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.

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.

Toxicity, genotoxicity and proinflammatory effects of amorphous nanosilica in the human intestinal Caco-2 cell line.

Silica (SiO2) in its nanosized form is now used in food applications although the potential risks for human health need to be evaluated in further detail. In the current study, the uptake of 15 and 55nm colloidal SiO2 NPs in the human intestinal Caco-2 cell line was investigated by transmission electron microscopy. The ability of these NPs to induce cytotoxicity (XTT viability test), genotoxicity (γH2Ax and micronucleus assay), apoptosis (caspase 3), oxidative stress (oxidation of 2,7-dichlorodihydrofluorescein diacetate probe) and proinflammatory effects (interleukin IL-8 secretion) was evaluated. Quartz DQ12 was used as particle control. XTT and cytokinesis-block micronucleus assays revealed size- and concentration-dependent effects on cell death and chromosome damage following exposure to SiO2 nanoparticles, concomitantly with generation of reactive oxygen species (ROS), SiO2-15nm particles being the most potent. In the same way, an increased IL-8 secretion was only observed with SiO2-15nm at the highest tested dose (32µg/ml). TEM images showed that both NPs were localized within the cytoplasm but did not enter the nucleus. SiO2-15nm, and to a lower extent SiO2-55nm, exerted toxic effects in Caco-2 cells. The observed genotoxic effects of these NPs are likely to be mediated through oxidative stress rather than a direct interaction with the DNA. Altogether, our results indicate that exposure to SiO2 NPs may induce potential adverse effects on the intestinal epithelium in vivo.

Genotoxicity of synthetic amorphous silica nanoparticles in rats following short-term exposure. Part 1: oral route.

Synthetic amorphous silica (SAS) in its nanosized form is now used in food applications although the potential risks for human health have not been evaluated. In this study, genotoxicity and oxidative DNA damage of two pyrogenic (NM-202 and 203) and two precipitated (NM-200 and -201) nanosized SAS were investigated in vivo in rats following oral exposure. Male Sprague Dawley rats were exposed to 5, 10, or 20 mg/kg b.w./day for three days by gavage. DNA strand breaks and oxidative DNA damage were investigated in seven tissues (blood, bone marrow from femur, liver, spleen, kidney, duodenum, and colon) with the alkaline and the (Fpg)-modified comet assays, respectively. Concomitantly, chromosomal damage was investigated in bone marrow and in colon with the micronucleus assay. Additionally, malondialdehyde (MDA), a lipid peroxidation marker, was measured in plasma. When required, a histopathological examination was also conducted. The results showed neither obvious DNA strand breaks nor oxidative damage with the comet assay, irrespective of the dose and the organ investigated. Similarly, no increases in chromosome damage in bone marrow or lipid peroxidation in plasma were detected. However, although the response was not dose-dependent, a weak increase in the percentage of micronucleated cells was observed in the colon of rats treated with the two pyrogenic SAS at the lowest dose (5 mg/kg b.w./day). Additional data are required to confirm this result, considering in particular, the role of agglomeration/aggregation of SAS NMs in their uptake by intestinal cells.

Polycyclic aromatic hydrocarbons in binary mixtures modulate the efficiency of benzo[a]pyrene to form DNA adducts in human cells.

Exposure to polycyclic aromatic hydrocarbons (PAHs) always involves complex mixtures that may induce synergistic or antagonistic effects on the genotoxic properties and make risk assessment more difficult. In this study, we evaluated how particulate PAHs modulated the formation of DNA damage induced by carcinogenic benzo[a]pyrene (B[a]P). Single strand breaks and alkali labile sites, as well as BPDE-N²-dGuo DNA adducts were measured in the competent HepG2 cells by Comet assay and HPLC-tandem mass spectrometry, respectively. B[a]P, alone or in binary mixture with other PAHs (1 µM each), led to low amounts of strand breaks. In contrast, formation of BPDE-N²-dGuo adducts was significant and found to be enhanced in HepG2 co-treated for 14 h by B[a]P in the presence of either benzo[b]fluoranthene (B[b]F), dibenz[a,h]anthracene (DB[a,h]A) or indeno[1,2,3-cd]pyrene (IP). Opposite results were obtained with benzo[k]fluoranthene (B[k]F). The same observations were made when cells were pre-incubated with PAH before incubation with B[a]P. These results show that the interactions between PAHs are not direct competition reactions. Emphasis was then placed on the modulation of B[a]P-induced DNA damage by B[b]F and B[k]F. No difference in the time-course formation of DNA damage was observed. However, dose-response relationship differed between these two PAHs with a concentration-dependent inhibition of BPDE-N²-dGuo DNA by B[k]F whereas a constant level of potentiation for B[b]F was observed for concentrations higher than 1 µM. Altogether, these results show that the genotoxicity of B[a]P in binary mixtures with other carcinogenic PAH may be modulated. In such cases, a potentiation of BPDE-N²-dGuo adduct formation is most often observed with exception of B[k]F. Several biological mechanisms may account for these observations, including binding of PAHs to the Ah receptor (AhR), their affinity toward CYP450 and competition for metabolism. These different interactions have to be considered when addressing the intricate issue of the toxicity of mixtures.

Relative contribution of DNA strand breaks and DNA adducts to the genotoxicity of benzo[a]pyrene as a pure compound and in complex mixtures.

Polycyclic aromatic hydrocarbons (PAH) produced upon incomplete combustion of organic matter are suspected to be carcinogenic to humans. In the present work, we especially studied the genotoxicity of benzo[a]pyrene (B[a]P), pure or in mixtures, with emphasis placed on the contribution of oxidative stress and alkylation. A comparison was made between the extent of DNA strand breaks as determined by the Comet assay and the number of DNA adducts to the diol epoxide metabolite of B[a]P measured by HPLC-mass spectrometry. HepG2 cultured human hepatocytes were treated with either pure B[a]P or particulate matter extracted from air samples collected in an urban peri-industrial site or in a metallurgic plant. Treatment with pure B[a]P did not induce increase in Comet measurements below a concentration of 1 microM whereas adducts were observed for concentrations as low as 0.025 microM. Very different results were obtained with environmental samples. Increase in the Comet score was observed with both urban and industrial mixtures containing 0.16 microM of B[a]P, especially for samples of urban origin. Comparison with the effect of the reconstituted PAH fraction of the mixtures allowed us to conclude that the induction of strand breaks results from the action of other components of the samples. In addition, a 30% potentialization and a 90% inhibition in the level of DNA adducts with respect to exposure to 0.16 microM pure B[a]P were observed for cells exposed to industrial and urban mixtures, respectively. These results contrast with the 6-fold enhancement in the yield of BPDE adducts in cells exposed to the reconstituted PAH fraction with respect to pure BaP. Altogether, our data emphasize that (i) a combination of analytical approaches is required to assess the genotoxicity of complex mixtures and (ii) risk assessment based on additivity consideration such as toxic equivalent factors may be misleading.

Influence of the metabolic properties of human cells on the kinetic of formation of the major benzo[a]pyrene DNA adducts.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants. Some of them, including benzo[a]pyrene (B[a]P), are tumorigenic due to their ability to generate DNA adducts. In order to define potential biomarkers of B[a]P exposure, the aim of the study was to identify the major stable DNA adducts in B[a]P-treated human cells. The role played by cellular metabolism on the nature and frequency of the DNA lesions was investigated using keratinocytes (HaCat) and actively metabolizing hepatocytes (HepG2) cell lines. Quantification of DNA damage was carried out by HPLC coupled to tandem mass spectrometry, a sensitive method making possible the selective detection of the different potential stable DNA adducts of B[a]P. These include two adducts of the 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) pathway and three adducts of the radical cation pathway. The results indicate that incubation of cells with B[a]P induces almost exclusively the formation of BPDE DNA adducts on purine bases. The amount of DNA adducts generated in hepatocytes was found to be two orders of magnitude higher than that measured in keratinocytes. Interestingly, the level of the DNA adducts produced in the cells incubated with (+/-)-anti-BPDE was similar in the two cell lines, indicating that the difference observed upon incubation with B[a]P could be attributed to different kinetics of B[a]P metabolism. The repair rate of BPDE DNA adducts was identical in the two cell lines with a half-life estimated to be around 20 h. These data support the use of the stable BPDE DNA adducts, as relevant biomarkers of exposure to B[a]P.