Hamid R. Habibi

Low-dose exposure to bisphenol A and replacement bisphenol S induces precocious hypothalamic neurogenesis in embryonic zebrafish

Significance Here we demonstrate that bisphenol A (BPA) exposure during a time point analogous to the second trimester in humans has real and measurable effects on brain development and behavior. Furthermore, our study is the first, to our knowledge, to show that bisphenol S, a replacement used in BPA-free products, equally affects neurodevelopment. These findings suggest that BPA-free products are not necessarily safe and support a societal push to remove all structurally similar bisphenol analogues and other compounds with endocrine-disruptive activity from consumer goods. Our data here, combined with over a dozen physiological and behavioral human studies that begin to point to the prenatal period as a BPA window of vulnerability, suggest that pregnant mothers limit exposure to plastics and receipts. Bisphenol A (BPA), a ubiquitous endocrine disruptor that is present in many household products, has been linked to obesity, cancer, and, most relevant here, childhood neurological disorders such as anxiety and hyperactivity. However, how BPA exposure translates into these neurodevelopmental disorders remains poorly understood. Here, we used zebrafish to link BPA mechanistically to disease etiology. Strikingly, treatment of embryonic zebrafish with very low-dose BPA (0.0068 μM, 1,000-fold lower than the accepted human daily exposure) and bisphenol S (BPS), a common analog used in BPA-free products, resulted in 180% and 240% increases, respectively, in neuronal birth (neurogenesis) within the hypothalamus, a highly conserved brain region involved in hyperactivity. Furthermore, restricted BPA/BPS exposure specifically during the neurogenic window caused later hyperactive behaviors in zebrafish larvae. Unexpectedly, we show that BPA-mediated precocious neurogenesis and the concomitant behavioral phenotype were not dependent on predicted estrogen receptors but relied on androgen receptor-mediated up-regulation of aromatase. Although human epidemiological results are still emerging, an association between high maternal urinary BPA during gestation and hyperactivity and other behavioral disturbances in the child has been suggested. Our studies here provide mechanistic support that the neurogenic period indeed may be a window of vulnerability and uncovers previously unexplored avenues of research into how endocrine disruptors might perturb early brain development. Furthermore, our results show that BPA-free products are not necessarily safer and support the removal of all bisphenols from consumer merchandise.

Impact of glyphosate and glyphosate‐based herbicides on the freshwater environment

Glyphosate [N‐(phosphonomethyl) glycine] is a broad spectrum, post emergent herbicide and is among the most widely used agricultural chemicals globally. Initially developed to control the growth of weed species in agriculture, this herbicide also plays an important role in both modern silviculture and domestic weed control. The creation of glyphosate tolerant crop species has significantly increased the demand and use of this herbicide and has also increased the risk of exposure to non‐target species. Commercially available glyphosate‐based herbicides are comprised of multiple, often proprietary, constituents, each with a unique level of toxicity. Surfactants used to increase herbicide efficacy have been identified in some studies as the chemicals responsible for toxicity of glyphosate‐based herbicides to non‐target species, yet they are often difficult to chemically identify. Most glyphosate‐based herbicides are not approved for use in the aquatic environment; however, measurable quantities of the active ingredient and surfactants are detected in surface waters, giving them the potential to alter the physiology of aquatic organisms. Acute toxicity is highly species dependant across all taxa, with toxicity depending on the timing, magnitude, and route of exposure. The toxicity of glyphosate to amphibians has been a major focus of recent research, which has suggested increased sensitivity compared with other vertebrates due to their life history traits and reliance on both the aquatic and terrestrial environments. This review is designed to update previous reviews of glyphosate‐based herbicide toxicity, with a focus on recent studies of the aquatic toxicity of this class of chemicals. Copyright

Functional Significance of Nuclear Estrogen Receptor Subtypes in the Liver of Goldfish

Estrogens work by binding to and activating specific estrogen receptors (ERs). Although mammals have two major nuclear ER subtypes (ERalpha and ERbeta), three subtypes have been shown in teleost fish (ERalpha, ERbeta-I, and ERbeta-II). 17beta-Estradiol stimulates the production of an egg yolk precursor protein (vitellogenin) in the liver of oviparous species, including the goldfish. However, the functional involvement of the ER subtypes in this process is not fully understood. Here, using primary goldfish hepatocytes, we test the hypothesis that all three ER subtypes are functionally involved in the liver of goldfish by using RNA interference to specifically knock-down the different ER subtypes. The results suggest that ERalpha is induced by estradiol through activation of the ERbeta subtypes. This induction serves to sensitize the liver to further stimulation by estradiol. The knock-down results were supported by use of ER subtype specific antagonists. Sensitization by up-regulation of ERalpha is likely to be important for seasonal spawners such as goldfish, to bring about a change from somatic growth to reproductive development, and vitellogenesis. The novel data presented in this study provide strong support for the hypothesis that the goldfish ER subtypes play functional roles in the regulation of vitellogenin and ERalpha and provide a framework for the better understanding of ER signaling in fish and other vertebrates.

Signal transduction in multifactorial neuroendocrine control of gonadotropin secretion and synthesis in teleosts-studies on the goldfish model.

In teleosts, gonadotropin (GTH) secretion and synthesis is controlled by multiple neuroendocrine factors from the hypothalamus, pituitary and peripheral sources. Pituitary gonadotropes must be able to differentiate and integrate information from these regulators at the cellular and intracellular level. In this article, the intracellular signal transduction mechanisms mediating the actions of some of these regulators, including GTH-releasing hormones, pituitary adenylate cyclase-activating polypeptide, dopamine, ghrelin, sex steroids, activin, and follistatin from experiments with goldfish are reviewed and discussed in relation with recent findings. Information from other teleost models is briefly compared. Goldfish gonadotropes possess multiple pharmacologically distinct intracellular Ca2+ stores that together with voltage-sensitive Ca2+ channels, Na+/H+ exchangers, protein kinase C, arachidonic acid, NO, protein kinase A, ERK/MAPK, and Smads allows for integrated control by different neuroendocrine factors.

Estrogen receptor function and regulation in fish and other vertebrates

Estrogens, steroid hormones critically involved in reproductive processes of vertebrates, signal primarily through their intracellular estrogen receptors (ERs). The ERs belong to a superfamily of nuclear receptors that act as ligand inducible transcription factors. Herein, we review what is known about ER structure, subtypes, mechanism(s) of action and auto-regulation by estrogens. Focus is placed on the ER in fish but comparisons are made to mammals and other vertebrates. Finally, we provide context and a proposed model integrating our knowledge on autoregulation of the receptor and its functions in the liver. Future areas of study are suggested, along with cautions when designing experiments, especially for the detection of endocrine disruptors.

Molecular characterization of LH-β and FSH-β subunits and their regulation by estrogen in the goldfish pituitary

Abstract The gonadal steroids, along with gonadotropin-releasing hormone (GnRH) are involved in the regulation of gonadotropin (GtH) production in vertebrates. Goldfish have an annual reproductive cycle, characterized by seasonal fluctuations in the circulating levels of the reproductive hormones, including 17β-estradiol (E2). The purpose of the present study was to investigate the effect of E2 on basal and GnRH-induced GtH subunit (α, FSH-β and LH-β) gene expression in the goldfish pituitary. Northern analyses were performed to determine changes in steady state mRNA levels. Both in vivo and in vitro treatment with E2 resulted in a stimulation of all three GtH subunit mRNA levels, although a higher concentration was required for the stimulation of the FSH-β subunit mRNA levels. The effect of E2 on GnRH-induced GtH mRNA level was also investigated and demonstrated that E2 influences the GnRH-induced GtH subunit mRNA levels in a seasonally dependent manner. Overall, the present results indicate that E2 stimulates GtH subunit mRNA levels directly at the level of the pituitary in a seasonally dependent manner in goldfish.

Seasonal effect of GnIH on gonadotrope functions in the pituitary of goldfish.

Gonadotropin-inhibitory hormone (GnIH) inhibits gonadotropin release in birds and mammals. To investigate its role in teleosts, we examined the effects of synthetic goldfish (g)GnIH on pituitary LH-β and FSH-β subunit, and gGnIH receptor (gGnIH-R) mRNA levels and LH secretion in goldfish. Intraperitoneal injections of gGnIH increased pituitary LH-β and FSH-β mRNA levels at early to late gonadal recrudescence, but reduced serum LH and pituitary gGnIH-R mRNA levels, respectively, at early to mid-recrudescence and later stages of recrudescence. Static incubation with gGnIH elevated LH secretion from dispersed pituitary cell cultures from prespawning fish, but not at other recrudescent stages; suppressed LH-β mRNA levels at early recrudescence and prespawning but elevated LH-β at mid-recrudescence; and consistently attenuated FSH-β mRNA in a dose-specific manner. Results indicate that in goldfish, regulation of LH secretion and gonadotropin subunit mRNA levels are dissociated in the presence of gGnIH and dependent on maturational status and administration route.

Activity of vertebrate gonadotropin-releasing hormones and analogs with variant amino acid residues in positions 5, 7 and 8 in the goldfish pituitary

All non-mammalian vertebrates as well as marsupial mammals have two or more forms of gonadotropin-releasing hormone (GnRH) in the brain. Goldfish brain and pituitary contains two molecular forms of GnRH, salmon GnRH ([Trp7, Leu8]m-GnRH; s-GnRH) and chicken GnRH-II ([His5, Trp7, Tyr8]m-GnRH; cII-GnRH). Both sGnRH and cII-GnRH stimulate gonadotropin (GtH) as well as growth hormone (GH) release from the goldfish pituitary. The purpose of the present study was to study the activity of the five known forms of GnRHs as well as analogs of mammalian GnRH (m-GnRH) with variant amino acid residues in positions 5, 7 and 8 in terms of binding to GnRH receptors, and release of GTH and GH from the perifused fragments of goldfish pituitary in vitro. All five vertebrate GnRH peptides stimulated both GtH and GH release in a dose-dependent manner, although their potencies were very different. cII-GnRH was somewhat more active than s-GnRH in releasing GtH, whereas s-GnRH tended to have a greater potency than cII-GnRH in terms of GH release. Both chicken GnRH-I (cI-GnRH) and lamprey GnRH (l-GnRH) were significantly less potent than mGnRH, s-GnRH and cII-GnRH in releasing GtH and GH. cII-GnRH binds with higher affinity for the high affinity binding sites compared to all other native peptides. The activity of [Trp7]-GnRH was similar to both s-GnRH and cII-GnRH in releasing GtH and GH. Substitution of His5 resulted in a significant decrease in GtH releasing potencies compared to mGnRH, sGnRH and cII-GnRH. [His5]-GnRH also had lower GH releasing potency than mGnRH and sGnRH. Tyr8, His8 and Leu8 substitutions caused significant decreases in GtH releasing potencies compared to mGnRH, s-GnRH and cII-GnRH, but did not cause a significant change in GH releasing potency. The combination of [His5, Trp7]-GnRH had GtH and GH releasing activities similar to m-GnRH, s-GnRH and cII-GnRH.(ABSTRACT TRUNCATED AT 400 WORDS)

Molecular characterization and sex-related seasonal expression of thyroid receptor subtypes in goldfish

The thyroid hormones, acting through the nuclear thyroid receptors (TRs), play important roles in the growth and development of vertebrates. The present study investigated the molecular structure and season-related expression of the TR isoforms in the male and female goldfish pituitary, brain, liver, gonads, gut, heart, and muscle. Based on sequence alignment with other species, the results demonstrate the presence of: (1) a TRalpha (TRalpha-1) consisting of 1496 nucleotides encoding a 466 amino acid protein, (2) a novel splice variant of TRalpha (TRalpha-2) containing an out-of-frame deletion of 246 nucleotides in the ligand-binding domain consisting of 1251 nucleotides encoding a 378 amino acid protein, (3) a novel transcript resembling TRalpha, except for non-homology in the hinge region and a premature stop codon prior to the ligand-binding domain (TRalpha-truncated; 1418 nucleotides, 206 amino acid protein), and (4) TRbeta consisting of 1823 nucleotides encoding a 395 amino acid protein. The findings provide the first demonstration of the presence of a truncated TR isoform in non-mammalian vertebrates. In goldfish, the expression patterns for all TRs subtypes were found to be remarkably similar in both male and female, changing significantly before and during reproductive season. The results provide a frame work for better understanding of the functional significance of novel TR forms and TR subtypes in fish and other vertebrates.

Thyroid receptor subtypes: Structure and function in fish

Thyroid hormones are important regulators of vertebrate growth and development, and are under the control of the hypothalamic-pituitary-thyroid axis. Nuclear thyroid receptors (TRs), which act as inducible transcription factors, mediate cellular functions of thyroid hormones. The molecular structure of several subtypes of TRs have been elucidated in vertebrate species, including N-terminal truncations as well as C-terminal variations in the domain responsible for binding hormone. In this paper, we review current information on the thyroid receptors studied in the vertebrate species with emphasis on recent findings in goldfish concerning functional significance of the thyroid receptor subtypes.