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Background Hexachloroethane (HC) based smokes and colored pyrotechnic smokes are widely used in the military field, as screening and signaling smokes, that are reported to cause pulmonary toxic effects in humans and in animal studies. Toxicity of pyrotechnic smokes might be caused by the inhalation of particles that composed the smoke and gives the desired pyrotechnic effect. In a previous study, we showed that smoke particles from a red signaling smoke (RSS) and from an HC obscuring smoke (HC-OS) have an intrinsic oxidative potential and induce an adaptive response in human small airway epithelial cells after an acute exposure. Thus, the aim of this study was to further explore the underlying mechanisms of toxicity linked to oxidative stress response of RSS and HC-OS particles, by using a 3D model of normal human bronchial epithelial cells (NHBE) cultured at the air-liquid interface (ALI). Results Acute exposure (24 h) to HC-OS particles induced a weak antioxidant response, characterized by increases of glutathione level and weak increase of one enzymatic antioxidant mRNA expression (NQO-1). Acute exposure to RSS particles induced oxidative stress characterized by production of reactive oxygen species (ROS), increases of glutathione level, increases mRNA expression of several enzymatic antioxidants (SOD-1, SOD-2, HO-1, NQO-1) and expression of NQO-1 protein as well as increase expression of IL-8 mRNA. We noticed that 24 h post-exposure to RSS particles, antioxidant response was still induced. Additionally, 24 h post-exposure to RSS particles revealed internalization of particles and morphological changes in 3D NHBE cells like loss of cilia and a cubic epithelium. Repeated exposures to RSS particles on 3D NHBE cells did not induce oxidative stress while cubic changing aspects of 3D NHBE cells were observed. Conclusions Thus, although further studies are needed to understand the mechanisms underlying these cubic changes, these results revealed differences in toxicity responses between the two types of particles, characterized by an RSS particles-induced oxidative stress as well as morphologic changes. Overall, this study provides a better overview of the toxic effects of pyrotechnic smoke particles whose toxic risks were very little studied.

Air pollution has significant health effects worldwide, and airborne particles play a significant role in these effects. Ultrafine particles (UFPs) have an aerodynamic diameter of 0.1 μm or less, can penetrate deep into the respiratory tree, and are more toxic due to their large specific surface area, which should adsorb organic compounds. The aim of this study is to show the toxicological effects of UFPs with high organic content at low dose on BEAS-2B cells through at air-liquid interface (ALI) exposure using a Vitrocell® technology and a miniCAST (Combustion Aerosol Standard) generator. In conjunction with this approach, chemical analysis of particles and gas phase was performed to evaluate the presence of polycyclic aromatic hydrocarbons (PAHs). Chemical analyses confirmed the presence of PAHs in UFPs. With this experimental setup, exposure of the BEAS-2B cells induced neither cytotoxicity nor mitochondrial dysfunction. However, an increase of oxidative stress was observed, as assessed through Nrf2, NQO1, HO-1, CuZnSOD, MnSOD, and Catalase gene expression, together with significant induction of genes related to xenobiotic metabolism CYP1A1 and CYP1B1. Negative regulation of inflammatory genes expression (IL-6 and IL-8) was present three hours after the exposition to the UFPs. Taken together, this experimental approach, using repeatable conditions, should help to clarify the mechanisms by which organic UFPs induce toxicological effects.

A new HPLC method for the simultaneous quantitative analysis of adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) was developed and validated. ATP, ADP, and AMP were extracted from human bronchial epithelial cells with a rapid extraction procedure and separated with a C18 column (3 × 150 mm, 2.7 µm) using isocratic elution with a mobile phase consisting of 50 mM of potassium hydrogen phosphate (pH 6.80). The absorbance was monitored at 254 nm. The calibration curves were linear in 0.2 to 10 µM, selective, precise, and accurate. This method allowed us to quantify the nucleotides from two cell models: differentiated NHBE primary cells grown at the air–liquid interface (ALI) and BEAS-2B cell line. Our study highlighted the development of a sensitive, simple, and green analytical method that is faster and less expensive than other existing methods to measure ATP, ADP, and AMP and can be carried out on 2D and 3D cell models.

International audience; Gloves represent an essential feature for hand protection because it is a requirement in the professional framework to comply with both hand hygiene standards and the principles of good laboratory practice. Despite their wide use, there is a knowledge gap regarding their composition, including phthalates. The purpose of the present study was to develop two orthogonal methods, GC–MS and HPLC–DAD, for the screening of plasticizers in gloves. Performances of these two methods were compared in terms of ease of use, number of analyzed plasticizers, and sample preparation. The two methods were validated and applied for the identification and quantification of plasticizers in ten gloves made with different materials (vinyl, nitrile, latex, and neoprene). Results revealed the presence of three main ones: DEHP, DEHT, and DINP. Additionally, the contents of plasticizers were extremely variable, depending on the glove material. As expected, the results point out a predominant use of plasticizers in vinyl gloves with an amount that should be of concern. While DEHP is classified as a toxic substance for reproduction 1B, it was, however, quantified in the ten different glove samples studied. This study provides new data regarding the plasticizers’ content in protective gloves, which could be useful for risk assessment.

This work describes the development of biophysical unbiased methods to study the interactions between new designed compounds and carbonic anhydrase II (CAII) enzyme. These methods have to permit both a screening of a series of sulfonamide derivatives and the identification of a lead compound after a thorough study of the most promising molecules. Interactions data were collected using surface plasmon resonance (SPR) and thermal shift assay (TSA). In the first step, experiments were performed with bovine CAII isoform and were extended to human CAII. Isothermal titration calorimetry (ITC) experiments were also conducted to obtain thermodynamics parameters necessary for the processing of the TSA data. Results obtained with this reference methodology demonstrate the effectiveness of SPR and TSA. KD values obtained from SPR data were in perfect accordance with ITC. For TSA, despite the fact that the absolute values of KD were quite different, the same affinity scale was obtained for all compounds. The binding affinities of the analytes studied vary by more than 50 orders of magnitude; for example, the KD value determined by SPR were 6 ± 4 and 299 ± 25 nM for compounds 1 and 3, respectively. This paper discusses some of the theoretical and experimental aspects of the affinity-based methods and evaluates the protein consumption to develop methods for the screening of further new compounds. The double interest of SPR, that is, for screening and for the quick thorough study of the interactions parameters (ka, kd, and KD), leads us to choose this methodology for the study of new potential inhibitors. Copyright © 2013 John Wiley & Sons, Ltd.

Carbonic anhydrase (CA) IX expression is increased upon hypoxia and has been proposed as a therapeutic target since it has been associated with poor prognosis, tumor progression and pH regulation. We report the synthesis and the pharmacological evaluation of a new class of human carbonic anhydrase (hCA) inhibitors, 4-(5-aryl-2-hydroxymethyl-pyrazol-1-yl)-benzenesulfonamides. A molecular modeling study was conducted in order to simulate the binding mode of this new family of enzyme inhibitors within the active site of hCA IX. Pharmacological studies revealed high hCA IX inhibitory potency in the parameters nanomolar range. This study showed that the position of sulfonamide group in meta of the 1-phenylpyrazole increase a selectivity hCA IX versus hCA II of our compounds. An in vitro antiproliferative screening has been performed on the breast cancer MDA-MB-231 cell using doxorubicin as cytotoxic agent and in presence of selected CA IX inhibitor. The results shown that the cytotoxic efficiency of doxorubicin in an hypoxic environment, expressed in IC 50 value, is restored at 20% level with 1 μM CA IX inhibitor. © 2012 Elsevier Ltd. All rights reserved.

International audience; This work was dedicated to the development of a reliable SPR method allowing the simultaneous and quick determination of the affinity and selectivity of designed sulfonamide derivatives for hCAIX and hCAXII versus hCAII, in order to provide an efficient tool to discover drugs for anticancer therapy of solid tumors. We performed for the first time a comparison of two immobilization approaches of hCA isoforms. First one relies on the use of an amine coupling strategy, using a CM7 chip to obtain higher immobilization levels than with a CM5 chip and consequently the affinity with an higher precision (CV% < 10%). The second corresponds to a capture of proteins on a streptavidin chip, named CAP chip, after optimization of biotinylation conditions (amine versus carboxyl coupling, biotin to protein ratio). Thanks to the amine coupling approach, only hCAII and hCAXII isoforms were efficiently biotinylated to reach relevant immobilization (3000 RU and 2700 RU, respectively) to perform affinity studies. For hCAIX, despite a successful biotinylation, capture on the CAP chip was a failure. Finally, concordance between affinities obtained for the three derivatives to CAs isozymes on both chips has allowed to valid the approaches for a further screening of new derivatives.

International audience; The complexes formed between six original chiral diaryl-pyrazole sulfonamide derivatives, displaying poor solubility, and various CDs (native α-, β- and γ-CDs, hydroxypropylated HP-β-CD, methylated Me-β-CD or amino NH2-β-CD) were studied by 1D and 2D 1H NMR at physiological pH in order to determine their apparent binding constant, stoichiometry and structure of the supramolecular assembly. For some complexes, the spectra obtained for free racemic compound and for racemic compound in presence of CD indicate a splitting of signal(s). Additional experiments with pure enantiomer and enriched enantiomer allow us to attribute this behavior to chiral discrimination. The complexing ability of the native β-CD towards our compounds appears the most promising since binding values around 7 × 102 M−1 are obtained. The two-dimensional ROESY (1H–1H) experiments prove the inclusion of the aliphatic part of the compound in the CD cavity. It is noteworthy that this inclusion occurs via the smaller opening of the cavity.

This work describes the development of biophysical unbiased methods to study the interactions between new designed compounds and carbonic anhydrase II (CAII) enzyme. These methods have to permit both a screening of a series of sulfonamide derivatives and the identification of a lead compound after a thorough study of the most promising molecules. Interactions data were collected using surface plasmon resonance (SPR) and thermal shift assay (TSA). In the first step, experiments were performed with bovine CAII isoform and were extended to human CAII. Isothermal titration calorimetry (ITC) experiments were also conducted to obtain thermodynamics parameters necessary for the processing of the TSA data. Results obtained with this reference methodology demonstrate the effectiveness of SPR and TSA. KD values obtained from SPR data were in perfect accordance with ITC. For TSA, despite the fact that the absolute values of KD were quite different, the same affinity scale was obtained for all compounds. The binding affinities of the analytes studied vary by more than 50 orders of magnitude; for example, the KD value determined by SPR were 6 ± 4 and 299 ± 25 nM for compounds 1 and 3, respectively. This paper discusses some of the theoretical and experimental aspects of the affinity-based methods and evaluates the protein consumption to develop methods for the screening of further new compounds. The double interest of SPR, that is, for screening and for the quick thorough study of the interactions parameters (ka , kd , and KD ), leads us to choose this methodology for the study of new potential inhibitors.