Jean Cambar

In vitro effects of nanoparticles on renal cells.

BackgroundThe 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 in vitro 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.ResultsNanoparticles induced a variety of cell responses. On both IP15 and LLC-PK1 cells, 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-PK1 cells 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-PK1 cells. 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.ConclusionThe present data constitute the first step towards determining in vitro dose effect of manufactured CB and TiO2 NPs 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 in vitro cellular systems will need to be further developed, standardized and validated (relative to in vivo effects) in order to provide useful screening data about the relative toxicity of nanoparticles.

Synthesis and study of antiproliferative activity of novel thienopyrimidines on glioblastoma cells

The receptor tyrosine kinases (for example EGFR, PDGFR, VEGFR) are a transmembrane protein family which plays a crucial role in tumor growth, survival, metastasis dissemination and angiogenesis. During the past 10 years, many tyrosine kinase inhibitors (TKIs) have been approved for cancer treatment (imatinib, gefitinib, erlotinib, sunitinib, sorafenib). These compounds generally possess a pyrrolo- or pyrimido- pyrimidine scaffold or approaching molecular structure. We synthesized 10 thienopyrimidine compounds (including 5 newly synthesized) whose scaffold is very similar to the agents cited above. The cytotoxicity of these agents was evaluated using a MTT assay and a flow cytometry technique on glioblastoma cell lines. Two compounds showed a similar cytotoxicity to the standard anti-EGFR gefitinib (IC50: gefitinib=51.9 microM, 6b=61.8 microM, 6c=41.2 microM), suggesting a blockade of the EGFR pathway by binding to the TK receptor.

Caldilinea tarbellica sp. nov., a filamentous, thermophilic, anaerobic bacterium isolated from a deep hot aquifer in the Aquitaine Basin

An anaerobic, thermophilic, filamentous (0.45 × >100 µm) bacterium, designated D1-25-10-4(T), was isolated from a deep hot aquifer in France. Cells were non-motile and Gram-negative. Growth was observed at 43-65 °C (optimum 55 °C), at pH 6.8-7.8 (optimum pH 7.0) and with 0-5 g NaCl l(-1) (optimum 0 g NaCl l(-1)). Strain D1-25-10-4(T) was a chemo-organotroph and fermented ribose, maltose, glucose, galactose, arabinose, fructose, mannose, sucrose, raffinose, xylose, glycerol, fumarate, peptone, starch and xylan. Yeast extract was required for growth. Sulfate, thiosulfate, sulfite, elemental sulfur, nitrate, nitrite and fumarate were not used as terminal electron acceptors. The G+C content of the DNA was 61.9 mol%. The major cellular fatty acids of strain D1-25-10-4(T) were C(17 : 0), C(18 : 0,) C(16 : 0) and iso-C(17 : 0). The closest phylogenetic relative of strain D1-25-10-4(T) was Caldilinea aerophila STL-6-O1(T) (97.9 % 16S rRNA gene sequence similarity). DNA-DNA relatedness between strain D1-25-10-4(T) and Caldilinea aerophila DSM 14535(T) was 8.7 ± 1 %. On the basis of phylogenetic, genotypic and phenotypic characteristics, strain D1-25-10-4(T) represents a novel species within the genus Caldilinea, class Caldilineae, phylum Chloroflexi, for which the name Caldilinea tarbellica sp. nov. is proposed. The type strain is D1-25-10-4(T) ( = DSM 22659(T)  = JCM 16120(T)).

Relationship between circadian changes in renal hemodynamics and circadian changes in urinary glycosaminoglycan excretion in normal rats

Circadian changes in renal hemodynamics and urinary glycosaminoglycan (GAG) excretion were studied in normal Sprague-Dawley rats to further investigate rhythms in kidney function. Urinary water, protein, and GAG excretion, as well as glomerular filtration rate (GFR) and renal plasma flow (RPF), were determined every 4h over the 24h cycle in an attempt to characterize any temporal changes. Urinary flow rate and proteinuria peaked during the dark activity period of the animals, consistently at the same hour, whereas the lowest values were detected during the resting phase. GAG are mucopolysaccharides entering the constitution of the glomerular basement membrane (GBM), which is the key component in the process of glomerular filtration. Similarly, the urinary excretion rate of GAG showed a circadian rhythmicity in phase with urinary water and protein excretion, with markedly increased values observed during the nocturnal phase of the animals. Moreover, GFR and RPF were demonstrated to exhibit large circadian variations in phase with renal excretory rhythmicity, showing nighttime values significantly greater compared to daytime ones. Strong correlations were found between GFR and RPF rhythms, as well as between GAG and GFR, and GAG and RPF rhythms, although the latter were not statistically significant. This pattern suggests that the circadian rhythmicity in urinary excretion rate of GAG in physiological conditions could presumably be secondary to the temporal changes in renal hemodynamics. In this respect, knowledge of renal chronobiology helpfully contributes to increase our understanding of renal physiology.

Circadian rhythms of renal hemodynamics in unanesthetized, unrestrained rats.

Catheters were placed in the jugular vein and femoral artery of male Sprague-Dawley rats and connected to a specially designed perfusor for continuous constant infusion of 0.9% NaCl and a syringe to perform simultaneous and intermittent blood collections. This permitted continuous 24-h study of renal hemodynamics, estimated by inulin (Cin) and p-amino-hippuric acid (CPAH) clearances; Cin represents glomerular filtration rate and CPAH renal plasma flow. Animals were individually housed in metabolism cages in a controlled environment with light/dark 12:12 h. Urine was collected every 4 h (12:00, 16:00, 20:00, 24:00, 04:00, and 08:00) and blood sampled at the midpoint of urine collection periods. Urine and plasma sodium, potassium, inulin, and PAH were spectrophotometrically assessed. During continuous infusion of isotonic saline, Cin exhibited circadian changes with large decrease between 12:00 and 20:00 h (0.9 +/- 0.2 ml/min) and acrophase at 00:30 h. Rhythmicity in CPAH was similar with the minimum between 16:00 and 20:00 h (2.5 +/- 0.3 ml/min) and peak between 00:00 and 04:00 h (acrophase at 00:25 h). Water and electrolyte excretion were also circadian rhythmic with a similar nighttime enhancement and daytime minimum. Such circadian changes persisted during continuous 0.9% NaCl infusion for several consecutive days. The unanesthetized, unrestrained rat model enables investigations in renal chronopharmacology and chronotoxicology.

Cadmium induces direct morphological changes in mesangial cell culture

The cadmium produced by industrial and agricultural practice represents a major environmental pollutant which may induce severe damage, especially in the kidney where cadmium accumulates. While cadmium is known to severely impair renal tubular functions, glomerular structures are also potential targets. The present study investigated the effects of cadmium on glomerular mesangial cell cultures after short- and long-term exposures, requiring for each endpoint specific culture conditions. After 30 min exposure to 1 microM CdCl(2), used as non-lethal concentration, 0.14 ng/microg proteins of cadmium was internalized by the cells as evaluated by atomic emision spectrometry and induced a significant, cell surface reduction (8.9+/-1.9%). These morphological changes could be correlated to smooth muscle alpha-actin disorganization, without quantitative change in its protein expression level as evaluated by Western-blot and Northern-blot analysis (SMAmRNA/28sRNA, 1.78 CdCl(2) vs. 1.42 control). For longer exposure times, in complex medium, cadmium uptake was efficient (0.36 ng/microg proteins) and induced changes in the actin cytoskeleton with no loss of cell membrane integrity. This study suggests that cultured mesangial cells provide an alternative model to study the effect of cadmium, and underlines the importance of using well-defined conditions to study further intracellular mechanisms.

Cytotoxicity of folpet fungicide on human bronchial epithelial cells

Folpet, a widely used dicarboximide fungicide, has been detected in the ambient air of several vine-growing regions of France. It is present in particle form in the environment; however, no study exploring its potential health impact on airways and the respiratory system has been published. Here, the biological effect of these particles was investigated in vitro on human bronchial epithelial cells (16HBE14o-). To be close to the real-life conditions of exposure, Folpan 80WG, a commercial form of folpet, was tested. Folpan 80WG particles showed dose- and time-dependent cytotoxic effects on 16HBE14o- cells. This effect was compared to that produced by technical-grade folpet and both were found to induce a toxicity with similar IC(50) values after 24h of exposure. After 4h and at least until 48h of exposure, the IC(50) values of Folpan 80WG particles were between 2.4 and 2.8 microg/cm(2). Investigation of the cytotoxicity found that Folpan 80WG particles at 1.85 microg/cm(2) induced an increase in ROS production from the first hour of exposure. Evidence that oxidative processes occur in folpet-exposed cells was confirmed by the presence of membrane lipid peroxidation. Furthermore, early apoptosis and late apoptosis/necrosis were both present after the first hour of exposure. These findings indicate that exposure to Folpan 80WG particles result in a rapid cytotoxic effect on human bronchial epithelial cells in vitro that could be in part explained by oxidative stress, characterised by membrane lipid peroxidation and ROS production.

Comparative Cytotoxicity of Low- and High-Osmolar Contrast Media to Human Fibroblasts and Rat Mesangial Cells in Culture

RATIONALE AND OBJECTIVES The authors investigate the relative sensitivity of rat mesangial cells to iodinated contrast media (CM) and control solutions versus less differentiated cells (ie, human fibroblasts) and compare the effects of low-osmolar ionic (ioxaglate) and nonionic (iopamidol) and high-osmolar ionic (diatrizoate) CM on rat mesangial cells. METHODS The cytotoxic effects of ioxaglate and control solutions of sodium chloride and mannitol were assessed by neutral red uptake in isolated rat mesangial cells and human fibroblasts. In a second series of studies, the cytotoxic effects of ioxaglate, iopamidol, and diatrizoate (0 to 100 mg I/mL) on rat mesangial cells were compared. RESULTS Rat mesangial cells were more sensitive to the cytotoxic effects of ioxaglate than the less differentiated human fibroblasts between 70 and 100 mg I/mL. A similar discrepancy was observed in the case of control solutions, sodium chloride, and mannitol. Ioxaglate and iopamidol induced a similar level of cytotoxicity in rat mesangial cells whereas the high-osmolar agent diatrizoate was significantly more cytotoxic. However, the calculated inhibitory concentrations of 50% of all three CM were associated with similar osmolalities, suggesting a major role for this parameter in the case of such media. CONCLUSIONS Rat mesangial cells are more sensitive to the cytotoxic effects of CM and hyperosmolar solutions than the less differentiated human fibroblasts. High-osmolar CM are more cytotoxic than ionic and nonionic low-osmolar CM to rat mesangial cells. Ionicity seems to play no deleterious role at similar iodine concentrations because ioxaglate and iopamidol had equivalent cytotoxic effects on mesangial cells.

Dosing time-dependent nephrotoxicity of cyclosporin A during 21-day administration to Wistar rats.

The incidence of cyclosporine A (CsA) nephrotoxicity with reference to the temporal stage of administration was studied during a chronic 21-day treatment in male Wistar rats. Oral administration (20 mg/kg/day) was given at four different times: 1, 7, 13, or 19 hours after light onset (HALO). Plasma creatinine and blood urea nitrogen (BUN) levels were determined at regular intervals over the 24 h: before treatment (day 0); 7, 14, and 21 days after the beginning of treatment (days 7, 14, and 21); and 7 and 14 days after CsA withdrawal (days 28 and 35). At the same times, creatinine clearance and g-glutamyl transferase urinary excretion were determined in the groups of animals treated at 7 and 19 HALO. Residual concentrations of CsA in the renal tissue were measured at the end of the treatment period (day 21) in all groups. Nephrotoxicity of CsA was dependent on the temporal stage of administration. The renal vasoconstriction showed by the increase in plasma creatinine and BUN levels and the decrease in creatinine clearance was maximal when the CsA was given at 7 and 19 HALO and was correlated to the tissue concentrations of CsA. Tubular injury seems to occur earlier and the return to normal function less rapidly in animals treated at 19 HALO compared with animals treated at 7 HALO.

Physicochemical characteristics and bronchial epithelial cell cytotoxicity of Folpan 80 WG® and Myco 500®, two commercial forms of folpet

BackgroundPesticides, in particular folpet, have been found in rural and urban air in France in the past few years. Folpet is a contact fungicide and has been widely used for the past 50 years in vineyards in France. Slightly water-soluble and mostly present as particles in the environment, it has been measured at average concentration of 40.1 μg/m3 during its spraying, 0.16–1.2 μg/m3 in rural air and around 0.01 μg/m3 in urban air, potentially exposing both the workers and the general population. However, no study on its penetration by inhalation and on its respiratory toxicity has been published. The objective of this study was to determine the physicochemical characteristics of folpet particles (morphology, granulometry, stability) in its commercial forms under their typical application conditions. Moreover, the cytotoxic effect of these particles and the generation of reactive oxygen species were assessed in vitro on respiratory cells.ResultsGranulometry of two commercial forms of folpet (Folpan 80WG® and Myco 500®) under their typical application conditions showed that the majority of the particles (>75%) had a size under 5 μm, and therefore could be inhaled by humans. These particles were relatively stable over time: more than 75% of folpet remained in the particle suspension after 30 days under the typical application conditions. The inhibitory concentration (IC50) on human bronchial epithelial cells (16HBE14o-) was found to be between 2.89 and 5.11 μg/cm2 for folpet commercial products after 24 h of exposure. Folpet degradation products and vehicles of Folpan 80 WG® did not show any cytotoxicity at tested concentrations. At non-cytotoxic and subtoxic concentrations, Folpan 80 WG® was found to increase DCFH-DA fluorescence.ConclusionThese results show that the particles of commercial forms of folpet are relatively stable over time. Particles could be easily inhaled by humans, could reach the conducting airways and are cytotoxic to respiratory cells in vitro. Folpet particles may mediate its toxicity directly or indirectly through ROS-mediated alterations. These data constitute the first step towards the risk assessment of folpet particles by inhalation for human health. This work confirms the need for further studies on the effect of environmental pesticides on the respiratory system.