Synergistic activation of genes promoting invasiveness by dual deprivation in oxygen and nutrients.

By depriving cancer cells of blood supplies of oxygen and nutrients, anti-angiogenic therapy is aimed at simultaneously asphyxiating and starving the cells. But in spite of its apparent logic, this strategy is generally counterproductive over the long term as the treatment seems to elicit malignancy. Since a defect of blood supply is expected to deprive tumours simultaneously of oxygen and nutrients naturally, we examine here these two deprivations, alone or in combination, on the phenotype and signalling pathways of moderately aggressive MCF7 cancer cells. Each deprivation induces some aspects of the aggressive and migratory phenotypes through activating several pathways, including HIF1-alpha as expected, but also SRF/MRTFA and TCF4/beta-catenin. Strikingly, the dual deprivation has strong cooperative effects on the upregulation of genes increasing the metastatic potential, such as four and a half LIM domains 2 (FHL2) and HIF1A-AS2 lncRNA, which have response elements for both pathways. Using anti-angiogenic agents as monotherapy is therefore questionable as it may give falsely promising short-term tumour regression, but could ultimately exacerbate aggressive phenotypes.

Millimeter-Wave Heating in In Vitro Studies: Effect of Convection in Continuous and Pulse-Modulated Regimes.

Shallow penetration of millimeter waves (MMW) and non-uniform illumination in in vitro experiments result in a non-uniform distribution of the specific absorption rate (SAR). These SAR gradients trigger convective currents in liquids affecting transient and steady-state temperature distributions. We analyzed the effect of convection on temperature dynamics during MMW exposure in continuous-wave (CW) and pulsed-wave (PW) amplitude-modulated regimes using micro-thermocouples. Temperature rise kinetics are characterized by the occurrence of a temperature peak that shifts to shorter times as the SAR of the MMW exposure increases and precedes initiation of convection in bulk. Furthermore, we demonstrate that the liquid volume impacts convection. Increasing the volume results in earlier triggering of convection and in a greater cooling rate after the end of the exposure. In PW regimes, convection strongly depends on the pulse duration that affects the heat pulse amplitude and cooling rate. The latter results in a change of the average temperature in PW regime. Bioelectromagnetics. 2019;40:553-568. © 2019 Bioelectromagnetics Society.

Untargeted Metabolomics Reveal Lipid Alterations upon 2-Deoxyglucose Treatment in Human HaCaT Keratinocytes.

The glucose analogue 2-deoxyglucose (2-DG) impedes cancer progression in animal models and is currently being assessed as an anticancer therapy, yet the mode of action of this drug of high clinical significance has not been fully delineated. In an attempt to better characterize its pharmacodynamics, an integrative UPLC-Q-Exactive-based joint metabolomic and lipidomic approach was undertaken to evaluate the metabolic perturbations induced by this drug in human HaCaT keratinocyte cells. R-XCMS data processing and subsequent multivariate pattern recognition, metabolites identification, and pathway analyses identified eight metabolites that were most significantly changed upon a 3 h 2-DG exposure. Most of these dysregulated features were emphasized in the course of lipidomic profiling and could be identified as ceramide and glucosylceramide derivatives, consistently with their involvement in cell death programming. Even though metabolomic analyses did not generally afford such clear-cut dysregulations, some alterations in phosphatidylcholine and phosphatidylethanolamine derivatives could be highlighted as well. Overall, these results support the adequacy of the proposed analytical workflow and might contribute to a better understanding of the mechanisms underlying the promising effects of 2-DG.

Microscale temperature and SAR measurements in cell monolayer models exposed to millimeter waves.

Due to shallow penetration of millimeter waves (MMW) and convection in liquid medium surrounding cells, the problem of accurate assessment of local MMW heating in in vitro experiments remains unsolved. Conventional dosimetric MMW techniques, such as infrared imaging or fiber optic (FO) sensors, face several inherent limits. Here we propose a methodology for accurate local temperature measurement and subsequent specific absorption rate (SAR) retrieval using microscale thermocouples (TC). SAR was retrieved by fitting the measured initial temperature rise to the numerical solution of an equivalent thermal model. It was found that the accuracy of temperature measurement depends on thermosensor size, that is, the smaller TC, the more accurate the temperature measurement. SAR determined using TC with lead diameters of 25 and 75 µm demonstrated 98.5% and 80.4% match with computed SAR, respectively. However, both TC provided the same temperature rises in long run (> 10 min). FO probe failed to measure adequately local heating both for short and long exposures due to the relatively large size of the probe sensor (400 µm) and time constant (0.6 s). Calculated SAR in the cell monolayer was almost two times lower than that in the surrounding liquid. It was shown that the impact of the cell monolayer on heating due to its small thickness (5 to 10 µm) can be considered as negligible. Moreover, we demonstrated the possibility of accurate measurement of MMW-induced thermal pulses (up to 10 °C) using 25 µm TC. Bioelectromagnetics. 38:11-21, 2017. © 2016 Wiley Periodicals, Inc.

Impact of 60-GHz millimeter waves on stress and pain-related protein expression in differentiating neuron-like cells.

Millimeter waves (MMW) will be increasingly used for future wireless telecommunications. Previous studies on skin keratinocytes showed that MMW could impact the mRNA expression of Transient Receptor Potential cation channel subfamily Vanilloid, member 2 (TRPV2). Here, we investigated the effect of MMW exposure on this marker, as well as on other membrane receptors such as Transient Receptor Potential cation channel subfamily Vanilloid, member 1 (TRPV1) and purinergic receptor P2X, ligand-gated ion channel, 3 (P2 × 3). We exposed the Neuroscreen-1 cell line (a PC12 subclone), in order to evaluate if acute MMW exposures could impact expression of these membrane receptors at the protein level. Proteotoxic stress-related chaperone protein Heat Shock Protein 70 (HSP70) expression level was also assessed. We used an original high-content screening approach, based on fluorescence microscopy, to allow cell-by-cell analysis and to detect any cell sub-population responding to exposure. Immunocytochemistry was done after 24 h MMW exposure of cells at 60.4 GHz, with an incident power density of 10 mW/cm(2) . Our results showed no impact of MMW exposure on protein expressions of HSP70, TRPV1, TRPV2, and P2 × 3. Moreover, no specific cell sub-populations were found to express one of the studied markers at a different level, compared to the rest of the cell populations. However, a slight insignificant increase in HSP70 expression and an increase in protein expression variability within cell population were observed in exposed cells, but controls showed that this was related to thermal effect. Bioelectromagnetics. 37:444-454, 2016. © 2016 Wiley Periodicals, Inc.

A functional analysis of MELK in cell division reveals a transition in the mode of cytokinesis during Xenopus development.

MELK is a serine/threonine kinase involved in several cell processes, including the cell cycle, proliferation, apoptosis and mRNA processing. However, its function remains elusive. Here, we explored its role in the Xenopus early embryo and show by knockdown that xMELK (Xenopus MELK) is necessary for completion of cell division. Consistent with a role in cell division, endogenous xMELK accumulates at the equatorial cortex of anaphase blastomeres. Its relocalization is highly dynamic and correlates with a conformational rearrangement in xMELK. Overexpression of xMELK leads to failure of cytokinesis and impairs accumulation at the division furrow of activated RhoA - a pivotal regulator of cytokinesis. Furthermore, endogenous xMELK associates and colocalizes with the cytokinesis organizer anillin. Unexpectedly, our study reveals a transition in the mode of cytokinesis correlated to cell size and that implicates xMELK. Collectively, our findings disclose the importance of xMELK in cytokinesis during early development and show that the mechanism of cytokinesis changes during Xenopus early development.

Millimeter-Wave Heating In Vitro: Local Microscale Temperature Measurements Correlated to Heat Shock Cellular Response.

OBJECTIVE: Cellular sensitivity to heat is highly variable depending on the cell line. The aim of this paper is to assess the cellular sensitivity of the A375 melanoma cell line to continuous (CW) millimeter-waves (MMW) induced heating at 58.4 GHz, between 37 °C and 47 °C to get a deeper insight into optimization of thermal treatment of superficial skin cancer. METHODS: Phosphorylation of heat shock protein 27 (HSP27) was mapped within an area of about 30 mm 2 to visualize the variation of heat-induced cellular stress as a function of the distance from the waveguide aperture (MMW radiation source). A multiphysics computational approach was then adopted to yield both electromagnetic and thermal field distributions as well as corresponding specific absorption rate (SAR) and temperature elevation. Induced temperature rise was experimentally measured using a micro-thermocouple ( µTC). RESULTS: Coupling of the incident electromagnetic (EM) field with µTC leads was first characterized, and optimal µTC placing was identified. HSP27 phosphorylation was induced at temperatures ≥ 41 °C, and its level increases as a function of the thermal dose delivered, remaining mostly focused within 3 mm 2. CONCLUSION: Phosphorylation of HSP27 represents a valuable marker of cellular stress of A375 melanoma cells under MMW exposure, providing both quantitative and spatial information about the distribution of the thermal stress. SIGNIFICANCE: These results may contribute to the design of thermal treatments of superficial melanoma through MMW-induced heating in the hyperthermic temperature range.

Hypoxia and ERα Transcriptional Crosstalk Is Associated with Endocrine Resistance in Breast Cancer.

Estrogen receptor-alpha (ERα) is the driving transcription factor in 70% of breast cancers and its activity is associated with hormone dependent tumor cell proliferation and survival. Given the recurrence of hormone resistant relapses, understanding the etiological factors fueling resistance is of major clinical interest. Hypoxia, a frequent feature of the solid tumor microenvironment, has been described to promote endocrine resistance by triggering ERα down-regulation in both in vitro and in vivo models. Yet, the consequences of hypoxia on ERα genomic activity remain largely elusive. In the present study, transcriptomic analysis shows that hypoxia regulates a fraction of ERα target genes, underlying an important regulatory overlap between hypoxic and estrogenic signaling. This gene expression reprogramming is associated with a massive reorganization of ERα cistrome, highlighted by a massive loss of ERα binding sites. Profiling of enhancer acetylation revealed a hormone independent enhancer activation at the vicinity of genes harboring hypoxia inducible factor (HIFα) binding sites, the major transcription factors governing hypoxic adaptation. This activation counterbalances the loss of ERα and sustains hormone-independent gene expression. We describe hypoxia in luminal ERα (+) breast cancer as a key factor interfering with endocrine therapies, associated with poor clinical prognosis in breast cancer patients.

Aromatase in the brain of teleost fish: expression, regulation and putative functions.

Unlike that of mammals, the brain of teleost fish exhibits an intense aromatase activity due to the strong expression of one of two aromatase genes (aromatase A or cyp19a1a and aromatase B or cyp19a1b) that arose from a gene duplication event. In situ hybridization, immunohistochemistry and expression of GFP (green fluorescent protein) in transgenic tg(cyp19a1b-GFP) fish demonstrate that aromatase B is only expressed in radial glial cells (RGC) of adult fish. These cells persist throughout life and act as progenitors in the brain of both developing and adult fish. Although aromatase B-positive radial glial cells are most abundant in the preoptic area and the hypothalamus, they are observed throughout the entire central nervous system and spinal cord. In agreement with the fact that brain aromatase activity is correlated to sex steroid levels, the high expression of cyp19a1b is due to an auto-regulatory loop through which estrogens and aromatizable androgens up-regulate aromatase expression. This mechanism involves estrogen receptor binding on an estrogen response element located on the cyp19a1b promoter. Cell specificity is achieved by a mandatory cooperation between estrogen receptors and unidentified glial factors. Given the emerging roles of estrogens in neurogenesis, the unique feature of the adult fish brain suggests that, in addition to classical functions on brain sexual differentiation and sexual behaviour, aromatase expression in radial glial cells could be part of the mechanisms authorizing the maintenance of a high proliferative activity in the brain of fish.

Neuroendocrine effects of endocrine disruptors in teleost fish.

Because a large proportion of potential endocrine disruptors (EDC) end up in surface waters, aquatic species are particularly vulnerable to their potential adverse effects. Recent studies identified a number of brain targets for EDC commonly present in environmentally relevant concentrations in surface waters. Among those neuronal systems disrupted by EDC are the gonadotropin-releasing hormone (GnRH) neurons, the dopaminergic and serotoninergic circuits, and more recently the Kiss/GPR54 system, which regulates gonadotropin release. However, one of the most striking effects of EDC, notably estrogen mimics, is their impact on the cyp19a1b gene that encodes the brain aromatase isoform in fish. Moreover, this is the only example in which the molecular basis of endocrine disruption is fully understood. The aims of this review were to (1) synthesize the most recent discoveries concerning the EDC effects upon neuroendocrine systems of fish and (2) provide, when possible, the underlying molecular basis of disruption for each system concerned. The potential adverse effects of EDC on neurogenesis, puberty, and brain sexualization are also described. It is important to point out the future environmental, social, and economical issues arising from endocrine disruption studies in the context of risk assessment.

Aromatase, brain sexualization and plasticity: the fish paradigm.

In contrast to mammals, teleost fish have a very labile genetic sex determination. Sex differentiation is influenced by a combination of hormonal, social and environmental factors and teleost fishes exhibit many examples of hermaphroditism. This means that the brain of fish is not irreversibly sexualized early in life. This review aims at highlighting some unique features of fish that may explain their brain sexual plasticity. Unlike mammals, in which brain aromatase activity decreases after birth, adult teleosts exhibit an intense aromatase activity due to strong expression of one of two aromatase genes (aromatase A or cyp19a1a and aromatase B or cyp19a1b) that arose from a gene duplication event. Interestingly, aromatase B is only expressed in radial glial cells (RGC) of adult fish. These cells persist throughout life and act as progenitors in the brain of both developing and adult fish. In agreement with the fact that brain aromatase activity is correlated with sex steroid levels, the high expression of cyp19a1b is due to an autoregulatory loop through which estrogens and aromatizable androgens upregulate aromatase expression. Given the well-established roles of estrogens and aromatase on brain sexualization, these features suggest that the brain of fish conserves properties of embryonic mammalian brain throughout life - high neurogenic activity and high aromatase expression in progenitor cells correlated with sex steroid levels. The permanent dialogue between the brain and the gonad would permit sex changes and thus the emergence of a variety of reproductive strategies. Other hypotheses are also discussed.

The Basal Level of Gene Expression Associated with Chromatin Loosening Shapes Waddington Landscapes and Controls Cell Differentiation.

The baseline level of transcription, which is variable and difficult to quantify, seriously complicates the normalization of comparative transcriptomic data, but its biological importance remains unappreciated. We show that this currently neglected ingredient is essential for controlling gene network multistability and therefore cellular differentiation. Basal expression is correlated to the degree of chromatin loosening measured by DNA accessibility and systematically leads to cellular dedifferentiation as assessed by transcriptomic signatures, irrespective of the molecular and cellular tools used. Modeling gene network motifs formally involved in developmental bifurcations reveals that the epigenetic landscapes of Waddington are restructured by the level of nonspecific expression, such that the attractors of progenitor and differentiated cells can be mutually exclusive. This mechanism is universal and holds beyond the particular nature of the genes involved, provided the multistable circuits are correctly described with autonomous basal expression. These results explain the relationships long established between gene expression noise, chromatin decondensation and cellular dedifferentiation, and highlight how heterochromatin maintenance is essential for preventing pathological cellular reprogramming, age-related diseases, and cancer.

Millimeter-wave pulsed heating in vitro: cell mortality and heat shock response.

Millimeter wave (MMW)-induced heating represents a promising alternative for non-invasive hyperthermia of superficial skin cancer, such as melanoma. Pulsed MMW-induced heating of tumors allows for reaching high peak temperatures without overheating surrounding tissues. Herein, for the first time, we evaluate apoptotic and heat shock responses of melanoma cells exposed in vitro to continuous (CW) or pulsed-wave (PW) amplitude-modulated MMW at 58.4 GHz with the same average temperature rise. Using an ad hoc exposure system, we generated 90 min pulse train with 1.5 s pulse duration, period of 20 s, amplitude of 10 °C, and steady-state temperature at the level of cells of 49.2 °C. The activation of Caspase-3 and phosphorylation of HSP27 were investigated using fluorescence microscopy to monitor the spatial variation of cellular response. Our results demonstrate that, under the considered exposure conditions, Caspase-3 activation was almost 5 times greater following PW exposure compared to CW. The relationship between the PW-induced cellular response and SAR-dependent temperature rise was non-linear. Phosphorylation of HSP27 was 58% stronger for PW compared to CW. It exhibits a plateau for the peak temperature ranging from 47.7 to 49.2 °C. Our results provide an insight into understanding of the cellular response to MMW-induced pulsed heating.

Untargeted metabolomics unveil alterations of biomembranes permeability in human HaCaT keratinocytes upon 60 GHz millimeter-wave exposure.

A joint metabolomic and lipidomic workflow is used to account for a potential effect of millimeter waves (MMW) around 60 GHz on biological tissues. For this purpose, HaCaT human keratinocytes were exposed at 60.4 GHz with an incident power density of 20 mW/cm², this value corresponding to the upper local exposure limit for general public in the context of a wide scale deployment of MMW technologies and devices. After a 24h-exposure, endo- and extracellular extracts were recovered to be submitted to an integrative UPLC-Q-Exactive metabolomic and lipidomic workflow. R-XCMS data processing and subsequent statistical treatment led to emphasize a limited number of altered features in lipidomic sequences and in intracellular metabolomic analyses, whatever the ionization mode (i.e 0 to 6 dysregulated features). Conversely, important dysregulations could be reported in extracellular metabolomic profiles with 111 and 99 frames being altered upon MMW exposure in positive and negative polarities, respectively. This unexpected extent of modifications can hardly stem from the mild changes that could be reported throughout transcriptomics studies, leading us to hypothesize that MMW might alter the permeability of cell membranes, as reported elsewhere.

Characterization of a cis-acting element involved in cell-specific expression of the zebrafish brain aromatase gene.

The cytochrome P450 Aromatase is the key enzyme catalyzing the conversion of androgens into estrogens. In zebrafish, the brain aromatase is encoded by cyp19b. Expression of cyp19b is restricted to radial glial cells bordering forebrain ventricles and is strongly stimulated by estrogens during development. At the promoter level, we have previously shown that an estrogen responsive element (ERE) is required for induction by estrogens. Here, we investigated the role of ERE flanking regions in the control of cell-specific expression. First, we show that a 20 bp length motif, named G x RE (glial x responsive element), acts in synergy with the ERE to mediate the estrogenic induction specifically in glial cells. Second, we demonstrate that, in vitro, this sequence binds factors exclusively present in glial or neuro-glial cells and is able to confer a glial specificity to an artificial estrogen-dependent gene. Taken together, these results contribute to the understanding of the molecular mechanisms allowing cyp19b regulation by estrogens and allowed to identify a promoter sequence involved in the strong estrogen inducibility of cyp19b which is specific for glial cells. The exceptional aromatase activity measured in the brain of teleost fish could rely on such mechanisms.

Identification of aromatase-positive radial glial cells as progenitor cells in the ventricular layer of the forebrain in zebrafish.

Compared with other vertebrates, the brain of adult teleost fish exhibits two unique features: it exhibits unusually high neurogenic activity and strongly expresses aromatase, a key enzyme that converts aromatizable androgens into estrogens. Until now, these two features, high neurogenic and aromatase activities, have never been related to each other. Recently, it was shown that aromatase is expressed in radial glial cells of the forebrain and not in neurons. Here, we further document that Aromatase B is never detected in cells expressing the markers of postmitotic neurons, Hu and acetylated tubulin. By using a combination of bromodeoxyuridine (BrdU) treatment and immunohistochemical techniques, we demonstrate for the first time to our knowledge that aromatase-positive radial cells actively divide to generate newborn cells in many forebrain regions. Such newborn cells can further divide, as shown by BrdU-proliferating cell nuclear antigen double staining. We also demonstrate that, over time, newborn cells move away from the ventricles, most likely by migrating along the radial processes. Finally, by using antisera to Hu and acetylated tubulin, we further document that some of the newborn cells derived from radial glia differentiate into neurons. These data provide new evidence for the mechanism of neurogenesis in the brain of adult fish. In addition, given that estrogens are well-known neurotrophic and neuroprotective factors affecting proliferation, apoptosis, migration, and differentiation, the expression of aromatase in the neural stem cells of the adult strongly demonstrates that the fish brain is an outstanding model for studying the effects of estrogens on adult neurogenesis and brain repair.

Expression of zebra fish aromatase cyp19a and cyp19b genes in response to the ligands of estrogen receptor and aryl hydrocarbon receptor.

Many endocrine-disrupting chemicals act via estrogen receptor (ER) or aryl hydrocarbon receptor (AhR). To investigate the interference between ER and AhR, we studied the effects of 17beta-estradiol (E2) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the expression of zebra fish cyp19a (zfcyp19a) and cyp19b (zfcyp19b) genes, encoding aromatase P450, an important steroidogenic enzyme. In vivo (mRNA quantification in exposed zebra fish larvae) and in vitro (activity of zfcyp19-luciferase reporter genes in cell cultures in response to chemicals and zebra fish transcription factors) assays were used. None of the treatments affected zfcyp19a, excluding the slight upregulation by E2 observed in vitro. Strong upregulation of zfcyp19b by E2 in both assays was downregulated by TCDD. This effect could be rescued by the addition of an AhR antagonist. Antiestrogenic effect of TCDD on the zfcyp19b expression in the brain was also observed on the protein level, assessed by immunohistochemistry. TCDD alone did not affect zfcyp19b expression in vivo or promoter activity in the presence of zebra fish AhR2 and AhR nuclear translocator 2b (ARNT2b) in vitro. However, in the presence of zebra fish ERalpha, AhR2, and ARNT2b, TCDD led to a slight upregulation of promoter activity, which was eliminated by either an ER or AhR antagonist. Studies with mutated reporter gene constructs indicated that both mechanisms of TCDD action in vitro were independent of dioxin-responsive elements (DREs) predicted in the promoter. This study shows the usefulness of in vivo zebra fish larvae and in vitro zfcyp19b reporter gene assays for evaluation of estrogenic chemical actions, provides data on the functionality of DREs predicted in zfcyp19 promoters and shows the effects of cross talk between ER and AhR on zfcyp19b expression. The antiestrogenic effect of TCDD demonstrated raises further concerns about the neuroendocrine effects of AhR ligands.

Effect of acute millimeter wave exposure on dopamine metabolism of NGF-treated PC12 cells.

Several forthcoming wireless telecommunication systems will use electromagnetic frequencies at millimeter waves (MMWs), and technologies developed around the 60-GHz band will soon know a widespread distribution. Free nerve endings within the skin have been suggested to be the targets of MMW therapy which has been used in the former Soviet Union. So far, no studies have assessed the impact of MMW exposure on neuronal metabolism. Here, we investigated the effects of a 24-h MMW exposure at 60.4 GHz, with an incident power density (IPD) of 5 mW/cm², on the dopaminergic turnover of NGF-treated PC12 cells. After MMW exposure, both intracellular and extracellular contents of dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) were studied using high performance liquid chromatography. Impact of exposure on the dopamine transporter (DAT) expression was also assessed by immunocytochemistry. We analyzed the dopamine turnover by assessing the ratio of DOPAC to DA, and measuring DOPAC accumulation in the medium. Neither dopamine turnover nor DAT protein expression level were impacted by MMW exposure. However, extracellular accumulation of DOPAC was found to be slightly increased, but not significantly. This result was related to the thermal effect, and overall, no evidence of non-thermal effects of MMW exposure were observed on dopamine metabolism.

Assessment of xenoestrogens using three distinct estrogen receptors and the zebrafish brain aromatase gene in a highly responsive glial cell system.

The brain cytochrome P450 aromatase (Aro-B) in zebrafish is expressed in radial glial cells and is strongly stimulated by estrogens (E2); thus, it can be used in vivo as a biomarker of xenoestrogen effects on the central nervous system. By quantitative real-time polymerase chain reaction, we first confirmed that the expression of Aro-B gene is robustly stimulated in juvenile zebrafish exposed to several xenoestrogens. To investigate the impact of environmental estrogenic chemicals on distinct estrogen receptor (ER) activity, we developed a glial cell-based assay using Aro-B as the target gene. To this end, the ER-negative glial cell line U251-MG was transfected with the three zebrafish ER subtypes and the Aro-B promoter linked to a luciferase reporter gene. E2 treatment of U251-MG glial cells cotransfected with zebrafish ER-alpha and the Aro-B promoter-luciferase reporter resulted in a 60- to 80-fold stimulation of luciferase activity. The detection limit was <0.05 nM, and the EC50 (median effective concentration) was 1.4 nM. Interestingly, in this glial cell context, maximal induction achieved with the Aro-B reporter was three times greater than that observed with a classical estrogen-response-element reporter gene (ERE-tk-Luc). Dose-response analyses with ethynylestradiol (EE2), estrone (E1), alpha-zeralenol, and genistein showed that estrogenic potency of these agents markedly differed depending on the ER subtype in the assay. Moreover, the combination of these agents showed an additive effect according to the concept of concentration addition. This confirmed that the combined additive effect of the xenoestrogens leads to an enhancement of the estrogenic potency, even when each single agent might be present at low effect concentrations. In conclusion, we demonstrate that our bioassay provides a fast, reliable, sensitive, and efficient test for evaluating estrogenic potency of endocrine disruptors on ER subtypes in a glial context.

Neurite analyzer: An original Fiji plugin for quantification of neuritogenesis in two-dimensional images.

BACKGROUND: In life sciences, there is a growing need for new informatics tools designed to provide automated solutions in order to analyze big amounts of images obtained from high-throughput imaging systems. Among the most widely used assays in neurotoxicity, endocrinology and brain diseases, the neurite outgrowth assay is popular. NEW METHOD: Cell-to-cell quantification of the main morphological features of neurite outgrowth assays remains very challenging. Here, we provide a new pipeline developed on Fiji software for analysis of series of two-dimensional images. It allows the automated analysis of most of these features. RESULTS: We tested the accuracy and usefulness of the software by confirming the effects of estradiol and hypoxia on in vitro neuronal differentiation, previously published by different authors with manual analysis methods. With this new method, we highlighted original interesting data. COMPARISON WITH EXISTING METHOD(S): The innovation brought by this plugin lies in the fact that it can process multiple images at the same time, in order to obtain: the number of nuclei, the number of neurites, the length of neurites, the number of neurites junctions, the number of neurites branches, the length of each branch, the position of the branch in the image, the angle of each branch, but also the area of each cell and the number of neurites per cell. CONCLUSIONS: This plugin is easy to use, highly sensitive, and allows the experimenter to acquire ready-to-use data coming from a vast amount of images.