Characterization of the elemental and particle load of patient exhaled breath condensate and comparison with pulmonary lavages.

In the field of biomonitoring, exhaled breath condensate (EBC) is described as a potentially useful matrix for assessing inhalation exposure biomarkers in a non-invasive way. However, it is still unclear to what extent EBC is representative of the deep lung. To address this knowledge gap, EBC, bronchial washes (BWs), and bronchoalveolar lavages (BALs) were collected from 82 patients suffering from interstitial lung diseases (ILDs). The particulate contents and elemental composition of EBC, BW, and BAL were then compared in the same patients. The size distribution of particles in EBC was assessed with dynamic light scattering while inductively coupled plasma mass spectrometry was used to quantify its elemental composition. In addition, transmission electron microscopy coupled with energy dispersive x-ray spectrometry were used to further characterize samples of interest. EBC was found to be representative of both the sub-micron and nano-sized particle fractions of BAL and BW, with lower overall levels of elements in EBC than in BW and BAL. Silicon (Si) was the main component for all respiratory matrices with median levels of 2525µg l-1, 5643µg l-1and 5169µg l-1in the nano/ion fractions of EBC, BAL and BW, respectively. Moreover, Si levels in EBC from patients in this study were elevated compared to the levels reported in the literature for healthy subjects. Interestingly, Si levels in the EBC of ILD patients were inversely related to those in BAL and BW. In conclusion, the particulate content of EBC is associated with the lung particle burden and potentially correlates with pathologies, rendering it a relevant biomonitoring technique for the occupational and clinical fields.

Kinetic time courses of inhaled silver nanoparticles in rats.

Silver nanoparticles (Ag NPs) are priority substances closely monitored by health and safety agencies. Despite their extensive use, some aspects of their toxicokinetics remain to be documented, in particular following inhalation, the predominant route of exposure in the workplace. A same experimental protocol and exposure conditions were reproduced two times (experiments E1 and E2) to document the kinetic time courses of inhaled Ag NPs. Rats were exposed nose-only to 20 nm Ag NPs during 6 h at a target concentration of 15 mg/m3 (E1: 218,341 ± 85,512 particles/cm3; E2, 154,099 ± 5728 particles/cm3). The generated aerosol showed a uniform size distribution of nanoparticle agglomerates with a geometric mean diameter ± SD of 79.1 ± 1.88 nm in E1 and 92.47 ± 2.19 nm in E2. The time courses of elemental silver in the lungs, blood, tissues and excreta were determined over 14 days following the onset of inhalation. Excretion profiles revealed that feces were the dominant excretion route and represented on average (± SD) 5.1 ± 3.4% (E1) and 3.3 ± 2.5% (E2) of the total inhaled exposure dose. The pulmonary kinetic profile was similar in E1 and E2; the highest percentages of the inhaled dose were observed between the end of the 6-h inhalation up to 6-h following the end of exposure, and reached 1.9 ± 1.2% in E1 and 2.5 ± 1.6% in E2. Ag elements found in the GIT followed the trend observed in lungs, with a peak observed at the end of the 6-h inhalation exposure and representing 6.4 ± 4.9% of inhaled dose, confirming a certain ingestion of Ag NPs from the upper respiratory tract. Analysis of the temporal profile of Ag elements in the liver showed two distinct patterns: (i) progressive increase in values with peak at the end of the 6-h inhalation period followed by a progressive decrease; (ii) second increase in values starting at 72 h post-exposure with maximum levels at 168-h followed by a progressive decrease. The temporal profiles of Ag elements in lymphatic nodes, olfactory bulbs, kidneys and spleen also followed a pattern similar to that of the liver. However, concentrations in blood and extrapulmonary organs were much lower than lung concentrations. Overall, results show that only a small percentage of the inhaled dose reached the lungs-most of the dose likely remained in the upper respiratory tract. The kinetic time courses in the gastrointestinal tract and liver showed that part of the inhaled Ag NPs was ingested; lung, blood and extrapulmonary organ profiles also suggest that a small fraction of inhaled Ag NPs progressively reached the systemic circulation by a direct translocation from the respiratory tract.

A Proteomic View of Cellular Responses to Anticancer Quinoline-Copper Complexes.

Metal-containing drugs have long been used in anticancer therapies. The mechansims of action of platinum-based drugs are now well-understood, which cannot be said of drugs containing other metals, such as gold or copper. To gain further insights into such mechanisms, we used a classical proteomic approach based on two-dimensional elelctrophoresis to investigate the mechanisms of action of a hydroxyquinoline-copper complex, which shows promising anticancer activities, using the leukemic cell line RAW264.7 as the biological target. Pathway analysis of the modulated proteins highlighted changes in the ubiquitin/proteasome pathway, the mitochondrion, the cell adhesion-cytoskeleton pathway, and carbon metabolism or oxido-reduction. In line with these prteomic-derived hypotheses, targeted validation experiments showed that the hydroxyquinoline-copper complex induces a massive reduction in free glutathione and a strong alteration in the actin cytoskeleton, suggesting a multi-target action of the hydroxyquinoline-copper complex on cancer cells.

Evaluation of a new method for the collection and measurement of 8-isoprostane in exhaled breath for future application in nanoparticle exposure biomonitoring.

BACKGROUND: In the field of nanoparticle exposure biomonitoring, oxidative stress biomarkers measured in exhaled breath condensate appear promising to detect early respiratory effects in workers handling nanomaterials. However, condensation is known for its poor efficiency in collecting non-volatiles in exhaled breath, leading to the low sensitivity of such measurements. Moreover, to be easily used in field studies on large groups of workers, the collection device must be disposable and convenient. OBJECTIVES: In this study, we have tested a totally disposable commercial device that allows for the easy dry collection of exhaled air after filtration on a patented filter. The suitability and efficiency of the SensAbues (SB) device for collecting 8-isoprostane were evaluated and compared to the RTube (RT). METHODS: Seven healthy volunteers performed two 15 min collections of exhaled breath, one with the SB and one with the RT. Blank devices were used to determine the background levels induced by each device. 8-isoprostane was measured in all samples using an EIA technique. RESULTS: The levels of 8-isoprostane in the exhaled breath of volunteers after collection with the SB were significantly higher than those after collection with the RT. Moreover, the levels obtained in volunteers with the SB were significantly higher than background levels obtained in blank devices, which was not the case for the RT. CONCLUSIONS: This is the first study to report the ability of the SB device to collect and measure 8-isoprostane in exhaled breath. The proposed method offers better sensitivity than a classical collection with the RT device and should be further explored before future application in biomonitoring studies.

Proteomic characterization of human exhaled breath condensate.

To improve biomedical knowledge and to support biomarker discovery studies, it is essential to establish comprehensive proteome maps for human tissues and biofluids, and to make them publicly accessible. In this study, we performed an in-depth proteomics characterization of exhaled breath condensate (EBC), a sample obtained non-invasively by condensation of exhaled air that contains submicron droplets of airway lining fluid. Two pooled samples of EBC, each obtained from 10 healthy donors, were processed using a straightforward protocol based on sample lyophilization, in-gel digestion and liquid chromatography tandem-mass spectrometry analysis. Two 'technical' control samples were processed in parallel to the pooled samples to correct for exogenous protein contamination. A total of 229 unique proteins were identified in EBC among which 153 proteins were detected in both EBC pooled samples. A detailed bioinformatics analysis of these 153 proteins showed that most of the proteins identified corresponded to proteins secreted in the respiratory tract (lung, bronchi). Eight proteins were salivary proteins. Our dataset is described and has been made accessible through the ProteomeXchange database (dataset identifier: PXD007591) and is expected to be useful for future MS-based biomarker studies using EBC as the diagnostic specimen.

Continuous in vitro exposure of intestinal epithelial cells to E171 food additive causes oxidative stress, inducing oxidation of DNA bases but no endoplasmic reticulum stress.

The whitening and opacifying properties of titanium dioxide (TiO2) are commonly exploited when it is used as a food additive (E171). However, the safety of this additive can be questioned as TiO2 nanoparticles (TiO2-NPs) have been classed at potentially toxic. This study aimed to shed some light on the mechanisms behind the potential toxicity of E171 on epithelial intestinal cells, using two in vitro models: (i) a monoculture of differentiated Caco-2 cells and (ii) a coculture of Caco-2 with HT29-MTX mucus-secreting cells. Cells were exposed to E171 and two different types of TiO2-NPs, either acutely (6-48 h) or repeatedly (three times a week for 3 weeks). Our results confirm that E171 damaged these cells, and that the main mechanism of toxicity was oxidation effects. Responses of the two models to E171 were similar, with a moderate, but significant, accumulation of reactive oxygen species, and concomitant downregulation of the expression of the antioxidant enzymes catalase, superoxide dismutase and glutathione reductase. Oxidative damage to DNA was detected in exposed cells, proving that E171 effectively induces oxidative stress; however, no endoplasmic reticulum stress was detected. E171 effects were less intense after acute exposure compared to repeated exposure, which correlated with higher Ti accumulation. The effects were also more intense in cells exposed to E171 than in cells exposed to TiO2-NPs. Taken together, these data show that E171 induces only moderate toxicity in epithelial intestinal cells, via oxidation.

Assessment of nanoparticles and metal exposure of airport workers using exhaled breath condensate.

Aircraft engine exhaust increases the number concentration of nanoparticles (NP) in the surrounding environment. Health concerns related to NP raise the question of the exposure and health monitoring of airport workers. No biological monitoring study on this profession has been reported to date. The aim was to evaluate the NP and metal exposure of airport workers using exhaled breath condensate (EBC) as a non-invasive biological matrix representative of the respiratory tract. EBC was collected from 458 French airport workers working either on the apron or in the offices. NP exposure was characterized using particle number concentration (PNC) and size distribution. EBC particles were analyzed using dynamic light scattering (DLS) and scanning electron microscopy coupled to x-ray spectroscopy (SEM-EDS). Multi-elemental analysis was performed for aluminum (Al), cadmium (Cd) and chromium (Cr) EBC contents. Apron workers were exposed to higher PNC than administrative workers (p < 0.001). Workers were exposed to very low particle sizes, the apron group being exposed to even smaller NP than the administrative group (p < 0.001). The particulate content of EBC was brought out by DLS and confirmed with SEM-EDS, although no difference was found between the two study groups. Cd concentrations were higher in the apron workers (p < 0.001), but still remained very low and close to the detection limit. Our study reported the particulate and metal content of airport workers airways. EBC is a potential useful tool for the non-invasive monitoring of workers exposed to NP and metals.

Long-term exposure of A549 cells to titanium dioxide nanoparticles induces DNA damage and sensitizes cells towards genotoxic agents.

Titanium dioxide nanoparticles (TiO2-NPs) are one of the most produced NPs in the world. Their toxicity has been studied for a decade using acute exposure scenarios, i.e. high exposure concentrations and short exposure times. In the present study, we evaluated their genotoxic impact using long-term and low concentration exposure conditions. A549 alveolar epithelial cells were continuously exposed to 1-50 µg/mL TiO2-NPs, 86% anatase/14% rutile, 24 ± 6 nm average primary diameter, for up to two months. Their cytotoxicity, oxidative potential and intracellular accumulation were evaluated using MTT assay and reactive oxygen species measurement, transmission electron microscopy observation, micro-particle-induced X-ray emission and inductively-coupled plasma mass spectroscopy. Genotoxic impact was assessed using alkaline and Fpg-modified comet assay, immunostaining of 53BP1 foci and the cytokinesis-blocked micronucleus assay. Finally, we evaluated the impact of a subsequent exposure of these cells to the alkylating agent methyl methanesulfonate. We demonstrate that long-term exposure to TiO2-NPs does not affect cell viability but causes DNA damage, particularly oxidative damage to DNA and increased 53BP1 foci counts, correlated with increased intracellular accumulation of NPs. In addition, exposure over 2 months causes cellular responses suggestive of adaptation, characterized by decreased proliferation rate and stabilization of TiO2-NP intracellular accumulation, as well as sensitization to MMS. Taken together, these data underline the genotoxic impact and sensitization effect of long-term exposure of lung alveolar epithelial cells to low levels of TiO2-NPs.

Modeling of the internal kinetics of benzo(a)pyrene and 3-hydroxybenzo(a)pyrene biomarker from rat data.

Measurements of 3-hydroxybenzo(a)pyrene (3-OHBaP) in urine has been proposed for the biomonitoring of exposure to benzo(a)pyrene (BaP) in workers. To allow a better understanding of the toxicokinetics of BaP and its key biomarker, a multicompartment model was developed based on rat data previously obtained by this group. According to the model, iv injected BaP is rapidly distributed from blood to tissues (t1/2 = 3.65 h), with particular affinity for tissue lipid components and liver and lung proteins. BaP is then rapidly distributed to lungs, where significant tissue uptake occurs, followed by the skin, liver, and adipose tissues. Once in liver, BaP is readily metabolized, and 3-OHBaP is formed with a t1/2 of 3.32 h. Lung metabolism of BaP was also accounted for, but its contribution to the whole kinetics was found to be negligible. Once formed, 3-OHBaP is distributed from blood to the various organs almost as fast as the parent compound (t1/2 = 2.26 h). In kidneys, 3-OHBaP builds up as a result of the smaller rate of 3-OHBaP urinary excretion (t1/2 = 4.52 h) as compared with its transfer rate from blood to kidneys (t1/2 = 27.8 min). However, overall clearance of 3-OHBaP from the body is driven by its biliary transfer from liver to the gastrointestinal tract (t1/2 = 3.81 h). The model provides a great fit to independent sets of published data on 3-OHBaP urinary excretion time course (χ² = 0.019). This model proves useful in establishing the main biological determinants of the overall kinetics of these compounds.

Evaluation of DNA adducts, DNA and RNA oxidative lesions, and 3-hydroxybenzo(a)pyrene as biomarkers of DNA damage in lung following intravenous injection of the parent compound in rats.

Biomarkers of exposure and effect were assessed in 40 male Sprague-Dawley rats injected intravenously with 40 micromol/kg of benzo(a)pyrene (BaP) to determine which biomarkers are more representative of BaP-induced DNA damage in lung. Lung, liver, blood, and urine were collected at t = 2, 4, 8, 16, 24, 33, 48, 72, and 360 h postdosing. Specific BaP-diol epoxide (BPDE)-DNA adducts, 8-hydroxy-7,8-dihydro-2'-deoxyguanosine (8-OHdGuo), were measured in lung, liver, and mononucleated blood cells by high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS). Urinary 8-OHdGuo and 8-hydroxy-7,8-dihydroguanosine (8-OHGuo) were also determined by HPLC-MS/MS, and urinary 3-hydroxybenzo(a)pyrene was measured by HPLC/fluorescence. Between 2 and 72 h postdosing, BPDE-DNA adducts were significantly increased in lung, liver, and mononucleated blood cells of BaP-treated rats as compared to controls, with the highest levels found in lung. 8-OHdGuo levels also increased in lung of BaP-treated rats with values reaching statistical significance at 2, 8, and 16 h postinjection. No influence of BaP treatment was found on 8-OHdGuo and 8-OHGuo urinary excretions. BPDE-DNA adducts in lung were strongly correlated to urinary 3-OHBaP (r = 0.936 and p < 0.001) and to a lesser extent to blood BPDE-DNA adducts (r = 0.636 and p < 0.001), the latter of which were correlated to each other (r = 0.573 and p = 0.002). Urinary 3-OHBaP and BPDE-DNA adducts in mononucleated blood cells appear as relevant biomarkers of BaP genotoxic exposure and are highly promising for health risk assessment in humans.