Metabolomics Provides Novel Insights into the Potential Toxicity Associated with Heated Tobacco Products, Electronic Cigarettes, and Tobacco Cigarettes on Human Bronchial Epithelial BEAS-2B Cells.

Smoking is an established risk factor for various pathologies including lung cancer. Electronic cigarettes (e-cigs) and heated tobacco products (HTPs) have appeared on the market in recent years, but their safety or, conversely, their toxicity has not yet been demonstrated. This study aimed to compare the metabolome of human lung epithelial cells exposed to emissions of e-cigs, HTPs, or 3R4F cigarettes in order to highlight potential early markers of toxicity. BEAS-2B cells were cultured at the air-liquid interface and exposed to short-term emissions from e-cigs set up at low or medium power, HTPs, or 3R4F cigarettes. Untargeted metabolomic analyses were performed using liquid chromatography coupled with mass spectrometry. Compared to unexposed cells, both 3R4F cigarette and HTP emissions affected the profiles of exogenous compounds, one of which is carcinogenic, as well as those of endogenous metabolites from various pathways including oxidative stress, energy metabolism, and lipid metabolism. However, these effects were observed at lower doses for cigarettes (2 and 4 puffs) than for HTPs (60 and 120 puffs). No difference was observed after e-cig exposure, regardless of the power conditions. These results suggest a lower acute toxicity of e-cig emissions compared to cigarettes and HTPs in BEAS-2B cells. The pathways deregulated by HTP emissions are also described to be altered in respiratory diseases, emphasizing that the toxicity of HTPs should not be underestimated.

Oxidative stress and inflammation induced by air pollution-derived PM2.5 persist in the lungs of mice after cessation of their sub-chronic exposure.

More than 7 million early deaths/year are attributable to air pollution. Current health concerns are especially focused on air pollution-derived particulate matter (PM). Although oxidative stress-induced airway inflammation is one of the main adverse outcome pathways triggered by air pollution-derived PM, the persistence of both these underlying mechanisms, even after exposure cessation, remained poorly studied. In this study, A/JOlaHsd mice were also exposed acutely (24 h) or sub-chronically (4 weeks), with or without a recovery period (12 weeks), to two urban PM2.5 samples collected during contrasting seasons (i.e., autumn/winter, AW or spring/summer, SS). The distinct intrinsic oxidative potentials (OPs) of AW and SS PM2.5, as evaluated in acellular conditions, were closely related to their respective physicochemical characteristics and their respective ability to really generate ROS over-production in the mouse lungs. Despite the early activation of the nuclear factor erythroid 2-related factor 2 (Nrf2) cell signaling pathway by AW and, in a lesser degree, SS PM2.5, in the murine lungs after acute and sub-chronic exposures, the critical redox homeostasis was not restored, even after the exposure cessation. Accordingly, an inflammatory response was reported through the activation of the nuclear factor-kappa B (NF-κB) cell signaling pathway activation, the secretion of cytokines, and the recruitment of inflammatory cells, in the murine lungs after the acute and sub-chronic exposures to AW and, in a lesser extent, to SS PM2.5, which persisted after the recovery period. Taken together, these original results provided, for the first time, new relevant insights that air pollution-derived PM2.5, with relatively high intrinsic OPs, induced oxidative stress and inflammation, which persisted admittedly at a lower level in the lungs after the exposure cessation, thereby contributing to the occurrence of molecular and cellular adverse events leading to the development and/or exacerbation of future chronic inflammatory lung diseases and even cancers.

Impact of Electronic Cigarettes, Heated Tobacco Products and Conventional Cigarettes on the Generation of Oxidative Stress and Genetic and Epigenetic Lesions in Human Bronchial Epithelial BEAS-2B Cells.

Electronic cigarettes (e-cig) and heated tobacco products (HTP) are often used as smoking cessation aids, while the harm reduction effects of these alternatives to cigarettes are still the subject of controversial debate, in particular regarding their carcinogenic potential. The objective of this study is to compare the effects of e-cig, HTP and conventional cigarette emissions on the generation of oxidative stress and genetic and epigenetic lesions in human bronchial epithelial BEAS-2B cells. Our results show that HTP were less cytotoxic than conventional cigarettes while e-cig were not substantially cytotoxic in BEAS-2B cells. E-cig had no significant effect on the Nrf2 pathway, whereas HTP and cigarettes increased the binding activity of Nrf2 to antioxidant response elements and the expression of its downstream targets HMOX1 and NQO1. Concordantly, only HTP and cigarettes induced oxidative DNA damage and significantly increased DNA strand breaks and chromosomal aberrations. Neither histone modulations nor global DNA methylation changes were found after acute exposure, regardless of the type of emissions. In conclusion, this study reveals that HTP, unlike e-cig, elicit a biological response very similar to that of cigarettes, but only after a more intensive exposure: both tobacco products induce cytotoxicity, Nrf2-dependent oxidative stress and genetic lesions in human epithelial pulmonary cells. Therefore, the health risk of HTP should not be underestimated and animal studies are required in order to determine the tumorigenic potential of these emerging products.

The relationship between residential exposure to atmospheric pollution and circulating miRNA in adults living in an urban area in northern France.

INTRODUCTION: MicroRNAs are epigenetic regulatory factors capable of silencing the expression of target genes and might mediate the effects of air pollution on health. The objective of the present population-based study was to investigate the association between microRNA expression and long-term, residential exposure to atmospheric PM10 and NO2. METHOD: We included 998 non-smoking adult participants from the cross-sectional ELISABET survey (2010-2014) in the Lille urban area of France. The mean residential annual pollution levels were estimated with an atmospheric dispersion modelling system. Ten microRNAs were selected on the basis of the literature data, together with two housekeeping microRNAs (miR-93-5p and miR-191-5p) and were quantified with RT-qPCRs. Multivariate linear regression models were used to study the association between microRNAs and air pollution. The threshold for statistical significance (after correction for the FDR) was set to p < 0.1. RESULTS: The mean annual exposure between 2011 and the year of inclusion was 26.4 ± 2.0 µg/m3 for PM10 and 24.7 ± 5.1 µg/m3 for NO2. Each 2 µg/m3 increment in PM10 exposure was associated with an 8.6% increment (95%CI [3.1; 14.3]; pFDR = 0.019) in miR-451a expression. A 5 µg/m3 increment in NO2 exposure was associated with a 5.3% increment ([0.7; 10]; pFDR = 0.056) in miR451a expression, a 3.6% decrement (95%CI [-6.1; -1.1]; pFDR = 0.052) in miR-223-3p expression, a 3.8% decrement (95%CI[-6.8; -0.7]; pFDR = 0.079) in miR-28-3p expression, a 4.3% decrement (95%CI [-7.7; -0.8]; pFDR = 0.055) in miR-146a-5p expression, and a 4.0% decrement (95% CI[-7.4; -0.4]; pFDR = 0.059) in miR-23a-5p expression. The difference between the two housekeeping microRNAs miR-93-5p and miR-191-5p was also associated with PM10 and NO2 exposure. CONCLUSION: Our results suggest that circulating miRNAs are potentially valuable biomarkers of the effects of air pollution.

Prediction of a Large-Scale Database of Collision Cross-Section and Retention Time Using Machine Learning to Reduce False Positive Annotations in Untargeted Metabolomics.

Metabolite identification in untargeted metabolomics is complex, with the risk of false positive annotations. This work aims to use machine learning to successively predict the retention time (Rt) and the collision cross-section (CCS) of an open-access database to accelerate the interpretation of metabolomic results. Standards of metabolites were tested using liquid chromatography coupled with high-resolution mass spectrometry. In CCSBase and QSRR predictor machine learning models, experimental results were used to generate predicted CCS and Rt of the Human Metabolome Database. From 542 standards, 266 and 301 compounds were detected in positive and negative electrospray ionization mode, respectively, corresponding to 380 different metabolites. CCS and Rt were then predicted using machine learning tools for almost 114,000 metabolites. R2 score of the linear regression between predicted and measured data achieved 0.938 and 0.898 for CCS and Rt, respectively, demonstrating the models' reliability. A CCS and Rt index filter of mean error ± 2 standard deviations could remove most misidentifications. Its application to data generated from a toxicology study on tobacco cigarettes reduced hits by 76%. Regarding the volume of data produced by metabolomics, the practical workflow provided allows for the implementation of valuable large-scale databases to improve the biological interpretation of metabolomics data.

Exposure of Caenorhabditis elegans to Dietary Nε-Carboxymethyllysine Emphasizes Endocytosis as a New Route for Intestinal Absorption of Advanced Glycation End Products.

The impact of dietary advanced glycation end products (dAGEs) on human health has been discussed in many studies but, to date, no consensual pathophysiological process has been demonstrated. The intestinal absorption pathways which have so far been described for dAGEs, the passive diffusion of free AGE adducts and transport of glycated di-tripeptides by the peptide transporter 1 (PEPT-1), are not compatible with certain pathophysiological processes described. To get new insight into the intestinal absorption pathways and the pathophysiological mechanisms of dAGEs, we initiated an in vivo study with a so-called simple animal model with a complete digestive tract, Caenorhabditis elegans. Dietary bacteria were chemically modified with glyoxylic acid to mainly produce Nε-carboxymethyllysine (CML) and used to feed the worms. We performed different immunotechniques using an anti-CML antibody for the relative quantification of ingested CML and localization of this AGE in the worms' intestine. The relative expression of genes encoding different biological processes such as response to stresses and intestinal digestion were determined. The physiological development of the worms was verified. All the results were compared with those obtained with the control bacteria. The results revealed a new route for the intestinal absorption of dietary CML (dCML), endocytosis, which could be mediated by scavenger receptors. The exposure of worms to dCML induced a reproductive defect and a transcriptional response reflecting oxidative, carbonyl and protein folding stresses. These data, in particular the demonstration of endocytosis of dCML by enterocytes, open up new perspectives to better characterize the pathophysiological mechanisms of dAGEs.

Comparison of the chemical composition of aerosols from heated tobacco products, electronic cigarettes and tobacco cigarettes and their toxic impacts on the human bronchial epithelial BEAS-2B cells.

The electronic cigarettes (e-cigs) and more recently the heated tobacco products (HTP) provide alternatives for smokers as they are generally perceived to be less harmful than conventional cigarettes. However, it is crucial to compare the health risks of these different emergent devices, in order to determine which product should be preferred to substitute cigarette. The present study aimed to compare the composition of emissions from HTP, e-cigs and conventional cigarettes, regarding selected harmful or potentially harmful compounds, and their toxic impacts on the human bronchial epithelial BEAS-2B cells. The HTP emitted less polycyclic aromatic hydrocarbons and carbonyls than the conventional cigarette. However, amounts of these compounds in HTP aerosols were still higher than in e-cig vapours. Concordantly, HTP aerosol showed reduced cytotoxicity compared to cigarette smoke but higher than e-cig vapours. HTP and e-cig had the potential to increase oxidative stress and inflammatory response, in a manner similar to that of cigarette smoke, but after more intensive exposures. In addition, increasing e-cig power impacted levels of certain toxic compounds and related oxidative stress. This study provides important data necessary for risk assessment by demonstrating that HTP might be less harmful than tobacco cigarette but considerably more harmful than e-cig.

Toxicity of fine and quasi-ultrafine particles: Focus on the effects of organic extractable and non-extractable matter fractions.

To date no study has been able to clearly attribute the observed toxicological effects of atmospheric particles (PM) to a specific class of components. The toxicity of both the organic extractable matter (OEM2.5-0.3) and non-extractable matter (NEM2.5-0.3) of fine particles (PM2.5-0.3) was compared to that of PM2.5-0.3 in its entirety on normal human epithelial bronchial BEAS-2B cells in culture. The specific effect of the quasi-ultrafine fraction (PM0.3) was assessed, by comparing the responses of cells exposed to the PM2.5-0.3 and PM0.3 organic extractable matter, OEM2.5-0.3 and OEM0.3 respectively. Chemically, PAH, O-PAH, and N-PAH were respectively 43, 17, and 4 times more concentrated in PM0.3 than in PM2.5-0.3, suggesting thereby a predominant influence of anthropogenic activities and combustion sources. BEAS-2B cells exposed to PM2.5-0.3, NEM2.5-0.3, EOM2.5-0.3 and OEM0.3 lead to different profiles of expression of selected genes and proteins involved in the metabolic activation of PAH, O-PAH, and N-PAH, and in the genotoxicity pathways. Specifically, OEM0.3 was the most inducer for phase I and phase II enzymes implicated in the metabolic activation of PAH (AHR, AHRR, ARNT, CYP1A1, CYP1B1, EPHX-1, GSTA-4) thereby producing the highest DNA damage, felt by ATR and, thereafter, a cascade of protein phosphorylation (CHK1/CHK2/MDM2) closely related to the cell cycle arrest (P21 and P53 induction). This study underlined the crucial role played by the organic chemicals present in PM0.3. These results should be considered in any future study looking for the main chemical determinants responsible for the toxicity of ambient fine PM.

Toxicological appraisal of the chemical fractions of ambient fine (PM2.5-0.3) and quasi-ultrafine (PM0.3) particles in human bronchial epithelial BEAS-2B cells.

New toxicological research is still urgently needed to improve the current knowledge about the induction of some underlying mechanisms of toxicity by the different chemical fractions of ambient particulate matter (PM). This in vitro study sought also to better evaluate and compare the respective toxicities of fine particles (PM2.5-0.3) and their inorganic and organic chemical fractions, and the respective toxicities of the organic chemical fractions of PM2.5-0.3 and quasi-ultrafine particles (PM0.3). Human bronchial epithelial BEAS-2B cells were also exposed for 6-48 h to relatively low doses of PM2.5-0.3 and their organic extractable (OEM2.5-0.3) and non-extractable (NEM2.5-0.3) fractions, and the organic extractable fraction (OEM0.3) of PM0.3. We reported that not only PM2.5-0.3, but also, to a lesser extent, its inorganic chemical fraction, NEM2.5-0.3, and organic chemical fraction, OEM2.5-0.3, were able to significantly induce ROS overproduction and oxidative damage notwithstanding the early activation of NRF2 signaling pathway. Moreover, for any exposure, inflammatory and apoptotic events were noticed. Similar results were observed in BEAS-2B cells exposed to OEM0.3, rich of polycyclic aromatic hydrocarbons and their nitrated and oxygenated derivatives. In BEAS-2B cells exposed for 24 and 48 h to OEM2.5-0.3 and OEM0.3, to a higher extent, there was an alteration of the levels of some critical proteins even though crucial for the autophagy rather than a real reduction of autophagy. It is noteworthy that the toxicological effects were equal or mostly higher in BEAS-2B cells exposed for 6 and/or 24 h to PM2.5-0.3 from those exposed to NEM2.5-0.3 or OEM2.5-0.3, and in BEAS-2B cells exposed for 6 and/or mostly 24 h to OEM0.3 from those exposed to OEM2.5-0.3. Taken together, these results revealed the higher potentials for toxicity, closely linked to their respective physical and chemical characteristics, of PM2.5-0.3 vs NEM2.5-0.3 and/or OEM2.5-0.3, and OEM0.3 vs OEM2.5-0.3.

Exposure to metal fumes and circulating miRNAs in Algerian welders.

PURPOSE: A cross-sectional study was conducted in a group of Algerian welders to study the relationship between the exposure to metal particles from welding fumes and the concentration of three circulating miRNAs, miR-21, miR-146a and miR-155, as markers of renal function injury. METHODS: Characteristics of the subjects and the curriculum laboris were determined by questionnaires. We measured the concentrations of metals in blood and urine samples using ICP-MS. The three circulating miRNAs studied were measured by quantitative PCR. Associations between miRNAs and internal exposure markers were assessed by simple and multiple regression analyses. RESULTS: miR-21 was significantly lower among welders (p = 0.017), compared with controls, adjusted for age, body mass index, smoking status and seniority. Significant adjusted associations were observed between miR-21 or miR-155 and urinary chromium (p = 0.005 or p = 0.041, respectively), miR-146a and urinary nickel (p = 0.019). The results of the multivariate analysis showed that duration of employment was the main factor responsible for the variation of miRNAs among welders. CONCLUSION: In conclusion, a recent exposure to certain metals, mainly chromium and nickel, appears to be associated to a decrease in plasma expression of miR-21, miR-146a and miR-155. Further larger studies would help to determine the mechanisms of action of metal particles on miRNA expression.

Exposure to Atmospheric Ultrafine Particles Induces Severe Lung Inflammatory Response and Tissue Remodeling in Mice.

Exposure to particulate matter (PM) is leading to various respiratory health outcomes. Compared to coarse and fine particles, less is known about the effects of chronic exposure to ultrafine particles, despite their higher number and reactivity. In the present study, we performed a time-course experiment in mice to better analyze the lung impact of atmospheric ultrafine particles, with regard to the effects induced by fine particles collected on the same site. Trace element and PAH analysis demonstrated the almost similar chemical composition of both particle fractions. Mice were exposed intranasally to FF or UFP according to acute (10, 50 or 100 µg of PM) and repeated (10 µg of PM 3 times a week during 1 or 3 months) exposure protocols. More particle-laden macrophages and even greater chronic inflammation were observed in the UFP-exposed mice lungs. Histological analyses revealed that about 50% of lung tissues were damaged in mice exposed to UFP for three months versus only 35% in FF-exposed mice. These injuries were characterized by alveolar wall thickening, macrophage infiltrations, and cystic lesions. Taken together, these results strongly motivate the update of current regulations regarding ambient PM concentrations to include UFP and limit their emission.

In vitro evaluation of organic extractable matter from ambient PM2.5 using human bronchial epithelial BEAS-2B cells: Cytotoxicity, oxidative stress, pro-inflammatory response, genotoxicity, and cell cycle deregulation.

A particular attention has been devoted to the type of toxicological responses induced by particulate matter (PM), since their knowledge is greatly complicated by the fact that it is a heterogeneous and often poorly described pollutant. However, despite intensive research effort, there is still a lack of knowledge about the specific chemical fraction of PM, which could be mainly responsible of its adverse health effects. We sought also to better investigate the toxicological effects of organic extractable matter (OEM) in normal human bronchial epithelial lung BEAS-2B cells. The wide variety of chemicals, including PAH and other related-chemicals, found in OEM, has been rather associated with early oxidative events, as supported by the early activation of the sensible NRF-2 signaling pathway. For the most harmful conditions, the activation of this signaling pathway could not totally counteract the ROS overproduction, thereby leading to critical oxidative damage to macromolecules (lipid peroxidation, oxidative DNA adducts). While NRF-2 is an anti-inflammatory, OEM exposure did not trigger any significant change in the secretion of inflammatory cytokines (i.e., TNFα, IL-1ß, IL-6, IL-8, MCP-1, and IFNγ). According to the high concentrations of PAH and other related organic chemicals found in this OEM, CYP1A1 and 1B1 genes exhibited high transcription levels in BEAS-2B cells, thereby supporting both the activation of the critical AhR signaling pathway and the formation of highly reactive ultimate metabolites. As a consequence, genotoxic events occurred in BEAS-2B cells exposed to this OEM together with cell survival events, with possible harmful cell cycle deregulation. However, more studies are required to implement these observations and to contribute to better decipher the critical role of the organic fraction of air pollution-derived PM2.5 in the activation of some sensitive signaling pathways closely associated with G1/S and intra-S checkpoint blockage, on the one hand, and cell survival, on the other hand.

Influence of puffing conditions on the carbonyl composition of e-cigarette aerosols.

Owing to their harmful effects on human health, the presence of carbonyl compounds in e-cigarette aerosols raises concerns. To date, the reported concentration levels in e-vapors vary greatly between studies and several factors that markedly influence carbonyl emission during vaping have been highlighted including the heating temperature, the power supply, the device architecture, the filling level of the tank and the main e-liquid constituents. This study investigated the impact of puffing regimen parameters on the carbonyl composition of e-cigarette aerosols with the aim of: (1) better estimating the variability of carbonyl emissions depending on puffing conditions; (2) highlighting puffing profiles that increase the exposure to carbonyls; and (3) estimating to what extent puffing topography could be implied in the variability of carbonyl concentrations reported in the current literature. E-vapors from a single e-liquid were generated from two e-cigarette models with a smoking machine. A total of 7 different puffing regimens were used to individually study the influence of the puff volume, duration and frequency. Carbonyls were collected by DNPH cartridges and analysed by HPLC-UV. E-liquid consumption and e-vapor temperature were also monitored. E-vapor concentrations of formaldehyde, acetaldehyde, acetone, acrolein, propionaldehyde and methylglyoxal were affected, sometimes differently, by the modification of the puffing regimen, as well as by the e-cigarette model. For example, formaldehyde concentration ranged from 20 to 255 ng/puff depending on the puffing conditions. The results of principal component analyses, applied to the concentration data sets for the 6 carbonyls, suggest that the studied parameters interact and highlight some "carbonyl-emitting" combinations of concern (e-cigarette model/puffing regimen). However, the highest concentrations measured in the present study remain far lower than those observed in conventional cigarette mainstream smoke. This study confirms that the chosen puffing regimen contributes a part of the observed variability in the carbonyl levels reported in the scientific literature, hampering comparisons between studies and making interpretation difficult. Thus, harmonized and realistic protocols for the assessment of e-cigarette toxicity by physicochemical or experimental approaches are clearly needed.

Reduced maternal behavior caused by gestational stress is predictive of life span changes in risk-taking behavior and gene expression due to altering of the stress/anti-stress balance.

Exposure of the mother to adverse events during pregnancy is known to induce pathological programming of the HPA axis in the progeny, thereby increasing the vulnerability to neurobehavioral disorders. Maternal care plays a crucial role in the programming of the offspring, and oxytocin plays a key role in mother/pup interaction. Therefore, we investigated whether positive modulation of maternal behavior by activation of the oxytocinergic system could reverse the long-term alterations induced by perinatal stress (PRS; gestational restraint stress 3 times/day during the last ten days of gestation) on HPA axis activity, risk-taking behavior in the elevated-plus maze, hippocampal mGlu5 receptor and gene expression in Sprague-Dawley rats. Stressed and control unstressed dams were treated during the first postpartum week with an oxytocin receptor agonist, carbetocin (1 mg/kg, i.p.). Remarkably, reduction of maternal behavior was predictive of behavioral disturbances in PRS rats as well as of the impairment of the oxytocin and its receptor gene expression. Postpartum carbetocin corrected the reduction of maternal behavior induced by gestational stress as well as the impaired oxytocinergic system in the PRS progeny, which was associated with reduced risk-taking behavior. Moreover, postpartum carbetocin had an anti-stress effect on HPA axis activity in the adult PRS progeny and increased hippocampal mGlu5 receptor expression in aging. In conclusion, the activation of the oxytocinergic system in the early life plays a protective role against the programming effect by adverse experiences and could be considered as a novel and powerful potential therapeutic target for stress-related disorders.

Chemical Evaluation of Electronic Cigarettes: Multicomponent Analysis of Liquid Refills and their Corresponding Aerosols.

Electronic cigarette use has raised concern worldwide regarding potential health risks and its position in tobacco cessation strategies. As part of any toxicity assessment, the chemical characterization of e-liquids and their related vapors are among fundamental data to be determined. Considering the lack of available reference methods, we developed and validated several analytical procedures in order to conduct a multicomponent analysis of six e-liquid refills and their resultant vapor emissions (generated by a smoking machine), and compared them with tobacco smoke. We combined several techniques including gas-chromatography, high and ultra-performance liquid chromatography and inductively coupled plasma with mass spectrometry or ultraviolet and flame ionization detection in order to identify the main e-liquid constituents (propylene glycol, glycerol and nicotine), as well as multiple potentially harmful components (trace elements, polycyclic aromatic hydrocarbons (PAHs), pesticides and carbonyl compounds). Regarding propylene glycol, glycerol and nicotine concentrations, the six tested e-liquids comply with the advertised composition and contain only traces of pollutants. Noticeable lower concentrations of trace elements (≤3.4 pg/mL puff), pesticides (

Particulate metal bioaccessibility in physiological fluids and cell culture media: Toxicological perspectives.

According to the literature, tiny amounts of transition metals in airborne fine particles (PM2.5) may induce proinflammatory cell response through reactive oxygen species production. The solubility of particle-bound metals in physiological fluids, i.e. the metal bioaccessibility is driven by factors such as the solution chemical composition, the contact time with the particles, and the solid-to-liquid phase ratio (S/L). In this work, PM2.5-bound metal bioaccessibility was assessed in various physiological-like solutions including cell culture media in order to evidence the potential impact on normal human bronchial epithelial cells (NHBE) when studying the cytotoxicity and inflammatory responses of PM2.5 towards the target bronchial compartment. Different fluids (H2O, PBS, LHC-9 culture medium, Gamble and human respiratory mucus collected from COPD patients), various S/L conditions (from 1/6000 to 1/100,000) and exposure times (6, 24 and 72h) were tested on urban PM2.5 samples. In addition, metals' total, soluble and insoluble fractions from PM2.5 in LHC-9 were deposited on NHBE cells (BEAS-2B) to measure their cytotoxicity and inflammatory potential (i.e., G6PDH activity, secretion of IL-6 and IL-8). The bioaccessibility is solution-dependent. A higher salinity or organic content may increase or inhibit the bioaccessibiliy according to the element, as observed in the complex mucus matrix. Decreasing the S/L ratio also affect the bioaccessibility depending on the solution tested while the exposure time appears less critical. The LHC-9 culture medium appears to be a good physiological proxy as it induces metal bioaccessibilities close to the mucus values and is little affected by S/L ratios or exposure time. Only the insoluble fraction can be linked to the PM2.5-induced cytotoxicity. By contrast, both soluble and insoluble fractions can be related to the secretion of cytokines. The metal bioaccessibility in LHC-9 of the total, soluble, and insoluble fractions of the PM2.5 under study did not explain alone, the cytotoxicity nor the inflammatory response observed in BEAS-2B cells. These findings confirm the urgent need to perform further toxicological studies to better evaluate the synergistic effect of both bioaccessible particle-bound metals and organic species.

Comparison of cellular and transcriptomic effects between electronic cigarette vapor and cigarette smoke in human bronchial epithelial cells.

The use of electronic cigarette (e-cig) can be considered as an alternative to smoking. However, due to a lack of thorough toxicological studies, absolute safety of these products cannot be guaranteed. The aim of this in vitro work was to investigate the potential toxicity of e-vapors generated by a smoking machine in human bronchial epithelial BEAS-2B cells cultured at air-liquid interface, in comparison to cigarette smoke (CS). Although CS decreased strongly cell viability from 48min exposure, e-vapors induced no cytotoxicity up to 288min exposure. Moreover, oxidative stress was evidenced only after exposure to CS, with a decrease secretion of GRO-ɑ from 8min and of IL-8 and MCP-1 after 48min exposure. Only a low increase of IL-6 secretion was measured in cells exposed to e-vapors. Finally, transcriptomic data of exposed cells indicated that a large number of genes were deregulated in response to CS, especially genes involved in important biological functions as oxidative stress and cell death, while e-vapors elicited very discrete modulation. These results strongly suggest a lower toxicity of e-vapors compared to CS in the BEAS-2B cell line and constitute a baseline for further experimental studies with a larger spectrum of e-liquids and e-cig models.

Expression profiles of genes involved in xenobiotic metabolism and disposition in human renal tissues and renal cell models.

Numerous xenobiotics have been shown to be harmful for the kidney. Thus, to improve our knowledge of the cellular processing of these nephrotoxic compounds, we evaluated, by real-time PCR, the mRNA expression level of 377 genes encoding xenobiotic-metabolizing enzymes (XMEs), transporters, as well as nuclear receptors and transcription factors that coordinate their expression in eight normal human renal cortical tissues. Additionally, since several renal in vitro models are commonly used in pharmacological and toxicological studies, we investigated their metabolic capacities and compared them with those of renal tissues. The same set of genes was thus investigated in HEK293 and HK2 immortalized cell lines in commercial primary cultures of epithelial renal cells and in proximal tubular cell primary cultures. Altogether, our data offers a comprehensive description of kidney ability to process xenobiotics. Moreover, by hierarchical clustering, we observed large variations in gene expression profiles between renal cell lines and renal tissues. Primary cultures of proximal tubular epithelial cells exhibited the highest similarities with renal tissue in terms of transcript profiling. Moreover, compared to other renal cell models, Tacrolimus dose dependent toxic effects were lower in proximal tubular cell primary cultures that display the highest metabolism and disposition capacity. Therefore, primary cultures appear to be the most relevant in vitro model for investigating the metabolism and bioactivation of nephrotoxic compounds and for toxicological and pharmacological studies.

Increased circulating miR-21 levels are associated with kidney fibrosis.

MicroRNAs (miRNAs) are a class of noncoding RNA acting at a post-transcriptional level to control the expression of large sets of target mRNAs. While there is evidence that miRNAs deregulation plays a causative role in various complex disorders, their role in fibrotic kidney diseases is largely unexplored. Here, we found a strong up-regulation of miR-21 in the kidneys of mice with unilateral ureteral obstruction and also in the kidneys of patients with severe kidney fibrosis. In addition, mouse primary fibroblasts derived from fibrotic kidneys exhibited higher miR-21 expression level compared to those derived from normal kidneys. Expression of miR-21 in normal primary kidney fibroblasts was induced upon TGFß exposure, a key growth factor involved in fibrogenesis. Finally, ectopic expression of miR-21 in primary kidney fibroblasts was sufficient to promote myofibroblast differentiation. As circulating miRNAs have been suggested as promising non-invasive biomarkers, we further assess whether circulating miR-21 levels are associated with renal fibrosis using sera from 42 renal transplant recipients, categorized according to their renal fibrosis severity, evaluated on allograft biopsies (Interstitial Fibrosis/Tubular Atrophy (IF/TA). Circulating miR-21 levels are significantly increased in patients with severe IF/TA grade (IF/TA grade 3: 3.0±1.0 vs lower grade of fibrosis: 1.5±1.2; p = 0.001). By contrast, circulating miR-21 levels were not correlated with other renal histological lesions. In a multivariate linear regression model including IF/TA grade and estimated GFR, independent associations were found between circulating miR-21 levels and IF/TA score (ß = 0.307, p = 0.03), and between miR-21 levels and aMDRD (ß = -0.398, p = 0.006). Altogether, these data suggest miR-21 has a key pathogenic role in kidney fibrosis and may represent a novel, predictive and reliable blood marker of kidney fibrosis.

miR-199a-5p Is upregulated during fibrogenic response to tissue injury and mediates TGFbeta-induced lung fibroblast activation by targeting caveolin-1.

As miRNAs are associated with normal cellular processes, deregulation of miRNAs is thought to play a causative role in many complex diseases. Nevertheless, the precise contribution of miRNAs in fibrotic lung diseases, especially the idiopathic form (IPF), remains poorly understood. Given the poor response rate of IPF patients to current therapy, new insights into the pathogenic mechanisms controlling lung fibroblasts activation, the key cell type driving the fibrogenic process, are essential to develop new therapeutic strategies for this devastating disease. To identify miRNAs with potential roles in lung fibrogenesis, we performed a genome-wide assessment of miRNA expression in lungs from two different mouse strains known for their distinct susceptibility to develop lung fibrosis after bleomycin exposure. This led to the identification of miR-199a-5p as the best miRNA candidate associated with bleomycin response. Importantly, miR-199a-5p pulmonary expression was also significantly increased in IPF patients (94 IPF versus 83 controls). In particular, levels of miR-199a-5p were selectively increased in myofibroblasts from injured mouse lungs and fibroblastic foci, a histologic feature associated with IPF. Therefore, miR-199a-5p profibrotic effects were further investigated in cultured lung fibroblasts: miR-199a-5p expression was induced upon TGFß exposure, and ectopic expression of miR-199a-5p was sufficient to promote the pathogenic activation of pulmonary fibroblasts including proliferation, migration, invasion, and differentiation into myofibroblasts. In addition, we demonstrated that miR-199a-5p is a key effector of TGFß signaling in lung fibroblasts by regulating CAV1, a critical mediator of pulmonary fibrosis. Remarkably, aberrant expression of miR-199a-5p was also found in unilateral ureteral obstruction mouse model of kidney fibrosis, as well as in both bile duct ligation and CCl4-induced mouse models of liver fibrosis, suggesting that dysregulation of miR-199a-5p represents a general mechanism contributing to the fibrotic process. MiR-199a-5p thus behaves as a major regulator of tissue fibrosis with therapeutic potency to treat fibroproliferative diseases.