosages on development, physiology, and social and anxiety-related behaviors later in life, and the consequences of a second juvenile "hit" following prenatal treatment. We exposed male and female Sprague-Dawley rats to PCBs (Aroclor 1221, 1mg/kg/day, ip injection) and/or vehicle during prenatal development (embryonic days 16, 18, 20), juvenile development (postnatal days 24, 26, 28), or both. These exposures had differential effects on behaviors in sex and age-dependent ways; while prenatal exposure had more effects than juvenile, juvenile exposure often modified or unmasked the effects of the first hit. Additionally, females exhibited altered social and anxiety behavior in adolescence, while males displayed small but significant changes in sociosexual preferences in adulthood. Thus, the brain continues to be sensitive to organizing effects of EDCs through juvenile development. As humans are exposed to EDCs throughout multiple periods in their life, these findings have implications for our understanding of EDC effects on physiology and behavior.

Age and Long-Term Hormone Treatment Effects on the Ultrastructural Morphology of the Median Eminence of Female Rhesus Macaques.

The median eminence (ME) of the hypothalamus comprises the hypothalamic nerve terminals, glia (especially tanycytes) and the portal capillary vasculature that transports hypothalamic neurohormones to the anterior pituitary gland. The ultrastructure of the ME is dynamically regulated by hormones and undergoes organizational changes during development and reproductive cycles in adult females, but relatively little is known about the ME during aging, especially in nonhuman primates. Therefore, we used a novel transmission scanning electron microscopy technique to examine the cytoarchitecture of the ME of young and aged female rhesus macaques in a preclinical monkey model of menopausal hormone treatments. Rhesus macaques were ovariectomized and treated for 2 years with vehicle, estradiol (E2), or estradiol + progesterone (E2 + P4). While the overall cytoarchitecture of the ME underwent relatively few changes with age and hormones, changes to some features of neural and glial components near the portal capillaries were observed. Specifically, large neuroterminal size was greater in aged compared to young adult animals, an effect that was mitigated or reversed by E2 alone but not by E2 + P4 treatment. Overall glial size and the density and tissue fraction of the largest subset of glia were greater in aged monkeys, and in some cases reversed by E2 treatment. Mitochondrial size was decreased by E2, but not E2 + P4, only in aged macaques. These results contrast substantially with work in rodents, suggesting that the ME of aging macaques is less vulnerable to age-related disorganization, and that the effects of E2 on monkeys' ME are age specific.

The effects of prenatal PCBs on adult social behavior in rats.

Endocrine disrupting chemical (EDC) exposures during critical periods of development may influence neuronal development and the manifestation of sexually dimorphic sociability and social novelty behaviors in adulthood. In this study, we assessed the effects of gestational exposure to PCBs on the social behavior of males and females later in adulthood. A weakly estrogenic PCB mixture, Aroclor 1221 (A1221, 0.5 or 1mg/kg) was administered to pregnant Sprague-Dawley rat dams. Both a positive control (estradiol benzoate; EB, 50µg/kg) and negative control (dimethylsulfoxide; DMSO in sesame oil vehicle) were similarly administered to separate sets of dams. The sexes responded differently in two tasks essential to sociality. Using a three-chamber apparatus that contained a caged, same-sex, gonadectomized stimulus animal and an empty stimulus cage, we found that both sexes showed a strong preference for affiliating with a stimulus animal (vs. an empty cage), an effect that was much more pronounced in the males. In the second task, a novel and a familiar stimulus animal were caged at opposite ends of the same apparatus. Females displayed a higher degree of novelty preference than the males. During both tests, females had significantly higher social approach behaviors while male engaged in significantly more interactive behaviors with the conspecific. Of particular interest, males born of dams that received prenatal A1221 (0.5mg/kg) exhibited an overall decrease in nose-to-nose investigations. These behavioral data suggest that the males are more sensitive to A1221 treatment than are females. In addition to behavioral analysis, serum corticosterone was measured. Females born of dams treated with A1221 (0.5mg/kg) had significantly higher concentrations of corticosterone than the DMSO female group; males were unaffected. Females also had significantly higher corticosterone concentrations than did males. Overall, our results suggest that the effects of gestational exposure to PCBs on adult social behavior are relatively limited within this particular paradigm.