In vitro effects of nanoparticles on renal cells

<p>Abstract</p> <p>Background</p> <p>The ability of nanoparticles to cross the lung-blood barrier suggests that they may translocate to blood and to targets distant from their portal of entry. Nevertheless, nanotoxicity in organs has received little attention. The purpose of this study was to evaluate nanotoxicity in renal cells using <it>in vitro </it>models. Various carbon black (CB) (FW2–13 nm, Printex60-21 nm and LB101-95 nm) and titanium dioxide (TiO₂-15 and TiO₂-50 nm) nanoparticles were characterized on size by electron microscopy. We evaluated theirs effects on glomerular mesangial (IP15) and epithelial proximal tubular (LLC-PK₁) renal cells, using light microscopy, WST-1 assay, immunofluorescence labeling and DCFH-DA for reactive oxygen species (ROS) assay.</p> <p>Results</p> <p>Nanoparticles induced a variety of cell responses. On both IP15 and LLC-PK₁cells, the smallest FW2 NP was found to be the most cytotoxic with classic dose-behavior. For the other NPs tested, different cytotoxic profiles were found, with LLC-PK₁cells being more sensitive than IP15 cells. Exposure to FW2 NPs, evidenced in our experiments as the most cytotoxic particle type, significantly enhanced production of ROS in both IP15 and LLC-PK₁cells. Immunofluorescence microscopy using latex beads indicated that depending on their size, the cells internalized particles, which accumulated in the cell cytoplasm. Additionally using transmission electronic microscope micrographs show nanoparticles inside the cells and trapped in vesicles.</p> <p>Conclusion</p> <p>The present data constitute the first step towards determining <it>in vitro </it>dose effect of manufactured CB and TiO₂NPs in renal cells. Cytotoxicological assays using epithelial tubular and glomerular mesangial cell lines rapidly provide information and demonstrated that NP materials exhibit varying degrees of cytotoxicity. It seems clear that <it>in vitro </it>cellular systems will need to be further developed, standardized and validated (relative to <it>in vivo </it>effects) in order to provide useful screening data about the relative toxicity of nanoparticles.</p

Carbon black and titanium dioxide nanoparticles elicit distinct apoptotic pathways in bronchial epithelial cells

<p>Abstract</p> <p>Background</p> <p>Increasing environmental and occupational exposures to nanoparticles (NPs) warrant deeper insight into the toxicological mechanisms induced by these materials. The present study was designed to characterize the cell death induced by carbon black (CB) and titanium dioxide (TiO₂) NPs in bronchial epithelial cells (16HBE14o- cell line and primary cells) and to investigate the implicated molecular pathways.</p> <p>Results</p> <p>Detailed time course studies revealed that both CB (13 nm) and TiO₂(15 nm) NP exposed cells exhibit typical morphological (decreased cell size, membrane blebbing, peripheral chromatin condensation, apoptotic body formation) and biochemical (caspase activation and DNA fragmentation) features of apoptotic cell death. A decrease in mitochondrial membrane potential, activation of Bax and release of cytochrome <it>c </it>from mitochondria were only observed in case of CB NPs whereas lipid peroxidation, lysosomal membrane destabilization and cathepsin B release were observed during the apoptotic process induced by TiO₂NPs. Furthermore, ROS production was observed after exposure to CB and TiO₂but hydrogen peroxide (H₂O₂) production was only involved in apoptosis induction by CB NPs.</p> <p>Conclusions</p> <p>Both CB and TiO₂NPs induce apoptotic cell death in bronchial epithelial cells. CB NPs induce apoptosis by a ROS dependent mitochondrial pathway whereas TiO₂NPs induce cell death through lysosomal membrane destabilization and lipid peroxidation. Although the final outcome is similar (apoptosis), the molecular pathways activated by NPs differ depending upon the chemical nature of the NPs.</p

Etude de l'expression de LRP (Lung Resistance-related protein) et des anomalies chromosomiques dans les cellules bronchiques normales et carcinomateuses pulmonaires humaines

Les carcinomes bronchiques non à petites cellules (CBNPC) sont caractérisés par une chimiorésistance intrinsèque. Actuellement, il n'existe pas de marqueur biologique prédictif de la réponse à la chimiothérapie. Les protéines principalement impliquées dans cette chimiorésistance sont MRP1, et LRP. Le but de ce travail de thèse a été d étudier, si LRP codée par le gène LRP situé près de MRP1, est hyperexprimée dans les CBNPC comme MRP1. Nos résultats montrent : une absence de modification aussi bien dans la distribution intracytoplasmique que dans l'expression de LRP au cours de la carcinogenèse en comparant cellules bronchiques normales et CBNPC, indépendamment de l ADN ploïdie. une augmentation de l expression de LRP accompagnée d une modification de sa localisation dans les CBNPC traités par chimiothérapie néo-adjuvante par rapport aux CBNPC non traités. En conclusion, dans le poumon normal ou tumoral, LRP pourrait avoir un rôle dans la détoxification, sans modification lors de la carcinogenèse. L augmentation de l expression de LRP est liée à la chimiothérapie, et non à l ADN ploïdie ou au gain de chromosomes 16.PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Expression de la protéine MRP1 (multidrug related protein) dans les sous-populations tumorales des cancers bronchiques non à petites cellules

PARIS5-BU Méd.Cochin (751142101) / SudocPARIS-BIUM (751062103) / SudocCentre Technique Livre Ens. Sup. (774682301) / SudocSudocFranceF

Can the centrosome be a marker for DNA ploidy in breast cancer?

Background: The role of DNA ploidy in genomic instability of cancer cells and prognosis has been described in a number of studies. The role of the centrosome in cell cycle has also been reported. Aim: In this study, we aimed to investigate the correlation between the centrosome and DNA ploidy in breast cancer in a search for a cytologic predictive and prognostic marker. Materials and Methods: Cell prints were prepared from cell culture of mesothelial cells, fibroblast cell line MRC5 and breast cancer cell lines MCF7 and T47D. Indirect immunofluorescence was used with anti-γ-tubulin and centrosomes were quantified using a fluorescence microscope. DNA ploidy was scored with the DNA index analyzed by flow cytometry. Results: The normal mesothelial cells (94% of the cells with one detected centrosome) and MRC5 diploid cells (68% with two centrosomes) were used as quality controls. A correlation between the number of centrosomes and DNA ploidy was found in MCF7 cell lines (64% of the cells with a number of centrosomes ≥ 3). It was not observed in invasive breast cancer samples; however, the frequency of cells with centrosomes ≥ 3 was found to be slightly higher in DNA aneuploid samples than in DNA diploid samples (15% vs 13.3%). Conclusion: Quantification of centrosome appears to be correlated to DNA ploidy in breast cancer cell lines and slightly associated to DNA aneuploidy in invasive breast cancer. Studies analyzing a larger number of samples as well as morphological abnormalities of the centrosome are needed

Endobronchial ultrasound-guided transbronchial needle aspiration is feasible, safe, and reaches a 90 % diagnostic yield in patients with hypoxemic acute respiratory failure

International audienceAdverse events are common during conventional invasive lung sampling procedures [1] and may be particularly detrimental in critically ill patients [2]. We sought to describe the feasibility, safety, and diagnostic accuracy of endobronchial ultrasound transbronchial needle aspiration (EBUS-TBNA) in the intensive care unit (ICU).Between May 2011 and May 2015, nine consecutive patients with acute respiratory failure (ARF) and mediastinal or hilar involvement were selected from a prospective cohort [3] that comprehensively describes all EBUS-TBNA procedures in Tenon hospital, a tertiary university teaching hospital in Paris, France. EBUS-TBNA was performed using a 6.7-mm-outer-diameter, real-time, bronchoscope with a 7.5-MHz linear ultrasound transducer

Inhibitors of vacuolar H+-ATPase impair the preferential accumulation of daunomycin in lysosomes and reverse the resistance to anthracyclines in drug-resistant renal epithelial cells.

It has been suggested that the inappropriate sequestration of weak-base chemotherapeutic drugs in acidic vesicles by multidrug-resistance (MDR) cells contributes to the mechanisms of drug resistance. The function of the acidic lysosomes can be altered in MDR cells, and so we investigated the effects of lysosomotropic agents on the secretion of lysosomal enzymes and on the intracellular distribution of the weak-base anthracycline daunomycin in drug-resistant renal proximal tubule PKSV-PR(col50) cells and their drug-sensitive PKSV-PR cell counterparts. Imaging studies using pH-dependent lysosomotropic dyes revealed that drug-sensitive and drug-resistant cells exhibited a similar acidic lysosomal pH (around 5.6-5.7), but that PKSV-PR(col50) cells contained more acidic lysosomes and secreted more of the lysosomal enzymes N -acetyl-beta-hexosaminidase and beta-glucuronidase than their parent PKSV-PR cells. Concanamycin A (CCM A), a potent inhibitor of the vacuolar H(+)-ATPase, but not the P-glycoprotein modulator verapamil, stimulated the secretion of N -acetyl-beta-hexosaminidase in both drug-sensitive and drug-resistant cells. Fluorescent studies and Percoll density gradient fractionation studies revealed that daunomycin accumulated predominantly in the lysosomes of PKSV-PR(col50) cells, whereas in PKSV-PR cells the drug was distributed evenly throughout the nucleo-cytoplasmic compartments. CCM A did not impair the cellular efflux of daunomycin, but induced the rapid nucleo-cytoplasmic redistribution of the drug in PKSV-PR(col50) cells. In addition, CCM A and bafilomycin A1 almost completely restored the sensitivity of these drug-resistant cells to daunomycin, doxorubicin and epirubicin. These findings indicate that lysosomotropic agents that impair the acidic-pH-dependent accumulation of weak-base chemotherapeutic drugs may reverse anthracycline resistance in MDR cells with an expanded acidic lysosomal compartment