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Experimentally induced depolarization of resting membrane potential in "instructor cells" in Xenopus laevis embryos causes hyperpigmentation in an all-or-none fashion in some tadpoles due to excess proliferation and migration of melanocytes. We showed that this stochastic process involved serotonin signaling, adenosine 3',5'-monophosphate (cAMP), and the transcription factors cAMP response element-binding protein (CREB), Sox10, and Slug. Transcriptional microarray analysis of embryos taken at stage 15 (early neurula) and stage 45 (free-swimming tadpole) revealed changes in the abundance of 45 and 517 transcripts, respectively, between control embryos and embryos exposed to the instructor cell-depolarizing agent ivermectin. Bioinformatic analysis revealed that the human homologs of some of the differentially regulated genes were associated with cancer, consistent with the induced arborization and invasive behavior of converted melanocytes. We identified a physiological circuit that uses serotonergic signaling between instructor cells, melanotrope cells of the pituitary, and melanocytes to control the proliferation, cell shape, and migration properties of the pigment cell pool. To understand the stochasticity and properties of this multiscale signaling system, we applied a computational machine-learning method that iteratively explored network models to reverse-engineer a stochastic dynamic model that recapitulated the frequency of the all-or-none hyperpigmentation phenotype produced in response to various pharmacological and molecular genetic manipulations. This computational approach may provide insight into stochastic cellular decision-making that occurs during normal development and pathological conditions, such as cancer.

In the last few years, it has become clear that a wide variety of environmental contaminants have specific effects on neuroendocrine systems in fish, amphibians, birds and mammals. While it is beyond the scope of this review to provide a comprehensive examination of all of these neuroendocrine disruptors, we will focus on select representative examples. Organochlorine pesticides bioaccumulate in neuroendocrine areas of the brain that directly regulate GnRH neurons, thereby altering the expression of genes downstream of GnRH signaling. Organochlorine pesticides can also agonize or antagonize hormone receptors, adversely affecting crosstalk between neurotransmitter systems. The impacts of polychlorinated biphenyls are varied and in many cases subtle. This is particularly true for neuroedocrine and behavioral effects of exposure. These effects impact sexual differentiation of the hypothalamic-pituitary-gonadal axis, and other neuroendocrine systems regulating the thyroid, metabolic, and stress axes and their physiological responses. Weakly estrogenic and anti-androgenic pollutants such as bisphenol A, phthalates, phytochemicals, and the fungicide vinclozolin can lead to severe and widespread neuroendocrine disruptions in discrete brain regions, including the hippocampus, amygdala, and hypothalamus, resulting in behavioral changes in a wide range of species. Behavioral features that have been shown to be affected by one or more these chemicals include cognitive deficits, heightened anxiety or anxiety-like, sociosexual, locomotor, and appetitive behaviors. Neuroactive pharmaceuticals are now widely detected in aquatic environments and water supplies through the release of wastewater treatment plant effluents. The antidepressant fluoxetine is one such pharmaceutical neuroendocrine disruptor. Fluoxetine is a selective serotonin reuptake inhibitor that can affect multiple neuroendocrine pathways and behavioral circuits, including disruptive effects on reproduction and feeding in fish. There is growing evidence for the association between environmental contaminant exposures and diseases with strong neuroendocrine components, for example decreased fecundity, neurodegeneration, and cardiac disease. It is critical to consider the timing of exposures of neuroendocrine disruptors because embryonic stages of central nervous system development are exquisitely sensitive to adverse effects. There is also evidence for epigenetic and transgenerational neuroendocrine disrupting effects of some pollutants. We must now consider the impacts of neuroendocrine disruptors on reproduction, development, growth and behaviors, and the population consequences for evolutionary change in an increasingly contaminated world. This review examines the evidence to date that various so-called neuroendocrine disruptors can induce such effects often at environmentally-relevant concentrations.

The global prevalence of depression is high during childbearing. Due to the associated risks to the mother and baby, the selective serotonin reuptake inhibitor fluoxetine (FLX) is often the first line of treatment. Given that FLX readily crosses the placenta, a fetus may be susceptible to the disruptive effects of FLX during this highly plastic stage of development. Here, we demonstrate that a 6-day FLX exposure to a fetus-relevant concentration at a critical developmental stage suppresses cortisol levels in the adult zebrafish (F0). This effect persists for three consecutive generations in the unexposed descendants (F1 to F3) without diminution and is more pronounced in males. We also show that the in vivo cortisol response of the interrenal (fish “adrenal”) to an i.p. injection of adrenocorticotropic hormone was also reduced in the males from the F0 and F3 FLX lineages. Transcriptomic profiling of the whole kidney containing the interrenal cells revealed that early FLX exposure significantly modified numerous pathways closely associated with cortisol synthesis in the male adults from the F0 and F3 generations. We also show that the low cortisol levels are linked to significantly reduced exploratory behaviors in adult males from the F0 to F2 FLX lineages. This may be a cause for concern given the high prescription rates of FLX to pregnant women and the potential long-term negative impacts on humans exposed to these therapeutic drugs.

Androgenic chemicals are present in the environment at concentrations that impair reproductive processes in fish. The objective of this experiment was to identify proteins and cell processes mediated through androgen receptor signaling using an androgen receptor agonist (17beta-trenbolone) and antagonist (flutamide) in the liver. Female fathead minnows were exposed to nominal concentrations of either 17beta-trenbolone (0.05, 0.5, or 5 microg/L), flutamide (50, 150, or 500 microg/L), or a mixture (500 microg flutamide/L and 0.5 microg 17beta-trenbolone/L) for 48 h. The iTRAQ method was used to label peptides after protein extraction and trypsin-digestion from livers of untreated controls or from fish treated with 17beta-trenbolone (5 microg/L), flutamide (500 microg/L), or a mixture of both compounds. Forty-five proteins were differentially altered by one or more treatments (p<0.05). Many altered proteins were involved in cellular metabolism (e.g., glyceraldehyde 3-phosphate dehydrogenase, phosphoglycerate mutase), general and oxidative stress response (e.g., superoxide dismutase and heat shock proteins), and the regulation of translation (e.g., ribosomal proteins). Cellular pathway analysis identified additional signaling cascades activated or inhibited by flutamide that may not be androgen receptor mediated. We also compared changes in select proteins to changes in their mRNA levels and observed, in general, that proteins and mRNA changes did not correlate, suggesting complex regulation at the level of both the transcriptome and proteome. It is concluded that both transcriptomic and proteomic approaches offer unique and complementary insights into mechanisms of regulation. We demonstrate the utility of proteomic profiling for use on a model species with value to ecotoxicology but having limited genomic information.

α,β-Unsaturated carbonyls are an important class of chemicals involved in environmental toxicity and disease processes. Whereas adduction of cysteine residues on proteins is a well-documented reaction of these chemicals, such a generic effect cannot explain the molecular mechanism of cytotoxicity. Instead, more detailed information is needed regarding the possible specificity and kinetics of cysteine targeting and the quantitative relationship between adduct burden and protein dysfunction. To address these datagaps, purified human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was incubated with acrylamide (ACR), acrolein or methylvinyl ketone (MVK). Results show that these α,β-unsaturated carbonyl toxicants inhibited GAPDH activity in a concentration-and time-dependent manner. The rank order of enzyme inhibition (KI); i.e., ACR << MVK < acrolein, was related to the calculated electrophilic reactivity of each compound and to the corresponding kinetics of cysteine adduct formation. Tandem mass spectrometry revealed that adduct formation was selective at lower concentrations; i.e., ACR preferentially formed adducts with Cys152 (residues 146-162). At higher concentrations, ACR also formed adducts with Cys156 and Cys247 (residues 235-248). Adduct formation at Cys152 was correlated to enzyme inhibition, which is consistent with the regulatory role of this residue in enzyme function and its location within the GAPDH active site. Further analyses indicated that Cys152 was present in a pKa-lowering microenvironment (pKa = 6.03) and, at physiological pH, the corresponding sulfhydryl group exists in the highly reactive nucleophilic thiolate-state. These data suggest a general cytotoxic mechanism where electrophilic α,β-unsaturated carbonyls selectively form adducts with reactive nucleophilic cysteine residues specifically associated with the active sites of proteins. These specialized cysteine residues are toxicologically relevant molecular targets, since chemical derivitization causes loss of protein function.