Assessing the consequences of the pesticide methoxychlor: neuroendocrine and behavioral measures as indicators of biological impact of an estrogenic environmental chemical

Japanese quail provide an advantageous avian model for assessing long-term biological consequences of endocrine disrupting chemicals (EDCs). These studies examined route of exposure and vulnerability to biological impact of EDCs over the life cycle in a precocial avian model, the Japanese quail. Embryonic exposure occurs with maternal deposition and methoxychlor (MXC) accumulated with maternal exposure. Egg injections of MXC or estradiol at selected stages of development impacted hypothalamic neuroendocrine systems in hatchlings and affected sexual maturation, with evidence for long-term effects on neurotransmitters and male behavior. Two-generation dietary studies were conducted to examine transgenerational effects of EDCs. Adult quail (P1) were exposed to dietary MXC (0, 0.5 and 5 ppm), with continued exposure in their offspring (F1), and control diet for all F2 chicks. Toxicological end points, including fertility, hatching success, and 14-day viability were unaffected. F1 and F2 male offspring from MXC-treated pairs MXC had impaired mating behavior and altered plasma hormones. These studies confirm neuroendocrine and behavioral measures as reliable indices of exposure to an estrogenic EDC. Moreover, maternal deposition remains a primary route of EDC exposure, with potential deleterious consequences for field birds, especially precocial species that appear to be particularly sensitive to embryonic EDC exposure.     

Effects of xenoestrogens on the differentiation of behaviorally-relevant neural circuits

It has become increasingly clear that environmental chemicals have the capability of impacting endocrine function. Moreover, these endocrine disrupting chemicals (EDCs) have long term consequences on adult reproductive function, especially if exposure occurs during embryonic development thereby affecting sexual differentiation. Of the EDCs, most of the research has been conducted on the effects of estrogen active compounds. Although androgen active compounds are also present in the environment, much less information is available about their action. However, in the case of xenoestrogens, there is mounting evidence for long-term consequences of early exposure at a range of doses. In this review, we present data relative to two widely used animal models: the mouse and the Japanese quail. These two species long have been used to understand neural, neuroendocrine, and behavioral components of reproduction and are therefore optimal models to understand how these components are altered by precocious exposure to EDCs. In particular we discuss effects of bisphenol A and methoxychlor on the dopaminergic and noradrenergic systems in rodents and the impact of these alterations. In addition, the effects of embryonic exposure to diethylstilbestrol, genistein or ethylene,1,1-dichloro-2,2-bis(p-chlorophenyl) is reviewed relative to behavioral impairment and associated alterations in the sexually dimorphic parvocellular vasotocin system in quail. We point out how sexually dimorphic behaviors are particularly useful to verify adverse developmental consequences produced by chemicals with endocrine disrupting properties, by examining either reproductive or non-reproductive behaviors.     

Effects of endocrine modulators on sexual differentiation and reproductive function in male Japanese quail

A number of environmental contaminants have been shown to interfere with the endocrine system. Many of these compounds bind to estrogen receptors, thereby potentially disrupting estrogen-regulated functions. In this paper, we review some background data on avian sexual differentiation and present some of the results from our studies on effects of estrogenic chemicals administered during sexual differentiation in the Japanese quail. Initially, our goal was to elucidate whether a decreased male sexual behavior in quail is a suitable endpoint for studying long-term effects of exposure to estrogenic compounds during sexual differentiation in ovo. We thereafter tested some environmental pollutants, suspected to act via mimicking estrogens, using the test system developed. Results from our studies on the synthetic estrogens ethinylestradiol and diethylstilbestrol, as well as the environmental pollutants bisphenol A, tetrabromobisphenol A, and o,p'-DDT are reviewed in this paper. We conclude that the Japanese quail is well suited as an animal model for studying various long-term effects after embryonic exposure to estrogenic compounds. Depressed sexual behavior proved to be the most sensitive of the variables studied in males and we find this endpoint appropriate for studying effects of endocrine modulating chemicals in the adult quail following embryonic exposure.     

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.     

Neuroendocrine Regulation of GnRH and Behavior During Aging in Birds

Avian species exhibit a great variety of life-long patterns in reproduction. Japanese quail are relatively short lived and undergo an age-related loss of reproductive function, making this species an excellent model for the study of the basic biology of aging. Because individuals age at variable rates, sexual behavior has provided a useful index to assess reproductive status of individuals of the same chronological age. Further, exogenous testosterone restores sexual behavior in reproductively senescent male quail, thereby providing evidence for a continued ability of the system to respond. In addition, we have been studying hypothalamic neuroendocrine systems that regulate the endocrine as well as behavioral components of reproduction. Overall, our findings point to the hypothalamic neuroendocrine systems as the site of initial age-related alterations that contribute to the reproductive deterioration. Specifically, we studied adrenergic, opioid peptide, vasotocin, and aromatase systems to understand their relationship to the cGnRH-I system and their potential role in the deterioration of the cGnRH-I system during aging. Our findings provide evidence for qualitative and quantitative alterations in the aromatase enzyme system, which can be partially restored with exogenous testosterone. In addition, other neuronal systems, including the vasotocin system, decline with the loss of gonadal steroids and are restimulated with exogenous testosterone. We will synthesize the data relative to these neuroendocrine systems with attention to the effects of gonadal steroids on these systems during aging.     

Introduction: neurobiological impact of environmental estrogens

This paper provides an introduction to a special issue dedicated to the action of environmental estrogens on neural circuits and behavior. The problem of endocrine disrupting chemicals (EDCs), i.e. chemicals that have the capacity to interfere with the endocrine system, has gained increasing attention as it has become clear that these environmental contaminants may be active in humans, as well as in wildlife and domestic animal species. The majority of the early investigations were aimed at the discovery of the toxicological effects of the EDCs, but biomedical observations were among some of the first indications that estrogenic compounds may exert deleterious effects, even some time after exposure. The data derived from women exposed prenatally to diethylstilbesterol provided powerful evidence for long-term effects and endocrine disruption associated with selected compounds. The examination of wild animal populations exposed to industrial chemicals showed that the chemical exposure, though nonlethal, left the individual impaired or even incapable of reproducing. Among the multiple targets of the action of EDCs, several researches performed in recent years have investigated subtle modifications of the animal behaviors (reproductive, aggressive) that are likely to be related to alterations of specific neural pathways. We have, therefore, focused here on the behavioral studies as one of the more powerful tools to investigate EDCs effects on specific neural circuits.     

Developmental programming and endocrine disruptor effects on reproductive neuroendocrine systems

The ability of a species to reproduce successfully requires the careful orchestration of developmental processes during critical time points, particularly the late embryonic and early postnatal periods. This article begins with a brief presentation of the evidence for how gonadal steroid hormones exert these imprinting effects upon the morphology of sexually differentiated hypothalamic brain regions, the mechanisms underlying these effects, and their implications in adulthood. Then, I review the evidence that aberrant exposure to hormonally-active substances such as exogenous endocrine-disrupting chemicals (EDCs), may result in improper hypothalamic programming, thereby decreasing reproductive success in adulthood. The field of endocrine disruption has shed new light on the discipline of basic reproductive neuroendocrinology through studies on how early life exposures to EDCs may alter gene expression via non-genomic, epigenetic mechanisms, including DNA methylation and histone acetylation. Importantly, these effects may be transmitted to future generations if the germline is affected via transgenerational, epigenetic actions. By understanding the mechanisms by which natural hormones and xenobiotics affect reproductive neuroendocrine systems, we will gain a better understanding of normal developmental processes, as well as develop the potential ability to intervene when development is disrupted.     

The parental brain and behavior: A target for endocrine disruption

During pregnancy, the sequential release of progesterone, 17β-estradiol, prolactin, oxytocin and placental lactogens reorganize the female brain. Brain structures such as the medial preoptic area, the bed nucleus of the stria terminalis and the motivation network including the ventral tegmental area and the nucleus accumbens are reorganized by this specific hormonal schedule such that the future mother will be ready to provide appropriate care for her offspring right at parturition. Any disruption to this hormone pattern, notably by exposures to endocrine disrupting chemicals (EDC), is therefore likely to affect the maternal brain and result in maladaptive maternal behavior. Development effects of EDCs have been the focus of intense study, but relatively little is known about how the maternal brain and behavior are affected by EDCs. We encourage further research to better understand how the physiological hormone sequence prepares the mother’s brain and how EDC exposure could disturb this reorganization.     

Foetal Hypothalamic and Pituitary Expression of Gonadotrophin‐Releasing Hormone and Galanin Systems is Disturbed by Exposure to Sewage Sludge Chemicals via Maternal Ingestion

Animals and humans are chronically exposed to endocrine disrupting chemicals (EDCs) that are ubiquitous in the environment. There are strong circumstantial links between environmental EDC exposure and both declining human/wildlife reproductive health and the increasing incidence of reproductive system abnormalities. The verification of such links, however, is difficult and requires animal models exposed to ‘real life’, environmentally relevant concentrations/mixtures of environmental contaminants (ECs), particularly in utero, when sensitivity to EC exposure is high. The present study aimed to determine whether the foetal sheep reproductive neuroendocrine axis, particularly gondotrophin‐releasing hormone (GnRH) and galaninergic systems, were affected by maternal exposure to a complex mixture of chemicals, applied to pasture, in the form of sewage sludge. Sewage sludge contains high concentrations of a spectrum of EDCs and other pollutants, relative to environmental concentrations, but is frequently recycled to land as a fertiliser. We found that foetuses exposed to the EDC mixture in utero through their mothers had lower GnRH mRNA expression in the hypothalamus and lower GnRH receptor (GnRHR) and galanin receptor (GALR) mRNA expression in the hypothalamus and pituitary gland. Strikingly, this, treatment had no significant effect on maternal GnRH or GnRHR mRNA expression, although GALR mRNA expression within the maternal hypothalamus and pituitary gland was reduced. The present study clearly demonstrates that the developing foetal neuroendocrine axis is sensitive to real‐world mixtures of environmental chemicals. Given the important role of GnRH and GnRHR in the regulation of reproductive function, its known role programming role in utero, and the role of galanin in the regulation of many physiological/neuroendocrine systems, in utero changes in the activity of these systems are likely to have long‐term consequences in adulthood and represent a novel pathway through which EC mixtures could perturb normal reproductive function.     

Cytokines inhibit sexual behavior in female rats: II. Prostaglandins mediate the suppressive effects of interleukin-1beta.

The proinflammatory cytokine interleukin-1 (IL-1) induces several behavioral alterations that are characteristic of illness, such as anorexia and reduced locomotor and social activity. We have recently demonstrated that IL-1 inhibits sexual activity, motivation and attractivity in female, but not in male rats following either central or peripheral administration. In the present study we examined the involvement of prostaglandin (PG) synthesis in mediating IL-1-induced suppression of female sexual behavior. Administration of the cyclooxygenase blockers indomethacin or ibuprofen completely prevented IL-1-induced suppression of female sexual behavior, including the reduction in proceptive behavior, the lordosis response to a male's mounts, and the preference for a sexually active partner. In a subsequent study, ex-vivo release of hypothalamic PGE2 and the secretion of corticosterone (CS) were measured in males and estrous females following IL-1 administration. At the same time and dose of IL-1 administration that significantly reduced sexual behavior in female but not male rats, IL-1 produced a significant increase in PGE2 release in female, but not in male rats. In contrast, IL-1 induced a significant elevation of serum CS levels in males but not in females. These findings suggest that PG synthesis is involved in mediating the effects of IL-1 on female sexual behavior. Furthermore, differential secretion of PGs and CS may underlie the gender difference in the effects of IL-1 on sexual behavior.     

Inhibition of Hypothalamic Aromatase Activity by 5 Beta-Dihydrotestosterone

A variable amount of circulating testosterone that reaches brain cells is converted to biologically inactive 5β-reduced metabolites, namely, 5β-dihydrotestosterone (5β-DHT) and 5β-androstane-3α,17β-diol (5β,3α-diol). In avian species, the production of inactive 5β-DHT and 5β,3α-diol is highest during embryonic and post-hatching life. In the present study, we have investigated the possibility that 5β-reduction may not only correspond to a steroid inactivation pathway, but that 5β-reduced metabolites of testosterone may exert direct inhibitory effects on enzymatic pathways producing biologically active steroids. When added to hypothalamic homogen-ates prepared from adult male doves, 5β-DHT but not 5β,3α-diol inhibits the activity of the aromatase enzyme, which converts testosterone to 17β-oestradiol. During the first days after hatching, when the production of 5β-reduced metabolites is high, the hypothalamic aromatase is also inhibited by 5β-DHT. We conclude that a high 5β-reductase activity during sensitive periods for sexual differentiation may protect the avian brain from the differentiating effects of circulating androgens by inhibiting the production of oestrogen.     

Aromatase in developing sensory systems of the rat brain

Sex differences in the rat brain are dependent, in part, on oestrogen exposure during specific developmental perinatal periods. The availability of oestrogen requires precursor androgen and the presence of intraneuronal aromatase. To examine sites of oestrogen formation and action in the brain, immunocytochemical and biochemical localization of aromatase in the rat brain were determined between embryonic day 14 and postnatal day 20. Aromatase-immunolabelled neuronal profiles were present in hypothalamic, cortical and limbic regions. Surprisingly, aromatase immunoreactivity was also observed in non-limbic regions of the immature brain where it was previously unsuspected. Among these regions, aromatase staining was robust in developing sensory systems, including primary afferents of the olfactory, trigeminal, vestibulocochlear, and visual systems. To determine whether this aromatase is functional in these systems, i.e. converts testosterone to estradiol, the trigeminal nerve was dissected from the hindbrain of perinatal animals and studied for enzyme activity by the tritium release method. The dpm/mg protein/h tritium release in these tissues equalled that of hypothalamic or limbic controls, indicating that these sensory areas are sites of in-situ estradiol synthesis. Our data suggests that aromatase (estradiol)-dependent mechanisms may play a role in the differentiation and maturation of sensory pathways, which, in turn, may contribute to sex differences in the activity of these systems.     

D-amphetamine-related reinforcing effects are reduced in mice exposed prenatally to estrogenic endocrine disruptors

Estrogenic endocrine disruptors are hormonally active compounds that can bind to estradiol receptors. Central dopamine pathways have been reported to be affected by early developmental exposure to estrogenic endocrine disruptors. In the present study, pregnant female CD-1 mice were allowed to drink spontaneously either oil or environmentally relevant low doses of two estrogenic compounds, methoxychlor (20 microg/kg) or bisphenol-A (10 microg/kg) during gestation days 11-18. Their adult offspring were assessed for conditioned place preference produced by D-amphetamine (0, 1 or 2 mg/kg). Interestingly, prenatal treatment effects were sex-dependent and no changes in conditioned place preference emerged for the male offspring. Conversely, a clear-cut profile of D-amphetamine-induced conditioned place preference was only shown by oil-exposed females, whereas exposure to bisphenol-A or methoxychlor resulted in little or no place conditioning. Locomotor effects of acute d-amphetamine were not affected by prenatal exposure to bisphenol-A or methoxychlor. As a whole, prenatal exposure to estrogenic endocrine disruptors affected some steps in the organization of the brain dopaminergic systems in the female offspring, thus leading to long-term alterations in neurobehavioral function. These data confirm that exposure to weak environmental estrogens in the period of brain sexual differentiation can influence adult behavior.     

Anabolic androgenic steroid abuse: multiple mechanisms of regulation of GABAergic synapses in neuroendocrine control regions of the rodent forebrain

Anabolic androgenic steroids (AAS) are synthetic derivatives of testosterone originally developed for clinical purposes but are now predominantly taken at suprapharmacological levels as drugs of abuse. To date, almost 100 different AAS compounds that vary in metabolic fate and physiological effects have been designed and synthesised. Although they are administered for their ability to enhance muscle mass and performance, untoward side effects of AAS use include changes in reproductive and sexual behaviours. Specifically, AAS, depending on the type of compound administered, can delay or advance pubertal onset, lead to irregular oestrous cyclicity, diminish male and female sexual behaviours, and accelerate reproductive senescence. Numerous brains regions and neurotransmitter signalling systems are involved in the generation of these behaviours, and are potential targets for both chronic and acute actions of the AAS. However, critical to all of these behaviours is neurotransmission mediated by GABA(A) receptors within a nexus of interconnected forebrain regions that includes the medial preoptic area, the anteroventral periventricular nucleus and the arcuate nucleus of the hypothalamus. We review how exposure to AAS alters GABAergic transmission and neural activity within these forebrain regions, taking advantage of in vitro systems and both wild-type and genetically altered mouse strains, aiming to better understand how these synthetic steroids affect the neural systems that underlie the regulation of reproduction and the expression of sexual behaviours.     

d-Amphetamine-related reinforcing effects are reduced in mice exposed prenatally to estrogenic endocrine disruptors

Estrogenic endocrine disruptors are hormonally active compounds that can bind to estradiol receptors. Central dopamine pathways have been reported to be affected by early developmental exposure to estrogenic endocrine disruptors. In the present study, pregnant female CD-1 mice were allowed to drink spontaneously either oil or environmentally relevant low doses of two estrogenic compounds, methoxychlor (20 μg/kg) or bisphenol-A (10 μg/kg) during gestation days 11–18. Their adult offspring were assessed for conditioned place preference produced by d-amphetamine (0, 1 or 2 mg/kg). Interestingly, prenatal treatment effects were sex-dependent and no changes in conditioned place preference emerged for the male offspring. Conversely, a clear-cut profile of d-amphetamine-induced conditioned place preference was only shown by oil-exposed females, whereas exposure to bisphenol-A or methoxychlor resulted in little or no place conditioning. Locomotor effects of acute d-amphetamine were not affected by prenatal exposure to bisphenol-A or methoxychlor. As a whole, prenatal exposure to estrogenic endocrine disruptors affected some steps in the organization of the brain dopaminergic systems in the female offspring, thus leading to long-term alterations in neurobehavioral function. These data confirm that exposure to weak environmental estrogens in the period of brain sexual differentiation can influence adult behavior.     

Epigenetic perspective on the developmental effects of bisphenol A

Bisphenol A (BPA) is an estrogenic environmental toxin widely used in the production of plastics and ubiquitous human exposure to this chemical has been proposed to be a potential risk to public health. Animal studies suggest that in utero and early postnatal exposure to this compound may produce a broad range of adverse effects, including impaired brain development, sexual differentiation, behavior, and immune function, which could extend to future generations. Molecular mechanisms that underlie the long-lasting effects of BPA continue to be elucidated, and likely involve disruption of epigenetic programming of gene expression during development. Several studies have provided evidence that maternal exposure to BPA results in postnatal changes in DNA methylation status and altered expression of specific genes in offspring. However, further studies are needed to extend these initial findings to other genes in different tissues, and to examine the correlations between BPA-induced epigenetic alterations, changes in gene expression, and various phenotypic outcomes. It will be also important to explore whether the epigenetic effects of BPA are related to its estrogenic activity, and to determine which downstream effector proteins could mediate changes in DNA methylation. In this review, we will highlight research indicating a consequence of prenatal BPA exposure for brain, behavior, and immune outcomes and discuss evidence for the role of epigenetic pathways in shaping these developmental effects. Based on this evidence, we will suggest future directions in the study of BPA-induced epigenetic effects and discuss the transgenerational implications of exposure to endocrine disrupting chemicals.     

A New Key Neurohormone Controlling Reproduction, Gonadotrophin-Inhibitory Hormone in Birds: Discovery, Progress and Prospects

In vertebrates, the neuropeptide control of gonadotrophin secretion is primarily through the stimulatory action of the hypothalamic decapeptide, gonadotrophin-releasing hormone (GnRH). Gonadal sex steroids and inhibin inhibit gonadotrophin secretion via feedback from the gonads, but a hypothalamic neuropeptide inhibiting gonadotrophin secretion was, until recently, unknown in vertebrates. In 2000, we discovered a novel hypothalamic dodecapeptide that directly inhibits gonadotrophin release in quail and termed it gonadotrophin-inhibitory hormone (GnIH). GnIH acts on the pituitary and GnRH neurones in the hypothalamus via a novel G-protein-coupled receptor for GnIH to inhibit gonadal development and maintenance by decreasing gonadotrophin release and synthesis. The pineal hormone melatonin is a key factor controlling GnIH neural function. GnIH occurs in the hypothalamus of several avian species and is considered to be a new key neurohormone inhibiting avian reproduction. Thus, the discovery of GnIH provides novel directions to investigate neuropeptide regulation of reproduction. This review summarises the discovery, progress and prospects of GnIH, a new key neurohormone controlling reproduction.     

Different effects by sex on hypothalamic-pituitary axis of prepubertal offspring rats produced by in utero and lactational exposure to di-(2-ethylhexyl) phthalate (DEHP)

This study investigated the effect of pre and perinatal exposure to di-(2-ethylhexyl) phthalate (DEHP) on the neuroendocrine parameters that regulate reproduction in prepubertal male and female rats. DEHP at doses of 3 and 30mg/kgbw/day was administered orally in the drinking water to dam rats since pregnancy onset until the moment of pups sacrifice at 15 days of age. In these animals gonadotropin serum level and the hypothalamic contents of the amino acids aspartate, glutamate and gamma-aminobutyric acid were determined. No changes in gonadotropin levels and amino acid neurotransmitters were detected at the low dose in both sexes. However, DEHP administered at high dose (30mg/kgbw/day) to dams produced a significant decrease in the inhibitory neurotransmitter GABA and an increase in the stimulatory neurotransmitter aspartate in prepubertal male offspring rats. These modifications were accompanied by gonadotropin serum levels increase. On the contrary, in treated female rats this chemical increased both, aspartate and GABA, which exert a characteristic stimulatory action on gonadotropin in 15-day-old normal females. This study provides new data about changes produced by DEHP on the hypothalamic amino acid neurotransmitters involved in the neuroendocrine reproductive regulation, in prepubertal male and female rat offspring from dams exposed during gestational and lactational periods. These alterations induced by DEHP exposure could be related to the gonadotropin modifications also described in this work, and with changes in the production of sexual hormones previously reported by other authors.     

Evidence that the deficit in sexual behavior in adult rats neonatally exposed to citalopram is a consequence of 5-HT1 receptor stimulation during development

Neonatal (postnatal days 8-21) exposure of rats to the selective serotonin reuptake inhibitor (SSRI), citalopram, results in persistent changes in behavior including decreased sexual activity in adult animals. We hypothesized that this effect was a consequence of abnormal stimulation of 5-HT(1A) and/or 5-HT(1B) receptors as a result of increased synaptic availability of serotonin during a critical period of development. We examined whether neonatal exposure to a 5-HT(1A) (8OH-DPAT) or a 5-HT(1B) (CGS 12066B) receptor agonist can mimic the effect of neonatal exposure to citalopram on adult sexual behavior. Results showed that neonatal treatment with 5-HT(1B) receptor agonist robustly impaired sexual behavior similar to the effect of citalopram, whereas exposure to 5-HT(1A) receptor agonist only moderately influenced male sexual activity in adult animals. These data support the hypothesis that stimulation of serotonin autoreceptors during development contributes to the adult sexual deficit in rats neonatally exposed to citalopram.