Characterization of the components of urban particulate matter mediating impairment of nitric oxide-dependent relaxation in intrapulmonary arteries.

We have previously shown that exposure to urban particulate matter (UPM) impairs endothelial nitric oxide (NO) bioactivity in intrapulmonary arteries. As UPM is composed of heterogeneous constituents, the aim of this study was to clarify the class of pollutants responsible for such effect. Extracts (aqueous, acidic or organic) were prepared from SRM1648, an UPM sample collected in St. Louis (MO, USA). The metal composition of extracts as well as endotoxin content was determined. The effects of each extract, metal mixture and endotoxin were evaluated on endothelium-dependent relaxation to acetylcholine (reflecting endothelial NO production) in rat isolated intrapulmonary arteries. Aqueous or organic SRM1648 pretreatment altered acetylcholine-induced relaxation, similar to that induced by native SRM1648. Organic extract induced similar attenuation of acetylcholine relaxation than organic-treated SRM1648, whereas aqueous extract had no effect. Acidic pretreatment, which impoverished metal and endotoxin content of SRM1648, prevented the impairment of acetylcholine-induced relaxation. However, neither the acidic extract enriched in metals, nor a metal mixture representative of SRM1648 content, modified acetylcholine relaxation, while endotoxin impaired it. Polymyxin B, which chelates endotoxin, prevented SRM1648-induced decrease in relaxation to acetylcholine. It is concluded that SRM1648-induced impairment of endothelial NO-dependent relaxation in intrapulmonary arteries unlikely involved a soluble factor released by vascular cells during UPM exposure, but rather an organic extractible and acidic-sensitive constituents of UPM. Endotoxin, but not metals, may be responsible for UPM-induced impairment of endothelial NO-dependent relaxation.

Cytotoxicity and oxidative stress induced by different metallic nanoparticles on human kidney cells.

BACKGROUND: Some manufactured nanoparticles are metal-based and have a wide variety of applications in electronic, engineering and medicine. Until now, many studies have described the potential toxicity of NPs on pulmonary target, while little attention has been paid to kidney which is considered to be a secondary target organ. The objective of this study, on human renal culture cells, was to assess the toxicity profile of metallic nanoparticles (TiO2, ZnO and CdS) usable in industrial production. Comparative studies were conducted, to identify whether particle properties impact cytotoxicity by altering the intracellular oxidative status. RESULTS: Nanoparticles were first characterized by size, surface charge, dispersion and solubility. Cytotoxicity of NPs was then evaluated in IP15 (glomerular mesangial) and HK-2 (epithelial proximal) cell lines. ZnO and CdS NPs significantly increased the cell mortality, in a dose-dependent manner. Cytotoxic effects were correlated with the physicochemical properties of NPs tested and the cell type used. Analysis of reactive oxygen species and intracellular levels of reduced and oxidized glutathione revealed that particles induced stress according to their composition, size and solubility. Protein involved in oxidative stress such as NF-κb was activated with ZnO and CdS nanoparticles. Such effects were not observed with TiO2 nanoparticles. CONCLUSION: On glomerular and tubular human renal cells, ZnO and CdS nanoparticles exerted cytotoxic effects that were correlated with metal composition, particle scale and metal solubility. ROS production and oxidative stress induction clearly indicated their nephrotoxic potential.

Identification of metallothionein subisoforms in HPLC using accurate mass and online sequencing by electrospray hybrid linear ion trap-orbital ion trap mass spectrometry.

A comprehensive approach to the characterization of metallothionein (MT) isoforms based on microbore HPLC with multimodal detection was developed. MTs were separated as Cd(7) complexes, detected by ICP MS and tentatively identified by molecular mass measured with 1-2 ppm accuracy using Orbital ion trap mass spectrometry. The identification was validated by accurate mass of the corresponding apo-MTs after postcolumn acidification and by their sequences acquired online by higher-energy collision dissociation MS/MS. The detection limits down to 10 fmol and 45 fmol could be obtained by ESI MS for apo- and Cd(7)-isoforms, respectively, and were lower than those obtained by ICP MS (100 fmol). The individual MT isoforms could be sequenced at levels as low as 200 fmol with the sequence coverage exceeding 90%. The approach was successfully applied to the identification of MT isoforms induced in a pig kidney cell line (LLC-PK(1)) exposed to CdS nanoparticles.

In vitro effects of nanoparticles on renal cells.

BACKGROUND: 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 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. RESULTS: Nanoparticles 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. CONCLUSION: The 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.

Physicochemical characteristics and bronchial epithelial cell cytotoxicity of Folpan 80 WG(R) and Myco 500(R), two commercial forms of folpet.

BACKGROUND: Pesticides, 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 mug/m3 during its spraying, 0.16-1.2 mug/m3 in rural air and around 0.01 mug/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. RESULTS: Granulometry of two commercial forms of folpet (Folpan 80WG(R) and Myco 500(R)) under their typical application conditions showed that the majority of the particles (>75%) had a size under 5 mum, 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 mug/cm2 for folpet commercial products after 24 h of exposure. Folpet degradation products and vehicles of Folpan 80 WG(R) did not show any cytotoxicity at tested concentrations. At non-cytotoxic and subtoxic concentrations, Folpan 80 WG(R) was found to increase DCFH-DA fluorescence. CONCLUSION: These 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.

ICP/OES application for assessing cadmium uptake (or toxicity) in glomerular cells: influence of extracellular calcium.

The risks of metals for health are highlighted by their chemical stability and their persistence in the environment. Chronic exposure to low cadmium (Cd) concentrations results in renal dysfunction mainly. Cd has been regarded primarily as a renal tubular toxicant, but glomerular structures may also be affected. Since the cellular environment may influence metal toxicity, differences concerning Cd uptake and toxicity were evaluated according to calcium (Ca) medium concentrations. An optimized inductively coupled plasma emission spectrometry method (ICP/OES) was developed under defined conditions, as a selective analytical tool to determine cadmium uptake in glomerular mesangial cells. The performance characteristics of the analytical system were evaluated for both Cd and Ca by calibration (50 to 250 microg/L and 1 to 5 mg/L), linearity (r2 .9968 and .9943), limits of detection (1 microg/L and 0.1 mg/L) and quantitation (3 microg/L and 0.3 mg/L), accuracy with spiking, and repeatability (1.2 and 2.9%) with matrix matched standards. Total intracellular Cd content was significantly threefold lower in 0.175 mM Ca medium (Ca-free Eagle's minimum essential medium [EMEM] medium with 5% fetal bovine serum [FBS]) than in EMEM medium (1.8 mM Ca) with respectively 0.16 and 0.37 microg/mg proteins after 24 h of Cd (1 microM) exposure. Similar differences were obtained in cytotoxicity studies with a fourfold reduction in the mortality index (IC50). Complementary assays using Ca-spiked medium reinforced that Cd cytotoxicity and uptake were significantly dependent on the concentration of extracellular Ca. These findings suggest direct link between Cd uptake and toxicity, underlining the relevance of the analytical method.

Influence of amoebae and physical and chemical characteristics of water on presence and proliferation of Legionella species in hospital water systems.

The reservoir for hospital-acquired Legionnaires' disease has been shown to be the potable water distribution system. The objectives of the present study were as follows: (1) to examine the possible relationship between physical-chemical characteristics of water such as temperature, pH, hardness, conductivity, and residual chlorine and the presence of amoebae as growth-promoting factors for Legionella species and (2) to determine eradication measures for water distribution systems to seek ways of reducing the risk of legionellosis. Ten hospitals in southwest France took part in this study. Water samples were collected from 106 hot water faucets, showers, hot water tanks, and cooling towers. Two analyses were performed to analyze the association between water characteristics and (1) the presence of Legionella species and (2) the proliferation of Legionella species. Of the 106 water samples examined, 67 (63.2%) were positive for Legionella species. Amoebae were detected in 73 of 106 (68.9%) samples and in 56 of 67 (86.6%) Legionella species-positive samples (P < 10(-6)). In these positive samples, conductivity was lower than 500 microOmega(-1).cm(-1) in 58.2% (P = .026), temperature was below 50 degrees C in 80.6% (P = .004), and hardness was significantly higher (P = 002) than in Legionella species-negative samples. Neither Legionella species nor amoebae were isolated from any sampling point in which the water temperature was above 58.8 degrees C. Multivariate analysis shows that high hardness and presence of amoebae were strongly correlated statistically with the presence of Legionella when showers, tanks, pH, and temperature promoted their proliferation. This study shows the importance of water quality evaluation in assessing environmental risk factors and in selecting the most appropriate prevention and control measures in hospital water systems.

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.