Arsenic inhibits mast cell degranulation via suppression of early tyrosine phosphorylation events

Exposure to arsenic is a global health concern. We previously documented an inhibitory effect of inorganic Arsenite on IgE‐mediated degranulation of RBL‐2H3 mast cells (Hutchinson et al., 2011; J. Appl. Toxicol. 31: 231–241). Mast cells are tissue‐resident cells that are positioned at the host–environment interface, thereby serving vital roles in many physiological processes and disease states, in addition to their well‐known roles in allergy and asthma. Upon activation, mast cells secrete several mediators from cytoplasmic granules, in degranulation. The present study is an investigation of Arsenite's molecular target(s) in the degranulation pathway. Here, we report that arsenic does not affect degranulation stimulated by either the Ca²⁺ ionophore A23187 or thapsigargin, which both bypass early signaling events. Arsenic also does not alter degranulation initiated by another non‐IgE‐mediated mast cell stimulant, the G‐protein activator compound 48/80. However, arsenic inhibits Ca²⁺ influx into antigen‐activated mast cells. These results indicate that the target of arsenic in the degranulation pathway is upstream of the Ca²⁺ influx. Phospho‐Syk and phospho‐p85 phosphoinositide 3‐kinase enzyme‐linked immunosorbent assays data show that arsenic inhibits early phosphorylation events. Taken together, this evidence indicates that the mechanism underlying arsenic inhibition of mast cell degranulation occurs at the early tyrosine phosphorylation steps in the degranulation pathway. Copyright © 2016 John Wiley & Sons, Ltd.     

Environmental mixture with estrogenic activity increases Hsd3b1 expression through estrogen receptors in immature rat granulosa cells

Humans are exposed not only to single endocrine disruptors, but also to chemical mixtures that can adversely affect their reproductive health. Steroidogenesis in reproductive tissues is emerging as the key target of endocrine disruptor action. Here, we analyzed the effect of environmental chemical mixtures with estrogenic activity on steroidogenic processes in immature rat granulosa cells and whether the observed steroidogenic effects were mediated through estrogen receptors. Extracts from untreated wastewater were prepared by solid‐phase extraction and silica gel fractionation. ER‐CALUX assay showed that the polar fractions of wastewater exerted different levels of estrogenic activity. Exposure of immature granulosa cells to the polar fraction exerting 9 ng of 17β‐estradiol equivalents per liter of water of estrogenic activity increased mRNA expression of the key enzymes of progesterone biosynthetic pathway Star and Hsd3b1, but did not alter the level of Cyp19a1 and Lhr. Addition of estrogen receptor inhibitor ICI 182 780 prevented the estrogenic mixture‐induced increase in Hsd3b1, but not Star mRNA level in immature granulosa cells. These results indicate that the environmental chemical mixtures with estrogenic activity exert endocrine disrupting effects by augmenting the progesterone biosynthetic pathway in immature rat granulosa cells, which is an effect achieved in part through activation of the estrogen receptors.     

Antibacterial agent triclosan suppresses RBL-2H3 mast cell function

Triclosan is a broad-spectrum antibacterial agent, which has been shown previously to alleviate human allergic skin disease. The purpose of this study was to investigate the hypothesis that the mechanism of this action of triclosan is, in part, due to effects on mast cell function. Mast cells play important roles in allergy, asthma, parasite defense, and carcinogenesis. In response to various stimuli, mast cells degranulate, releasing allergic mediators such as histamine. In order to investigate the potential anti-inflammatory effect of triclosan on mast cells, we monitored the level of degranulation in a mast cell model, rat basophilic leukemia cells, clone 2H3. Having functional homology to human mast cells, as well as a very well defined signaling pathway leading to degranulation, this cell line has been widely used to gain insight into mast-cell driven allergic disorders in humans. Using a fluorescent microplate assay, we determined that triclosan strongly dampened the release of granules from activated rat mast cells starting at 2 μM treatment, with dose-responsive suppression through 30 μM. These concentrations were found to be non-cytotoxic. The inhibition was found to persist when early signaling events (such as IgE receptor aggregation and tyrosine phosphorylation) were bypassed by using calcium ionophore stimulation, indicating that the target for triclosan in this pathway is likely downstream of the calcium signaling event. Triclosan also strongly suppressed F-actin remodeling and cell membrane ruffling, a physiological process that accompanies degranulation. Our finding that triclosan inhibits mast cell function may explain the clinical data mentioned above and supports the use of triclosan or a mechanistically similar compound as a topical treatment for allergic skin disease, such as eczema.     

Endocrine disruptors and brain estrogen receptors: the current state of behavioral, neurochemical, and molecular biological studies]

Based on epidemiological studies and animal studies, endocrine disrupters have received considerable attention as exerting disrupting actions on the developing brain. On the other hand, there has been increasing evidence that sex hormones and thyroid hormones play important roles in the development of the brain, including sexual dimorphism during the perinatal stage. Thus it seems probable that perinatal exposure to endocrine disruptors, which may have an affect on biosynthesis, transport, action, and metabolism of the hormones, may disrupt brain development enough to impair the brain functions. In this review, we introduce the current state of studies on brain disrupting actions of endocrine disruptors, addressing their actions on the estrogen system, including our own findings. The outline of the findings thus far reported are as follows: (1) Perinatal exposure to relatively low concentrations of endocrine disrupters may cause an impairment of higher brain functions, such as sexual behavior and learning behavior, (2) There seems to be sexual difference about the impairment described above, (3) Endocrine disruptors may cause an increase in volume of some nuclei, such as the sexual dimorphic nucleus of the preoptic area and locus coeruleus of the brain, (4) The disruptor might change the level of some substances that are considered to be involved in synaptic functions. Much remained to be studied about how does each finding reported link the others, and about detailed mechanisms of the disrupting actions of endocrine disruptors on the developing brain.     

Endocrine disruptors: a new scientific role for clinical pharmacologists? Impact on human health, wildlife, and the environment.

It is important for the clinical pharmacologist to understand the potential human health implications of exposure to environmental chemicals that may act as hormonally active agents. It is necessary to have an understanding of how pharmaceutical and personal care products and other chemicals affect the ecosystem of planet Earth and to understand how they may negatively contribute to human disease. Clinical pharmacologists must understand the various definitions of endocrine disruptors and be able to "decipher" these terms for their patients. Understanding the need for the EPA endocrine disruptor screening program and possessing knowledge of the screening assays used to assess endocrine activity potential are two essential components relevant to the topic of endocrine disruptors. Clinical pharmacologists have an opportunity to play an important role in resolving the question of what role endocrine disruptors play in initiating human disease since some scientists argue that the present evidence is not compelling. Clinical pharmacologists can also play an important role in the evaluation of the risk assessment and use of risk management and risk communication tools required to address public health concerns related to actions of endocrine disruptors. It is important that clinical pharmacologists work with veterinary clinical pharmacologists, toxicologists, industrial chemists, regulators, the scientific community, the general public, and environmental groups to understand the impact of endocrine disruptors on human health, wildlife, and the environment with an ultimate goal to minimize and/or alleviate the unwanted, detrimental effects of the endocrine disruptors.     

Antimicrobial agent triclosan is a proton ionophore uncoupler of mitochondria in living rat and human mast cells and in primary human keratinocytes

Triclosan (TCS) is an antimicrobial used widely in hospitals and personal care products, at ~10 mm . Human skin efficiently absorbs TCS. Mast cells are ubiquitous key players both in physiological processes and in disease, including asthma, cancer and autism. We previously showed that non‐cytotoxic levels of TCS inhibit degranulation, the release of histamine and other mediators, from rat basophilic leukemia mast cells (RBL‐2H3), and in this study, we replicate this finding in human mast cells (HMC‐1.2). Our investigation into the molecular mechanisms underlying this effect led to the discovery that TCS disrupts adenosine triphosphate (ATP) production in RBL‐2H3 cells in glucose‐free, galactose‐containing media (95% confidence interval EC₅₀ = 7.5–9.7 µm ), without causing cytotoxicity. Using these same glucose‐free conditions, 15 µm TCS dampens RBL‐2H3 degranulation by 40%. The same ATP disruption was found with human HMC‐1.2 cells (EC₅₀ 4.2–13.7 µm ), NIH‐3 T3 mouse fibroblasts (EC₅₀ 4.8–7.4 µm ) and primary human keratinocytes (EC₅₀ 3.0–4.1 µm ) all with no cytotoxicity. TCS increases oxygen consumption rate in RBL‐2H3 cells. Known mitochondrial uncouplers (e.g., carbonyl cyanide 3‐chlorophenylhydrazone) previously were found to inhibit mast cell function. TCS‐methyl, which has a methyl group in place of the TCS ionizable proton, affects neither degranulation nor ATP production at non‐cytotoxic doses. Thus, the effects of TCS on mast cell function are due to its proton ionophore structure. In addition, 5 µm TCS inhibits thapsigargin‐stimulated degranulation of RBL‐2H3 cells: further evidence that TCS disrupts mast cell signaling. Our data indicate that TCS is a mitochondrial uncoupler, and TCS may affect numerous cell types and functions via this mechanism. Copyright © 2015 John Wiley & Sons, Ltd.     

Effects of dopaminergic drugs on the mast cell degranulation and nitric oxide generation in RAW 264.7 cells

Effects of dopaminergic drugs on the degranulation of mast cells (RBL-2H3 cells) and the nitric oxide production from macrophage cells (RAW 264.7) were studied. Among the dopaminergic agonists and antagonists tested, bromocriptine, 7-OH-DPAT, haloperidol, and clozapine showed potent inhibitions of mast cell degranualtion (IC50 value, 5 microM). However, these dopaminergic agents did not affect the tyrosine phosphorylations of the signaling components of the high affinity IgE receptor (FcepsilonRI), such as Syk, PLCgamma1, and PLCgamma2.; This suggested that these signaling components were not involved in the inhibition of the mast cell degranulation by these compounds. On the other hand, dopamine, bromocriptine, 7-OH-DAPT, and haloperidol markedly inhibited the nitric oxide production from RAW 264.7 cells (IC50 values, 10-20 microM). Bromocriptine, a dopamine agonist that is routinely used for the treatment of Parkinsons disease, inhibited the expression of the inducible nitric oxide synthase at an early stage of the LPS-induced protein expression in a dose-dependent manner. The results suggested that these dopaminergic agents, when used for the treatment of dopamine receptors-related diseases, such as Schizophrenia or Parkinsons disease, might have additional beneficial effects.     

The phenoxazine derivative Phx-1 suppresses IgE-mediated degranulation in rat basophilic leukemia RBL-2H3 cells

Antigen-induced aggregation of the high affinity IgE receptor (FcepsilonRI) on mast cells induces degranulation to release chemical mediators, leading to acute allergic inflammation. We have demonstrated that the treatment of rat mast cells, RBL-2H3, with a phenoxazine derivative Phx-1 (2-amino-4,4alpha-dihydro-4alpha,7-dimethyl-3H-phenoxazine-3-one) suppresses the antigen-induced degranulation. Biochemical analysis reveals that the complementary signaling pathway through Gab2 and Akt is inhibited by this compound in mast cells. These findings suggest that phenoxazine derivatives may have a therapeutic potential for allergic diseases by inhibiting mast cell degranulation.     

Acrolein induction of oxidative stress and degranulation in mast cells

Increases in asthma worldwide have been associated epidemiologically with expanding urban air pollution. The mechanistic relationship between airway hyper‐responsiveness, inflammation, and ambient airborne triggers remains ambiguous. Acrolein, a ubiquitous aldehyde pollutant, is a product of incomplete combustion reactions. Acrolein is abundant in cigarette smoke, effluent from industrial smokestacks, diesel exhaust, and even hot oil cooking vapors. Acrolein is a potent airway irritant and can induce airway hyper‐responsiveness and inflammation in the lungs of animal models. In the present study, we utilized the mast cell analog, RBL‐2H3, to interrogate the responses of cells relevant to airway inflammation and allergic responses as a model for the induction of asthma‐like conditions upon exposure to acrolein. We hypothesized that acrolein would induce oxidative stress and degranulation in airway mast cells. Our results indicate that acrolein at 1 ppm initiated degranulation and promoted the generation of reactive oxygen species (ROS). Introduction of antioxidants to the system significantly reduced both ROS generation and degranulation. At higher levels of exposure (above 100 ppm), RBL‐2H3 cells displayed signs of severe toxicity. This experimental data indicates acrolein can induce an allergic inflammation in mast cell lines, and the initiation of degranulation was moderated by the application of antioxidants. © 2012 Wiley Periodicals, Inc. Environ Toxicol 29: 908–915, 2014.     

Targeting mast cells: Uncovering prolific therapeutic role in myriad diseases

The mast cells are integral part of immune system and they have pleiotropic physiological functions in our body. Any type of abnormal stimuli causes the mast cells receptors to spur the otherwise innocuous mast cells to degranulate and release inflammatory mediators like histamine, cytokines, chemokines and prostaglandins. These mediators are involved in various diseases like allergy, asthma, mastocytosis, cardiovascular disorders, etc. Herein, we describe the receptors involved in degranulation of mast cells and are broadly divided into four categories: G-protein coupled receptors, ligand gated ion channels, immunoreceptors and pattern recognition receptors. Although, activation of pattern recognition receptors do not cause mast cell degranulation, but result in cytokines production. Degranulation itself is a complex process involving cascade of events like membrane fusion events and various proteins like VAMP, Syntaxins, DOCK5, SNAP-23, MARCKS. Furthermore, we described these mast cell receptors antagonists or agonists useful in treatment of myriad diseases. Like, omalizumab anti-IgE antibody is highly effective in asthma, allergic disorders treatment and recently mechanistic insight of IgE uncovered; matrix mettaloprotease inhibitor marimistat is under phase III trial for inflammation, muscular dystrophy diseases; ZPL-389 (H4 receptor antagonist) is in Phase 2a Clinical Trial for atopic dermatitis and psoriasis; JNJ3851868 an oral H4 receptor antagonist is in phase II clinical development for asthma, rheumatoid arthritis. Therefore, research is still in inchoate stage to uncover mast cell biology, mast cell receptors, their therapeutic role in myriad diseases.     

Inhibition of rat mast cell degranulation by verapamil

Calcium antagonists, e.g. verapamil, prevent exercise-induced asthma. This protective effect may proceed from inhibition of contraction of bronchial smooth muscle, release of mediators by primary effector cells, e.g. mast cells, or both. Therefore, we studied the inhibitory effect of increasing concentrations of verapamil on both in vitro antigen-induced degranulation and ionophore A23187-induced release of labelled serotonin by rat peritoneal mast cells. There was a dose-dependent inhibition by verapamil of both ovalbumin-induced degranulation of mast cells passively sensitized by incubation with mice IgE-rich serum and ionophore-induced release of tritiated serotonin by mast cells previously incubated with (3H)-5HT; the 50% inhibiting concentration was 1.4 X 10(-4) mol I-1 and 5.2 X 10(-5) mol I-1, respectively. An attractive explanation of our results is that verapamil inhibits the antigen-induced release of mediators by mast cells through its calcium antagonist effect. Our results also suggest that the preventing effect of calcium antagonists on asthma may be multi-factorial since other authors have clearly shown that these drugs inhibit contraction of guinea-pig tracheal smooth muscle in vitro.     

Effects on guppy brain aromatase activity following short‐term steroid and 4‐nonylphenol exposures

Brain estrogen production, performed by the enzyme aromatase, can be disrupted/affected in teleost fish exposed to endocrine disruptors found in polluted aquatic environments. The guppy (Poecilia reticulata) was previously studied and confirmed to suffer negative effects on reproductive behaviors following inhibition of the brain aromatase reaction. Here adult guppies (Poecilia reticulata) of both genders were subjected to known endocrine disruptors: the androgen androstenedione (A), the synthetic estrogen 17α‐ethinylestradiol (EE₂), and the estrogenic surfactant 4‐nonylphenol (NP), at high (50 μg/L) and at environmentally relevant concentrations (10 ng/L EE₂, 5 μg/L NP, and 0.7 μg/L A) for 2 weeks followed by measurements of brain aromatase activity (bAA). In the adult males, bAA was stimulated by A and EE₂ at 50 μg/L. Female activity was also stimulated by the higher estrogenic treatment. At environmentally relevant concentrations only the EE₂ treatment affected bAA, and only in males. The alkylphenolic substance NP produced no effect in either of the experiments, not on males nor females. The results indicate that short‐term steroid treatments have stimulatory effects on guppy brain aromatase even at concentrations that can be found in the environment. We thus suggest bAA of adult guppies to be a suitable bioindicator of endocrine disruptors. © 2009 Wiley Periodicals, Inc. Environ Toxicol, 2010.     

Epigenetic transgenerational actions of endocrine disruptors

Environmental factors have a significant impact on biology. Therefore, environmental toxicants through similar mechanisms can modulate biological systems to influence physiology and promote disease states. The majority of environmental toxicants do not have the capacity to modulate DNA sequence, but can alter the epigenome. In the event an environmental toxicant such as an endocrine disruptor modifies the epigenome of a somatic cell, this may promote disease in the individual exposed, but not be transmitted to the next generation. In the event a toxicant modifies the epigenome of the germ line permanently, then the disease promoted can become transgenerationaly transmitted to subsequent progeny. The current review focuses on the ability of environmental factors such as endocrine disruptors to promote transgenerational phenotypes.     

Exposure to Zearalenone Leads to Metabolic Disruption and Changes in Circulating Adipokines Concentrations in Pigs

Zearalenone (ZEN) is a mycotoxin classified as an endocrine disruptor. Many endocrine disruptors are also metabolic disruptors able to modulate energy balance and inflammatory processes in a process often involving a family of protein hormones known as adipokines. The aim of our study was to elucidate the role of ZEN as metabolic disruptor in pigs by investigating the changes in energy balance and adipokines levels in response to different treatment diets. To this end, weaned piglets (n = 10/group) were exposed to either basal feed or feed contaminated with 680 and 1620 µg/kg ZEN for 28 days. Serum samples collected at days 7 and 21 were subjected to biochemistry analysis, followed by determination of adipokine levels using a combined approach of protein array and ELISA. Results indicate that ZEN has an impact on lipid and glucose metabolism that was different depending on the dose and time of exposure. In agreement with these changes, ZEN altered circulating adipokines concentrations, inducing significant changes in adiponectin, resistin, and fetuin B. Our results suggest that ZEN may function as a natural metabolism-disrupting chemical.     

Update of OECD DART guidelines with endocrine disruptor relevant endpoints: Practical considerations

In 1998, the OECD initiated a high-priority project aimed at revising existing test guidelines and developing new test guidelines for screening of potential endocrine disruptors. In 2011, OECD 443 was adopted, and in 2015 OECD 421 and OECD 422 were updated with endocrine disruptor relevant endpoints. A feasibility study for the enhancement of OECD 414 with endocrine disruptor relevant endpoints is currently ongoing. The addition of these endpoints is considered crucial for gaining more information on endocrine disruptor potency of tested chemicals, however it should be noted that these additions have a major impact on the study designs and give rise to several practical challenges. The aim of this review is to discuss important aspects of these challenging study designs and to share our knowledge on their implementation in our laboratory. Together, this review can be used as guidance for other laboratories, study monitors and registration officers.     

Stem cell-derived in vitro models for investigating the effects of endocrine disruptors on developing neurons and neuroendocrine cells

Neuroendocrine cells are a set of specialized hormone-releasing neurons that control most vital functions in humans and wildlife, such as growth, reproduction, metabolism, and stress responses. Increasing evidence points to neuroendocrine cells as the primary neuronal target of endocrine disruptors. Endocrine disruption appears to be most significant during prenatal and early postnatal development. However, limitations with traditional cell culture models of neuronal development led to a lack of understanding regarding the mechanisms by which endocrine disruptors affect neurodevelopment. In recent years, Stem Cell-derived neuronal models have become available and may offer distinct advantages over other in vitro model systems for investigating the effects of endocrine disruptors on the developing brain. Further, recently new models of Stem Cell-derived neuroendocrine cells that may provide more effective ways for studying the effects of endocrine disruptors directly on developing neuroendocrine cells in vitro were developed. This constitutes a review of currently available cell models of developing neurons that have been used to investigate in vitro effects of endocrine disruptors on developing brain. The review also presents recently developed models of Stem Cell-derived neuroendocrine cells that might be used to investigate in vitro effects of endocrine disruptors and their mechanisms of action directly on the developing neuroendocrine cells.     

Effect of nonpersistent pesticides on estrogen receptor,androgen receptor,and aryl hydrocarbon receptor

Nonpersistent pesticides are considered less harmful for the environment, but their impact as endocrine disruptors has not been fully explored. The pesticide Switch was applied to grape vines, and the maximum residue concentration of its active ingredients was quantified. The transactivation potential of the pesticides Acorit, Frupica, Steward, Reldan, Switch, Cantus, Teldor, and Scala and their active compounds (hexythiazox, mepanipyrim, indoxacarb, chlorpyrifos‐methyl, cyprodinil, fludioxonil, boscalid, fenhexamid, and pyrimethanil) were tested on human estrogen receptor α (ERα), androgen receptor (AR) and arylhydrocarbon receptor (AhR) in vitro. Relative binding affinities of the pure pesticide constituents for AR and their effect on human breast cancer and prostate cancer cell lines were evaluated. Residue concentrations of Switch's ingredients were below maximum residue limits. Fludioxonil and fenhexamid were ERα agonists (EC₅₀‐values of 3.7 and 9.0 μM, respectively) and had time‐dependent effects on endogenous ERα‐target gene expression (cyclin D1, progesterone receptor, and nuclear respiratory factor 1) in MCF‐7 human breast cancer cells. Fludioxonil, mepanipyrim, cyprodinil, pyrimethanil, and chlorpyrifos‐methyl were AhR‐agonists (EC₅₀s of 0.42, 0.77, 1.4, 4.6, and 5.1 μM, respectively). Weak AR binding was shown for chlorpyrifos‐methyl, cyprodinil, fenhexamid, and fludioxonil. Assuming a total uptake which does not take metabolism and clearance rates into account, our in vitro evidence suggests that pesticides could activate pathways affecting hormonal balance, even within permitted limits, thus potentially acting as endocrine disruptors. © 2013 Wiley Periodicals, Inc. Environ Toxicol 29: 1201–1216, 2014.