Analysis of Sublethal Toxicity in Developing Zebrafish Embryos Exposed to a Range of Petroleum Substances.

The Organisation for Economic Co-operation and Development (OECD) test guideline 236 (fish embryo acute toxicity test; 2013) relies on 4 endpoints to describe exposure-related effects (coagulation, lack of somite formation, tail-bud detachment from the yolk sac, and the presence of a heartbeat). Danio rerio (zebrafish) embryos were used to investigate these endpoints along with a number of additional sublethal effects (cardiac dysfunction, pericardial edema, yolk sac edema, tail curvature, hatch success, pericardial edema area, craniofacial malformation, swim bladder development, fin development, and heart rate) following 5-d exposures to 7 petroleum substances. The substances investigated included 2 crude oils, 3 gas oils, a diluted bitumen, and a petrochemical containing a mixture of branched alcohols. Biomimetic extraction-solid-phase microextraction (BE-SPME) was used to quantify freely dissolved concentrations of test substances as the exposure metric. The results indicated that the most prevalent effects observed were pericardial and yolk sac edema, tail curvature, and lack of embryo viability. A BE-SPME threshold was determined to characterize sublethal morphological alterations that preceded embryo mortality. Our results aid in the understanding of aquatic hazards of petroleum substances to developing zebrafish beyond traditional OECD test guideline 236 endpoints and show the applicability of BE-SPME as a simple analytical tool that can be used to predict sublethal embryo toxicity. Environ Toxicol Chem 2019;38:1302-1312. © 2019 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.

Editor's Highlight: Development of an In vitro Assay Measuring Uterine-Specific Estrogenic Responses for Use in Chemical Safety Assessment.

A toxicity pathway approach was taken to develop an in vitro assay using human uterine epithelial adenocarcinoma (Ishikawa) cells as a replacement for measuring an in vivo uterotrophic response to estrogens. The Ishikawa cell was determined to be fit for the purpose of recapitulating in vivo uterine response by verifying fidelity of the biological pathway components and the dose-response predictions to women of child-bearing age. Expression of the suite of estrogen receptors that control uterine proliferation (ERα66, ERα46, ERα36, ERß, G-protein coupled estrogen receptor (GPER)) were confirmed across passages and treatment conditions. Phenotypic responses to ethinyl estradiol (EE) from transcriptional activation of ER-mediated genes, to ALP enzyme induction and cellular proliferation occurred at concentrations consistent with estrogenic activity in adult women (low picomolar). To confirm utility of this model to predict concentration-response for uterine proliferation with xenobiotics, we tested the concentration-response for compounds with known uterine estrogenic activity in humans and compared the results to assays from the ToxCast and Tox21 suite of estrogen assays. The Ishikawa proliferation assay was consistent with in vivo responses and was a more sensitive measure of uterine response. Because this assay was constructed by first mapping the key molecular events for cellular response, and then ensuring that the assay incorporated these events, the resulting cellular assay should be a reliable tool for identifying estrogenic compounds and may provide improved quantitation of chemical concentration response for in vitro-based safety assessments.

Multi-well plate immunoassays for measuring signaling protein activations/deactivations and membrane vs. intracellular receptor levels.

We developed fixed-cell multi-well plate immunoassays that increase the throughput and ease of quantification for questions formerly assessed by immunoblot scanning. The assays make use of the now abundant antibodies designed to recognize receptor subtypes and posttranslationally modified signaling proteins. By optimizing permeabilization and fixation conditions, mainly based on specific cell types, the assay can be adapted to the study of many different antigens of importance to hormonal and neurotransmitter signaling scenarios.

Are we ready to consider transgenerational epigenetic effects in human health risk assessment?

Recently, there has been a growing concern that chemically or nutritionally mediated epigenetic changes might lead to adverse health outcomes. The natural question is whether the existing chemical safety assessment paradigm is or is not protective of epigenetic-mediated effects, and if there is a need to incorporate new endpoints to specifically address epigenetics. Of particular interest are transgenerational epigenetic effects, which can be passed on through multiple generations. To investigate these questions, a comparison was performed between OECD guideline rat toxicology studies versus several rat transgenerational epigenetic studies. This analysis focused on vinclozolin owing to the availability of a comprehensive suite of dose-response data (NOAEL, reference dose, and human exposure estimates) for both conventional and epigenetic endpoints. This analysis revealed that vinclozolin transgenerational effects were demonstrated at a dose level (100 mg/kg/day) that was: (1) ∼40-fold higher than the overall lowest-observed-adverse-effect level (LOAEL) from rat guideline studies, (2) ∼80-fold higher than the lowest NOAEL from rat guideline studies, (3) ∼80,000-fold higher than the reference dose for the molecule, and (4) ∼1.2-million fold above human exposure estimates. Through this analysis, we conclude that additional research across a spectrum of doses is necessary to elucidate the interplay between epigenetics and apical endpoints before considering epigenetics in human health risk assessment. Therefore, we recommend focusing future research toward (1) examining for potential causal relationships between epigenetic alterations and adverse apical endpoints, and (2) understanding the dose-response relationship of these causal epigenetic alterations when compared with those of the apical endpoints.

Characterization of membrane receptor binding activity for cortisol in the liver and kidney of the euryhaline teleost, Mozambique tilapia (Oreochromis mossambicus).

Glucocorticoids (GCs) regulate an array of physiological responses in vertebrates. Genomic GC actions mediated by nuclear steroid receptors require a lag time on the order of hours to days to generate an appreciable physiological response. Experimental evidence has accumulated that GCs, can also act rapidly through a nongenomic mechanism to modulate cellular physiology in vertebrates. Causal evidence in the Mozambique tilapia (Oreochromis mossambicus) suggests that the GC cortisol exerts rapid, nongenomic actions in the gills, liver, and pituitary of this euryhaline teleost, but the membrane receptor mediating these actions has not been characterized. Radioreceptor binding assays were conducted to identify a putative GC membrane receptor site in O. mossambicus. The tissue distribution, binding kinetics, and pharmacological signature of the GC membrane-binding activity were characterized. High affinity (Kd=9.527±0.001 nM), low-capacity (Bmax=1.008±0.116 fmol/mg protein) [(3)H] cortisol binding was identified on plasma membranes prepared from the livers and a lower affinity (Kd=30.08±2.373 nM), low capacity (Bmax=4.690±2.373 fmol/mg protein) binding was found in kidney membrane preparations. Competitors with high binding affinity for nuclear GC receptors, mifepristone (RU486), dexamethasone, and 11-deoxycorticosterone, displayed no affinity for the membrane GC receptor. The association and dissociation kinetics of [(3)H] cortisol binding to membranes were orders of magnitude faster (t1/2=1.7-2.6 min) than those for the intracellular (nuclear) GC receptor (t1/2=10.2h). Specific [(3)H] cortisol membrane binding was also detected in the gill and pituitary but not in brain tissue. This study represents the first characterization of a membrane GC receptor in fishes and one of only a few characterized in vertebrates.

Is the current product safety assessment paradigm protective for epigenetic mechanisms?

INTRODUCTION: The emerging field of epigenetics has revealed a new layer of gene regulation that is only now being fully explored. Concomitant with the increase in our understanding of epigenetic regulation are questions as to the role environmental factors may play in altering the epigenome. As these correlations between epigenetic changes and toxicity are made, the natural next question is if the current safety assessment paradigm utilizing a no-observed-adverse-effect level (NOAEL) is protective of public health for an epigenetic mechanism. METHODS: To begin to answer this question, several case studies were examined where apical end point dose response curves were compared to dose response data on epigenetic end points for 1,3-butadiene, arsenic, and diethylstilbesterol. RESULTS: This limited examination of the available literature for these three molecules revealed that epigenetic alterations largely fell within the dose response curve for apical effects. Perhaps more importantly, this analysis also revealed some key data gaps that should be addressed such as incongruent study designs and limited epigenetic dose response data for only a small subset of known epigenetic marks. Taken together, the answer to the question of whether the current product safety assessment paradigm is protective of epigenetic alterations is "yes, based on our current understanding of epigenetics". That is, this paradigm would be protective of any mechanism that resulted in adverse effects typically observed in guideline studies, because product safety assessment is based upon observed apical effects to drive an overall NOAEL that is the basis to set reference doses for a risk assessment. DISCUSSION: These adverse apical effects are the culmination of all molecular events, regardless of mechanism and may include alterations in the epigenome secondary to the actions of those mechanism(s). The epigenome is in a constant state of flux throughout cellular growth and development, and this dynamic variability is not completely characterized. Thus given the state of our current scientific understanding, a change in itself cannot be contextualized as adverse in the absence of a phenotypic anchor. Clearly, more research is needed in this area to perform additional epigenetic studies that include apical end points with full dose response curves in order to gain a more comprehensive understanding of adverse health outcomes that could be causally linked to epigenetic changes.

Estrogens of multiple classes and their role in mental health disease mechanisms.

Gender and sex hormones can influence a variety of mental health states, including mood, cognitive development and function, and vulnerability to neurodegenerative diseases and brain damage. Functions of neuronal cells may be altered by estrogens depending upon the availability of different physiological estrogenic ligands; these ligands and their effects vary with life stages, the genetic or postgenetic regulation of receptor levels in specific tissues, or the intercession of competing nonphysiological ligands (either intentional or unintentional, beneficial to health or not). Here we review evidence for how different estrogens (physiological and environmental/dietary), acting via different estrogen receptor subtypes residing in alternative subcellular locations, influence brain functions and behavior. We also discuss the families of receptors and transporters for monoamine neurotransmitters and how they may interact with the estrogenic signaling pathways.

Nongenomic mechanisms of physiological estrogen-mediated dopamine efflux.

BACKGROUND: Neurological diseases and neuropsychiatric disorders that vary depending on female life stages suggest that sex hormones may influence the function of neurotransmitter regulatory machinery such as the dopamine transporter (DAT). RESULTS: In this study we tested the rapid nongenomic effects of several physiological estrogens [estradiol (E2), estrone (E1), and estriol (E3)] on dopamine efflux via the DAT in a non-transfected, NGF-differentiated, rat pheochromocytoma (PC12) cell model that expresses membrane estrogen receptors (ERs) alpha, beta, and GPR30. We examined kinase, ionic, and physical interaction mechanisms involved in estrogenic regulation of the DAT function. E2-mediated dopamine efflux is DAT-specific and not dependent on extracellular Ca2+-mediated exocytotic release from vesicular monoamine transporter vesicles (VMATs). Using kinase inhibitors we also showed that E2-mediated dopamine efflux is dependent on protein kinase C and MEK activation, but not on PI3K or protein kinase A. In plasma membrane there are ligand-independent associations of ERalpha and ERbeta (but not GPR30) with DAT. Conditions which cause efflux (a 9 min 10(-9) M E2 treatment) cause trafficking of ERalpha (stimulatory) to the plasma membrane and trafficking of ERbeta (inhibitory) away from the plasma membrane. In contrast, E1 and E3 can inhibit efflux with a nonmonotonic dose pattern, and cause DAT to leave the plasma membrane. CONCLUSION: Such mechanisms explain how gender biases in some DAT-dependent diseases can occur.

Differential regulation of dopamine transporter function and location by low concentrations of environmental estrogens and 17beta-estradiol.

BACKGROUND: The effects of 17beta-estradiol (E2) and xenoestrogens (XEs) on dopamine transport may have important implications for the increased incidence of neurologic disorders, especially in women during life stages characterized by frequent hormonal fluctuations. OBJECTIVE: We examined low concentrations of XEs [dieldrin, endosulfan, o', p'-dichlorodiphenyl-ethylene (DDE), nonylphenol (NP), and bisphenol A (BPA)] for nongenomic actions via action of membrane estrogen receptors (ERs). METHODS: We measured activity of the dopamine transporter (DAT) by the efflux of 3H-dopamine in nontransfected nerve growth factor-differentiated PC12 rat pheochromocytoma cells expressing membrane DAT, ER-alpha, ER-beta, and G-protein-coupled receptor 30. We used a plate immunoassay to monitor trafficking of these proteins. RESULTS: All compounds at 1 nM either caused efflux or inhibited efflux, or both; each compound evoked a distinct oscillatory pattern. At optimal times for each effect, we examined different concentrations of XEs. All XEs were active at some concentration < 10 nM, and dose responses were all nonmonotonic. For example, 10(-14) to 10(-11) M DDE caused significant efflux inhibition, whereas NP and BPA enhanced or inhibited efflux at several concentrations. We also measured the effects of E2/XE combinations; DDE potentiated E(2)-mediated dopamine efflux, whereas BPA inhibited it. In E2-induced efflux, 15% more ER-alpha trafficked to the membrane, whereas ER-beta waned; during BPA-induced efflux, 20% more DAT was trafficked to the plasma membrane. CONCLUSIONS: Low levels of environmental estrogen contaminants acting as endocrine disruptors via membrane ERs can alter dopamine efflux temporal patterning and the trafficking of DAT and membrane ERs, providing a cellular mechanism that could explain the disruption of physiologic neurotransmitter function.

Surface-expressed enolase contributes to the pathogenesis of clinical isolate SSU of Aeromonas hydrophila.

In this study, we demonstrated that the surface-expressed enolase from diarrheal isolate SSU of Aeromonas hydrophila bound to human plasminogen and facilitated the latter's tissue-type plasminogen activator-mediated activation to plasmin. The bacterial surface-bound plasmin was more resistant to the action of its specific physiological inhibitor, the antiprotease alpha(2)-antiplasmin. We found that immunization of mice with purified recombinant enolase significantly protected the animals against a lethal challenge dose of wild-type (WT) A. hydrophila. Minimal histological changes were noted in organs from mice immunized with enolase and then challenged with WT bacteria compared to severe pathological changes found in the infected and nonimmunized group of animals. This correlated with the smaller bacterial load of WT bacteria in the livers and spleens of enolase-immunized mice than that found in the nonimmunized controls. We also showed that the enolase gene could potentially be important for the viability of A. hydrophila SSU as we could delete the chromosomal copy of the enolase gene only when another copy of the targeted gene was supplied in trans. By site-directed mutagenesis, we altered five lysine residues located at positions 343, 394, 420, 427, and 430 of enolase in A. hydrophila SSU; the mutated forms of enolase were hyperexpressed in Escherichia coli, and the proteins were purified. Our results indicated that lysine residues at positions 420 and 427 of enolase were crucial in plasminogen-binding activity. We also identified a stretch of amino acid residues ((252)FYDAEKKEY(260)) in the A. hydrophila SSU enolase involved in plasminogen binding. To our knowledge, this is the first report of the direct involvement of surface-expressed enolase in the pathogenesis of A. hydrophila SSU infections and of any gram-negative bacteria in general.

The roles of membrane estrogen receptor subtypes in modulating dopamine transporters in PC-12 cells.

The effects of 17beta-estradiol (E(2)) on dopamine (DA) transport could explain gender and life-stage differences in the incidence of some neurological disorders. We tested the effects of E(2) at physiological concentrations on DA efflux in nerve growth factor-differentiated rat pheochromocytoma cells that express estrogen receptors (ER) alpha, ERbeta, and G-protein coupled receptor 30 (GPR30), and DA transporter (DAT). DAT efflux was determined as the transporter-specific loss of (3)H-DA from pre-loaded cells; a 9-15 min 10(-9 )M E(2) treatment caused maximal DA efflux. Such rapid estrogenic action suggests a non-genomic response, and an E(2)-dendrimer conjugate (limited to non-nuclear actions) caused DA efflux within 5 min. Efflux dose-responses for E(2) were non-monotonic, also characteristic of non-genomic estrogenic actions. ERalpha siRNA knockdown abolished E(2)-mediated DA efflux, while ERbeta knockdown did not, and GPR30 knockdown increased E(2)-mediated DA efflux (suggesting GPR30 is inhibitory). Use of ER-selective agonists/antagonists demonstrated that ERalpha is the predominant mediator of E(2)-mediated DA efflux, with inhibitory contributions from GPR30 and ERbeta. E(2) also caused trafficking of ERalpha to the plasma membrane, trafficking of ERbeta away from the plasma membrane, and unchanged membrane GPR30 levels. Therefore, ERalpha is largely responsible for non-genomic estrogenic effects on DAT activity.

Xenoestrogens are potent activators of nongenomic estrogenic responses.

Studies of the nuclear transcriptional regulatory activities of non-physiological estrogens have not explained their actions in mediating endocrine disruption in animals and humans at the low concentrations widespread in the environment. However, xenoestrogens have rarely been tested for their ability to participate in the plethora of nongenomic steroid signaling pathways elucidated over the last several years. Here we review what is known about such responses in comparison to our recent evidence that xenoestrogens can rapidly and potently elicit signaling through nongenomic pathways culminating in functional endpoints. Both estradiol (E(2)) and compounds representing various classes of xenoestrogens (diethylstilbestrol, coumestrol, bisphenol A, DDE, nonylphenol, endosulfan, and dieldrin) act via a membrane version of the estrogen receptor-alpha on pituitary cells, and can provoke Ca(2+) influx via L-type channels, leading to prolactin (PRL) secretion. These hormones and mimetics can also cause the oscillating activation of extracellular regulated kinases (ERKs). However, individual estrogen mimetics differ in their potency and temporal phasing of these activations compared to each other and to E(2). It is perhaps in these ways that they disrupt some endocrine functions when acting in combination with physiological estrogens. Our quantitative assays allow comparison of these outcomes for each mimetic, and let us build a detailed picture of alternative signaling pathway usage. Such an understanding should allow us to determine the estrogenic or antiestrogenic potential of different types of xenoestrogens, and help us to develop strategies for preventing xenoestrogenic disruption of estrogen action in many tissues.

Estradiol effects on the dopamine transporter - protein levels, subcellular location, and function.

BACKGROUND: The effects of estrogens on dopamine (DA) transport may have important implications for the increased incidence of neurological disorders in women during life stages when hormonal fluctuations are prevalent, e.g. during menarche, reproductive cycling, pregnancy, and peri-menopause. RESULTS: The activity of the DA transporter (DAT) was measured by the specific uptake of 3H-DA. We found that low concentrations (10(-14) to 10(-8) M) of 17beta-estradiol (E2) inhibit uptake via the DAT in PC12 cells over 30 minutes, with significant inhibition taking place due to E2 exposure during only the last five minutes of the uptake period. Such rapid action suggests a non-genomic, membrane-initiated estrogenic response mechanism. DAT and estrogen receptor-alpha (ERalpha) were elevated in cell extracts by a 20 ng/ml 2 day NGFbeta treatment, while ERbeta was not. DAT, ERalpha and ERbeta were also detectable on the plasma membrane of unpermeabilized cells by immunocytochemical staining and by a fixed cell, quantitative antibody (Ab)-based plate assay. In addition, PC12 cells contained RNA coding for the alternative membrane ER GPR30; therefore, all 3 ER subtypes are candidates for mediating the rapid nongenomic actions of E2. At cell densities above 15,000 cells per well, the E2-induced inhibition of transport was reversed. Uptake activity oscillated with time after a 10 nM E2 treatment; in a slower room temperature assay, inhibition peaked at 9 min, while uptake activity increased at 3 and 20-30 min. Using an Ab recognizing the second extracellular loop of DAT (accessible only on the outside of unpermeabilized cells), our immunoassay measured membrane vs. intracellular/nonvesicular DAT; both were found to decline over a 5-60 min E2 treatment, though immunoblot analyses demonstrated no total cellular loss of protein. CONCLUSION: Our results suggest that physiological levels of E2 may act to sequester DAT in intracellular compartments where the transporter's second extramembrane loop is inaccessible (inside vesicles) and that rapid estrogenic actions on this differentiated neuronal cell type may be regulated via membrane ERs of several types.