Investigation of the effects of short-term inhalation of carbon nanoparticles on brains and lungs of c57bl/6j and p47(phox-/-) mice.

Recent studies indicate that the brain is a target for toxic carbonaceous nanoparticles present in ambient air. It has been proposed that the neurotoxic effects of such particles are driven by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase mediated generation of reactive oxygen species (ROS) in activated microglia. In the present study, we have evaluated the effects of short term (4h) nose-only inhalation exposure to carbon NP (CNP) in the brains and lungs of C57BL/6J mice and in p47(phox-/-) mice that lack a functional NADPH oxidase. It was shown that the lungs of the p47(phox-/-) mice are less responsive to CNP inhalation than lungs of the corresponding C57BL/6J control animals. Lung tissue mRNA expression of the oxidative stress/DNA damage response genes 8-oxoguanine glycosylase (OGG1) and apurinic/apyrimidinic endonuclease 1 (APE1) were induced by CNP exposure in C57BL/6J but not in the p47(phox-/-) mice. In contrast, the expression of these genes, as well as Tumor Necrosis Factor-α (TNFα), Cyclooxygenase-2 (COX-2) and Heme Oxygenase-1 (HO-1) was not altered in the olfactory bulb, cerebellum or remaining brain tissue part of either mouse background. This indicates that neuroinflammation was not induced by this exposure. CNP inhalation for 4h or for 4h on three consecutive days also did not affect brain tissue protein expression of interleukin (IL)-1ß, while a clear significant difference in constitutive expression level of this pro-inflammatory cytokine was found between C57BL/6J and p47(phox-/-) mice. In conclusion, short-term inhalation exposure to pure carbon nanoparticles can trigger mild p47(phox) dependent oxidative stress responses in the lungs of mice whereas in their brains at the same exposure levels signs of oxidative stress and inflammation remain absent. The possible role of p47(phox) in the neuro-inflammatory effects of nanoparticles in vivo remains to be clarified.

Apoptotic, inflammatory, and fibrogenic effects of two different types of multi-walled carbon nanotubes in mouse lung.

There is increasing concern about the toxicity of inhaled multi-walled carbon nanotubes (MWCNTs). Pulmonary macrophages represent the primary cell type involved in the clearance of inhaled particulate materials, and induction of apoptosis in these cells has been considered to contribute to the development of lung fibrosis. We have investigated the apoptotic, inflammogenic, and fibrogenic potential of two types of MWCNTs, characterised by a contrasting average tube length and entanglement/agglomeration. Both nanotube types triggered H2O2 formation by RAW 264.7 macrophages, but in vitro toxicity was exclusively seen with the longer MWCNT. Both types of nanotubes caused granuloma in the mouse lungs. However, the long MWCNT induced a more pronounced pro-fibrotic (mRNA expression of matrix metalloproteinase-8 and tissue inhibitor of metalloproteinase-1) and inflammatory (serum level of monocyte chemotactic protein-1) response. Masson trichrome staining also revealed epithelial cell hyperplasia for this type of MWCNT. Enhanced apoptosis was detected by cleaved caspase 3 immunohistochemistry in lungs of mice treated with the long and rigid MWCNT and, to a lesser extent, with the shorter, highly agglomerated MWCNT. However, staining was merely localised to granulomatous foci, and neither of the MWCNTs induced apoptosis in vitro, evaluated by caspase 3/7 activity in RAW 264.7 cells. In addition, our study reveals that the inflammatory and pro-fibrotic effects of MWCNTs in the mouse lung can vary considerably depending on their composition. The in vitro analysis of macrophage apoptosis appears to be a poor predictor of their pulmonary hazard.

Carbon nanotubes: an insight into the mechanisms of their potential genotoxicity.

After the health catastrophe resulting from the widespread use of asbestos which was once hailed as a new miracle material, the increasing use of carbon nanotubes (CNTs) has spawned major concern due to their similarities in terms of size, shape and poor solubility. Assessment of genotoxicity has shown that CNTs can damage DNA in vitro and in vivo. The genotoxic potential of different CNT samples varies considerably, however, with negative findings reported in a number of studies, probably due to the enormous heterogeneity of CNTs. The observed spectrum of genotoxic effects shows similarities with those reported for asbestos fibres. Mutagenicity has been found in vivo but in bacterial assays both asbestos and CNTs have mostly tested negative. An overview of key experimental observations on CNT-induced genotoxicity is presented in the first half of this review. In the second part, the potential mechanisms of CNT-elicited genotoxicity are discussed. Whereas CNTs possess intrinsic ROS-scavenging properties they are capable of generating intracellular ROS upon interaction with cellular components, and can cause antioxidant depletion. These effects have been attributed to their Fenton-reactive metals content. In addition, CNTs can impair the functionality of the mitotic apparatus. A noteworthy feature is that frustrated phagocytosis, which is involved in asbestos-induced pathology, has been observed for specific CNTs as well. The involvement of other mechanisms generally implicated in particle toxicity, such as phagocyte activation and impairment of DNA repair, is largely unknown at present and needs further investigation.

Nanoparticle-driven DNA damage mimics irradiation-related carcinogenesis pathways.

The epidemiological association between cancer and exposure to ambient air pollution particles (particles with a 50% cut-off aerodynamic diameter of 10 microm (PM(10))) has been related to the ability of PM(10) and its constituent nanoparticles (NPs) to cause reactive oxidative species (ROS)-driven DNA damage. However, there are no data on the molecular response to these genotoxic effects. In order to assess whether PM(10), NP and ROS-driven DNA damage induce carcinogenesis pathways, A549 cells were treated with tert-butyl-hyperperoxide (Tbh), urban dust (UD), carbon black (CB), nanoparticulate CB (NPCB), benzo(a)pyrene (BaP) and NPCB coated with BaP for

Nanoparticle carbon black driven DNA damage induces growth arrest and AP-1 and NFkappaB DNA binding in lung epithelial A549 cell line.

To assess whether nanoparticle (NP) driven DNA damage induces the expression of proinflammatory transcription factors such as NFB and AP-1 A549, lung epithelial cells were treated with Carbon Black (CB), nanoparticulate CB (NPCB), NPCB coated with BaP (BaP-NPCB) for various times ranging from 30 min to 24 h. DNA strand break was determined by the comet assay and cell cycle status was analyzed using flow cytometry. Nuclear extracts were used for WB analysis of P approximately Ser15-p53. EMSA was used to detect DNA binding. Tested NP caused single strand breaks and significantly altered cell cycle kinetics. NF-kappaB and AP-1 DNA binding were increased at early time points (2.3 and 2.6 fold at 1 hour, respectively). Effects were also found on Ser15-p53 phosphorylation. N-acetylcysteine blocked NP driven effects. In conclusion, NPCB and BaP-NPCB induce DNA damage, activating p53, proteins related to DNA repair and proinflammatory transcription factors.

Association of neuropeptides with Th1/Th2 balance and allergic sensitization in children.

BACKGROUND: Among neurogenic factors, the neuropeptides have an important regulatory influence on immune system activity and may lead to allergic sensitization. OBJECTIVE: The aim of our study was to investigate the relationship of the neuropeptides vasoactive intestinal peptide (VIP), somatostatin (SOM) and substance P (SP) on modulation of Th1/Th2 balance and allergic sensitization in children. METHODS: Within the LISAplus (Life style-Immune system-Allergy) study, blood samples of 321 six-year-old children were analysed for concentration of neuropeptides, Th1 and Th2 cytokines, transcription factors for T cell regulation and suppressors of cytokine signalling. In addition, samples were screened for specific IgE against inhalant and food allergens. RESULTS: Children with high SOM values showed a Th2 polarization and a reduced expression of FOXP3, the marker for regulatory T cells. High (VIP) levels correlated inversely with the expression of T cell transcription factors (Tbet and SOCS3). In contrast, elevated levels of SP were associated with reduced GATA3 and SOCS3 expression and with increased IFN-gamma concentrations. Allergic sensitization was more prevalent in children with higher SOM and VIP concentrations but not associated with SP levels. CONCLUSION: Our data reveal an association between neuropeptides and modulatory effects on immune cells in vivo, especially on Th1/Th2 balance with a correlation to allergic sensitization in children. We suggest that elevated SOM and VIP concentrations and the inducing factors should be considered as allergy risk factors.

Platinum levels in nasal lavage fluid as a biomarker for traffic-related exposure and inflammation in children.

Platinum (Pt) is a well-known constituent of particles emitted by catalytic converters during car operation. To evaluate Pt as a potential marker for traffic related particle exposure, we investigated Pt content along with metals vanadium (V) and chromium (Cr) in coarse and fine particulate matter (PM), sampled in four areas with different traffic density, as well as in the nasal lavage (NAL) of 67 children (average age: 6 years) living in these areas. The different sites were characterised by significant differences in air pollutants including PM, NO, NO(2), CO and Cr, but differences in V or Pt were absent. No significant differences in neutrophil and epithelial cell counts or concentrations of the neutrophil chemoattractant interleukin-8 (IL-8) were found in the NAL of children living in the different areas. In addition, the concentrations of V, Cr and Pt, which were detectable in 64%, 73% and 93% of the individuals, respectively, did not differ between the different locations. However, in the NAL of the children, a significant correlation between Pt and the number of neutrophils/ml (r=0.40, p<0.001) as well as of epithelial cells/ml (r=0.41, p<0.001) was found. No relation was present between nasal inflammation and nasal Cr levels, whereas a relatively weak association was observed between V and epithelial cells counts (r=0.30, p=0.018). In conclusion, our data suggests a role for nasal lavage Pt as a candidate biomarker for traffic-related PM, which is able to induce inflammation in the upper respiratory tract.

Temporal variation of hydroxyl radical generation and 8-hydroxy-2'-deoxyguanosine formation by coarse and fine particulate matter.

AIMS: To determine the induction of 8-hydroxy-2'-deoxyguanosine (8-OHdG) by fine (<2.5 microm) and coarse (10-2.5 microm) particulate matter (PM) sampled over time at one sampling location, and to relate the observed effects to the hydroxyl radical (*OH) generating activities and transition metal content of these samples, and to meteorological parameters. METHODS: Weekly samples of coarse and fine PM were analysed for H(2)O(2) dependent *OH formation using electron spin resonance (ESR) and formation of 8-OHdG in calf thymus DNA using an immuno-dotblot assay. Immunocytochemistry was used to determine 8-OHdG formation in A549 human epithelial lung cells. To determine temporal effects, samples from six weeks in summer and six weeks in autumn/winter were compared using ESR and the dotblot assay. Concentrations of leachable V, Cr, Fe, Ni, and Cu were determined by inductively coupled plasma mass spectrometry. RESULTS: Both PM fractions elicited *OH generation as well as 8-OHdG formation in calf thymus DNA and in A549 cells. 8-OHdG formation in the naked DNA was significantly related to *OH generation, but not to metal concentrations except for copper. A significantly higher *OH generation was observed for coarse PM, but not fine PM collected during the autumn/winter season; this was not due to differences in sampled mass or metal content. Specific weather conditions under which increased *OH formation in the coarse mode was observed suggest that other, as yet unknown, anthropogenic components might affect the radical generating capacity of PM. CONCLUSIONS: Both coarse and fine PM are able to generate *OH, and induce formation of 8-OHdG. When considered at equal mass, *OH formation shows considerable variability with regard to the fraction of PM, as well as the sampling season. The toxicological implications of this heterogeneity in *OH formation by PM, as can be easily determined by ESR, need further investigation.