Combined in silico and in vitro approaches to identify P-glycoprotein-inhibiting pesticides.

The P-glycoprotein (P-gp) efflux pump plays a major role in xenobiotic detoxification. The inhibition of its activity by environmental contaminants remains however rather little characterised. The present study was designed to develop a combination of different approaches to identify P-gp inhibitors among a large number of pesticides using in silico and in vitro models. First, the prediction performance of four web tools was evaluated alone or in combination using a set of recently marketed drugs. The best combination of web tools-AdmetSAR2.0/PgpRules/pkCSM-was next used to predict P-gp activity inhibition by 762 pesticides. Among the 187 pesticides predicted to be P-gp inhibitors, 11 were tested in vitro for their ability to inhibit the efflux of reference substrates (rhodamine 123 and Hoechst 33342) in P-gp overexpressing MCF7R cells and to inhibit the efflux of the reference substrate rhodamine 123 in the Caco-2 cell monolayer. In MCF7R cell assays, ivermectin B1a, emamectin B1 benzoate, spinosad, dimethomorph and tralkoxydim inhibited P-gp activity; ivermectin B1a, emamectin B1 benzoate and spinosad were determined to be stronger inhibitors (half-maximal inhibitory concentration [IC50 ] of 3 ± 1, 5 ± 1 and 7 ± 1 µM, respectively) than dimethomorph and tralkoxydim (IC50 of 102 ± 7 and 88 ± 7 µM, respectively). Ivermectin B1a, emamectin B1 benzoate, spinosad and dimethomorph also inhibited P-gp activity in Caco-2 cell monolayer assays, with dimethomorph being a weaker P-gp inhibitor. These combined approaches could be used to identify P-gp inhibitors among food contaminants, but need to be optimised and adapted for high-throughput screening.

Key parameter optimization using multivariable linear model for the evaluation of the in vitro estrogenic activity assay in T47D cell lines (CXCL-test).

In comparison with analytical tools, bioassays provide higher sensitivity and more complex evaluation of environmental samples and are indispensable tools for monitoring increasing in anthropogenic pollution. Nevertheless, the disadvantage in cellular assays stems from the material variability used within the assays, and an interlaboratory adaptation does not usually lead to satisfactory test sensitivities. The aim of this study was to evaluate the influence of material variability on CXCL12 secretion by T47D cells, the outcome of the CXCL-test (estrogenic activity assay). For this purpose, the cell line sources, sera suppliers, experimental and seeding media, and the amount of cell/well were tested. The multivariable linear model (MLM), employed as an innovative approach in this field for parameter evaluation, identified that all the tested parameters had significant effects. Knowledge of the contributions of each parameter has permitted step-by-step optimization. The most beneficial approach was seeding 20,000 cells/well directly in treatment medium and using DMEM for the treatment. Great differences in both basal and maximal cytokine secretions among the three tested cell lines and different impacts of each serum were also observed. Altogether, both these biologically based and highly variable inputs were additionally assessed by MLM and a subsequent two-step evaluation, which revealed a lower variability and satisfactory reproducibility of the test. This analysis showed that not only parameter and procedure optimization but also the evaluation methodology must be considered from the perspective of interlaboratory method adaptation. This overall methodology could be applied to all bioanalytical methods for fast multiparameter and accurate analysis.

Comparative in silico prediction of P-glycoprotein-mediated transport for 2010-2020 US FDA-approved drugs using six Web-tools.

P-glycoprotein (P-gp) is an efflux pump implicated in pharmacokinetics and drug-drug interactions. The identification of its substrates is consequently an important issue, notably for drugs under development. For such a purpose, various in silico methods have been developed, but their relevance remains to be fully established. The present study was designed to get insight about this point, through determining the performance values of six freely accessible Web-tools (ADMETlab, AdmetSAR2.0, PgpRules, pkCSM, SwissADME and vNN-ADMET), computationally predicting P-gp-mediated transport. Using an external test set of 231 marketed drugs, approved over the 2010-2020 period by the US Food and Drug Administration and fully in vitro characterized for their P-gp substrate status, various performance parameters (including sensitivity, specificity, accuracy, Matthews correlation coefficient and area under the receiver operating characteristics curve) were determined. They were found to rather poorly meet criteria commonly required for acceptable prediction, whatever the Web-tools were used alone or in combination. Predictions of being P-gp substrate or non-substrate by these online in silico methods may therefore be considered with caution.

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.

Estrogen receptor preparation effects on the receptor-DNA interaction by surface plasmon resonance.

Up to now, several studies have investigated estrogen receptor (ER)-estrogen response element (ERE) interaction using biosensors such as surface plasmon resonance. These strategies have aimed to understand the molecular mechanism of such interaction as well as the effect of the ligand on this interaction. These approaches start to be used to determine the mechanisms of protein/DNA interaction, in particular in the context of drug discovery or environmental applications. However, some physical and biochemical parameters (incubation time, temperature, protease inhibitor cocktail, and bovine serum albumin (BSA)) are not completely described in the literature and could deeply modify the obtained results. This paper aims to focus not only on the preliminary steps of sample preparation such as protein thawing and incubation conditions (time and temperature) but also on the evaluation of protease inhibitor cocktail and BSA effect on the measurement of ER-ERE interactions.

Impact of biochemical design on estrogen receptor/estrogen response element interaction by surface plasmon resonance technology.

The estrogen receptor (ER) is a transcription factor that binds under 17-ß-estradiol (E2) stimulation as homodimer to a short DNA consensus sequence named estrogen response element (ERE). The ER/ERE interaction has been assessed by several research groups through different methodologies notably by surface plasmon resonance (SPR) techniques. The biochemical parameters and conditions (solvent, ER concentration, salt, time and temperature) used to prepare samples before analysis were very different from one study to another. But no studies have aimed to compare the effect of these modifications on ER/ERE interaction. Therefore the main objective of the present paper was to assess the influence of biochemical parameters onto the ER/ERE interaction with the final aim to improve the comprehension of this interaction. Our results highlighted that parameters like solvent, ER concentration, salt and surfactant concentration, temperature and time deeply modify ER/ERE interaction. Nevertheless, the dimer formation under E2 stimulation occurred with all tested conditions. Altogether, incubation parameters of ER with E2, deeply modify its binding level onto ERE. These data constitute an important key point to consider for the improvement of ER/ERE detection method depending upon the aim of the study (interaction measurement, environmental detection, development of new technologies or devices).

Impact of 60-GHz millimeter waves and corresponding heat effect on endoplasmic reticulum stress sensor gene expression.

Emerging high data rate wireless communication systems, currently under development, will operate at millimeter waves (MMW) and specifically in the 60 GHz band for broadband short-range communications. The aim of this study was to investigate potential effects of MMW radiation on the cellular endoplasmic reticulum (ER) stress. Human skin cell lines were exposed at 60.4 GHz, with incident power densities (IPD) ranging between 1 and 20 mW/cm(2) . The upper IPD limits correspond to the ICNIRP local exposure limit for the general public. The expression of ER-stress sensors, namely BIP and ORP150, was then examined by real-time RT-PCR. Our experimental data demonstrated that MMW radiations do not change BIP or ORP150 mRNA basal levels, whatever the cell line, the exposure duration or the IPD level. Co-exposure to the well-known ER-stress inducer thapsigargin (TG) and MMW were then assessed. Our results show that MMW exposure at 20 mW/cm(2) inhibits TG-induced BIP and ORP150 over expression. Experimental controls showed that this inhibition is linked to the thermal effect resulting from the MMW exposure.

Transcriptional landscape of human keratinocyte models exposed to 60-GHz millimeter-waves.

The use of millimeter waves (MMW) will exponentially grow in the coming years due to their future utilization in 5G/6G networks. The question of possible biological effects at these frequencies has been raised. In this present study, we aimed to investigate gene expression changes under exposure to MMW using the Bulk RNA Barcoding and sequencing (BRB-seq) technology. To address this issue, three exposure scenarios were performed aiming at: i) comparing the cellular response of two primary culture of keratinocytes (HEK and NHEK) and one keratinocyte derivate cell line (HaCaT) exposed to MMW; ii) exploring the incident power density dose-effect on gene expression in HaCaT cell line; and, iii) studying the exposure duration at the new ICNIRP exposure limit for the general population. With the exception of heat effect induced by high power MMW (over 10 mW/cm2), those exposure scenarios have not enabled us to demonstrate important gene expression changes in the different cell populations studied. Very few differentially genes were observed between MMW exposed samples and heat shock control, and most of them were significantly associated with heat shock response that may reflect small differences in the heat generation. Together these results show that acute exposure to MMW has no effects on the transcriptional landscape of human keratinocyte models under athermal conditions.

Prioritization of mycotoxins based on mutagenicity and carcinogenicity evaluation using combined in silico QSAR methods.

Mycotoxins and their metabolites are a family of compounds that contains a great diversity of both structure and biological properties. Information on their toxicity is spread within several databases and in scientific literature. Due to the number of molecules and their structure diversity, the cost and time required for hazard evaluation of each compound is unrealistic. In that purpose, new approach methodologies (NAMs) can be applied to evaluate such large set of molecules. Among them, quantitative structure-activity relationship (QSAR) in silico models could be useful to predict the mutagenic and carcinogenic properties of mycotoxins. First, a complete list of 904 mycotoxins and metabolites was built. Then, some known mycotoxins were used to determine the best QSAR tools for mutagenicity and carcinogenicity predictions. The best tool was further applied to the whole list of 904 mycotoxins. At the end, 95 mycotoxins were identified as both mutagen and carcinogen and should be prioritized for further evaluation.

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.

Effects of estrogens and endocrine-disrupting chemicals on cell differentiation-survival-proliferation in brain: contributions of neuronal cell lines.

Estrogens and estrogen receptors (ER) are key actors in the control of differentiation and survival and act on extrareproductive tissues such as brain. Thus, estrogens may display neuritogenic effects during development and neuroprotective effects in the pathophysiological context of brain ischemia and neurodegenerative pathologies like Alzheimer's disease or Parkinson's disease. Some of these effects require classical transcriptional "genomic" mechanisms through ER, whereas other effects appear to rely clearly on "membrane-initiated mechanisms" through cytoplasmic signal transduction pathways. Disturbances of these mechanisms by endocrine-disrupting chemicals (EDC) may exert adverse effects on brain. Some EDC may act via ER-independent mechanisms but might cross-react with endogenous estrogen. Other EDC may act through ER-dependent mechanisms and display agonistic/antagonistic estrogenic properties. Because of these potential effects of EDC, it is necessary to establish sensitive cell-based assays to determine EDC effects on brain. In the present review, some effects of estrogens and EDC are described with focus on ER-mediated effects in neuronal cells. Particular attention is given to PC12 cells, an interesting model to study the mechanisms underlying ER-mediated differentiating and neuroprotective effects of estrogens.

Effects of radiofrequency field exposure on proteotoxic-induced and heat-induced HSF1 response in live cells using the bioluminescence resonance energy transfer technique.

As of today, only acute effects of RF fields have been confirmed to represent a potential health hazard and they are attributed to non-specific heating (≥ 1 °C) under high-level exposure. Yet, the possibility that environmental RF impact living matter in the absence of temperature elevation needs further investigation. Since HSF1 is both a thermosensor and the master regulator of heat-shock stress response in eukaryotes, it remains to assess HSF1 activation in live cells under exposure to low-level RF signals. We thus measured basal, temperature-induced, and chemically induced HSF1 trimerization, a mandatory step on the cascade of HSF1 activation, under RF exposure to continuous wave (CW), Global System for Mobile (GSM), and Wi-Fi-modulated 1800 MHz signals, using a bioluminescence resonance energy transfer technique (BRET) probe. Our results show that, as expected, HSF1 is heat-activated by acute exposure of transiently transfected HEK293T cells to a CW RF field at a specific absorption rate of 24 W/kg for 30 min. However, we found no evidence of HSF1 activation under the same RF exposure condition when the cell culture medium temperature was fixed. We also found no experimental evidence that, at a fixed temperature, chronic RF exposure for 24 h at a SAR of 1.5 and 6 W/kg altered the potency or the maximal capability of the proteasome inhibitor MG132 to activate HSF1, whatever signal used. We only found that RF exposure to CW signals (1.5 and 6 W/kg) and GSM signals (1.5 W/kg) for 24 h marginally decreased basal HSF1 activity.

Critical parameters in surface plasmon resonance biosensor development: The interaction between estrogen receptor and estrogen response element as model.

Estrogenic compounds are contaminants that may be active at low concentrations and are a major concern for environmental quality. They interact with organisms via Estrogen Receptors (ER). Some detection methods which have been developed use the ability of ER to interact with short consensus DNA sequences known as Estrogen Response Elements (ERE). Surface Plasmon Resonance (SPR) based techniques allow detection of interaction without labelled molecule use. Such optical transductors are widely used to convert the biological recognition signals into electric quantifiable signals. In this study, SPR is used to assess signal variation in the presence of estrogenic compounds. The combination of physical properties and biological recognition events (e.g. ER/ERE) permits the development of biosensors. These require several steps: activation of the surface, DNA sequence binding, ERE sequence evaluation, ER preparation, characterization of binding properties and regeneration of the surface. This article focuses on the mode of surface activation, protein-DNA binding conditions and the regeneration of ERE. After giving a summary of the literature concerning the usual conditions employed in these steps, an evaluation of some key parameters is given.

Evaluation of the Effect of Chronic 94 GHz Exposure on Gene Expression in the Skin of Hairless Rats In Vivo.

Millimeter waves (MMW) are broadband frequencies that have recently been used in several applications in wireless communications, medical devices and nonlethal weapons [i.e., the nonlethal weapon, Active Denial Systems, (ADS) operating at 94-95 GHz, CW]. However, little information is available on their potential effects on humans. These radio-frequencies are absorbed and stopped by the first layer of the skin. In this study, we evaluated the effects of 94 GHz on the gene expression of skin cells. Two rat populations consisting of 17 young animals and 14 adults were subjected to chronic long-term 94 GHz MMW exposure. Each group of animals was divided into exposed and sham subgroups. The two independent exposure experiments were conducted for 5 months with rats exposed 3 h per day for 3 days per week to an incident power density of 10 mW/cm2, which corresponded to twice the ICNIRP limit of occupational exposure for humans. At the end of the experiment, skin explants were collected and RNA was extracted. Then, the modifications to the whole gene expression profile were analyzed with a gene expression microarray. Without modification of the animal's temperature, long-term chronic 94 GHz-MMW exposure did not significantly modify the gene expression of the skin on either the young or adult rats.

Nuclear accumulation of MKL1 in luminal breast cancer cells impairs genomic activity of ERα and is associated with endocrine resistance.

Estrogen receptor (ERα) is central in driving the development of hormone-dependent breast cancers. A major challenge in treating these cancers is to understand and overcome endocrine resistance. The Megakaryoblastic Leukemia 1 (MKL1, MRTFA) protein is a master regulator of actin dynamic and cellular motile functions, whose nuclear translocation favors epithelial-mesenchymal transition. We previously demonstrated that nuclear accumulation of MKL1 in estrogen-responsive breast cancer cell lines promotes hormonal escape. In the present study, we confirm through tissue microarray analysis that nuclear immunostaining of MKL1 is associated with endocrine resistance in a cohort of breast cancers and we decipher the underlining mechanisms using cell line models. We show through gene expression microarray analysis that the nuclear accumulation of MKL1 induces dedifferentiation leading to a mixed luminal/basal phenotype and suppresses estrogen-mediated control of gene expression. Chromatin immunoprecipitation of DNA coupled to high-throughput sequencing (ChIP-Seq) shows a profound reprogramming in ERα cistrome associated with a massive loss of ERα binding sites (ERBSs) generally associated with lower ERα-binding levels. Novel ERBSs appear to be associated with EGF and RAS signaling pathways. Collectively, these results highlight a major role of MKL1 in the loss of ERα transcriptional activity observed in certain cases of endocrine resistances, thereby contributing to breast tumor cells malignancy.

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.

Determination of estrogen presence in water by SPR using estrogen receptor dimerization.

Estrogenic compounds are a class of pharmaceutical products harmful to animals and a cause of environmental damage. The biological activity of these compounds is high since they have been designed to act at low concentrations. Thus, even at the low concentrations found in the environment, they may produce deleterious effects on aquatic organisms as well as on humans, who might be contaminated in a number of ways (via drinking water or contaminated food, for example). We used the property of these compounds to bind a specific protein (estrogen receptor, ER) to develop a quantification method of these chemical entities. Estrogenic compound detection was performed using ER dimerization properties monitored by surface plasmon resonance (SPR). The ligand-activated ER dimer was detected by its interaction with a specific DNA consensus sequence estrogen response element. The concentration and the nature of the estrogenic compounds modified the SPR signal and were characteristic of the ligand-dependent homodimerization of ER. For 17beta-estradiol, dimerization of ER was experimentally determined at an ER to 17beta-estradiol ratio near 1:1. Estrogenic compounds (17beta-estradiol, estriol, estrone, ethynyl estradiol) activated the dimerization process at different concentration levels, while some others (tamoxiphen, resveratrol, genistein, bisphenol A) did not seem to have any effects on it. We demonstrated that this method allows the direct detection of 17beta-estradiol at concentrations above 1.4 microg/L (5 nM).

Emerging Estrogenic Pollutants in the Aquatic Environment and Breast Cancer.

The number and amount of man-made chemicals present in the aquatic environment has increased considerably over the past 50 years. Among these contaminants, endocrine-disrupting chemicals (EDCs) represent a significant proportion. This family of compounds interferes with normal hormonal processes through multiple molecular pathways. They represent a potential risk for human and wildlife as they are suspected to be involved in the development of diseases including, but not limited to, reprotoxicity, metabolic disorders, and cancers. More precisely, several studies have suggested that the increase of breast cancers in industrialized countries is linked to exposure to EDCs, particularly estrogen-like compounds. Estrogen receptors alpha (ERα) and beta (ERß) are the two main transducers of estrogen action and therefore important targets for these estrogen-like endocrine disrupters. More than 70% of human breast cancers are ERα-positive and estrogen-dependent, and their development and growth are not only influenced by endogenous estrogens but also likely by environmental estrogen-like endocrine disrupters. It is, therefore, of major importance to characterize the potential estrogenic activity from contaminated surface water and identify the molecules responsible for the hormonal effects. This information will help us understand how environmental contaminants can potentially impact the development of breast cancer and allow us to fix a maximal limit to the concentration of estrogen-like compounds that should be found in the environment. The aim of this review is to provide an overview of emerging estrogen-like compounds in the environment, sum up studies demonstrating their direct or indirect interactions with ERs, and link their presence to the development of breast cancer. Finally, we emphasize the use of in vitro and in vivo methods based on the zebrafish model to identify and characterize environmental estrogens.

Rapid assessment of estrogenic compounds by CXCL-test illustrated by the screening of the UV-filter derivative benzophenones.

CXCL-test is a method that uses the estrogen-dependent secretion of the natural endogenous chemokine CXCL12 to evaluate the estrogenic activity of molecules. CXCL12 chemokine is involved in the estrogen dependent proliferation of breast cancer cells. Its measure is an indicator of cell proliferation and is used as an alternative test to classical proliferation test. Here we aimed to optimize this test, first to increase the number of tested molecules in a single assay and then to decrease the number of intermediate steps. The optimized CXCL-test was finally used for the evaluation of the estrogenic potency of emerging chemical pollutants: the UV filter benzophenones (BPs). The effect of BPs on CXCL12 secretion was also validated by real time quantitative RT-PCR. The optimized CXCL-test allowed a fast and direct assessment of estrogenic potency of molecules. The estrogenic activities of benzophenones were characterized and divided in two groups. The first one contains weak estrogenic compounds (BP, BP1, BP2, BP3, 234BP and 2344'BP). The second one contains medium estrogenic compounds (4BP, 44'BP, BP8, THB).