Lung Damage in Mice after Inhalation of Nanofilm Spray Products: The Role of Perfluorination and Free Hydroxyl Groups

Exposures to two commercial nanofilm spray products (NFPs), a floor sealant (NFP 1) and a coating product for tiles (NFP 2), were investigated for airway irritation, airway inflammation, and lung damage in a mouse inhalation model. The particle exposure was characterized by particle number, particle size distribution, and gravimetric analysis. BALB/cJ mice were exposed for 60 min to the aerosolized products at 3.3–60 mg/m3 (105–106 fine particles/cm3) measured in the breathing zone of the mice. Lung inflammation and lung damage were assessed by study of bronchoalveolar lavage fluid (BALF) cytology, protein in BALF, and histology. Mass spectral analysis showed that NFP 1 and NFP 2 contained hydrolysates and condensates of a perfluorosilane and alkylsilane, respectively. NFP 1 induced a concentration-dependent decrease of the tidal volume lasting for at least 1 day. Exposure concentrations above 16.1 mg/m3 (2.1 × 106 fine particles/cm3) gave rise to significant increases of protein level in BALF and reduced body weight, and histological examination showed atelectasis, emphysema, and hemorrhages. A narrow interval between the no-effect level (16.1 mg/m3) and the lethal concentrations (18.4 mg/m3) was observed. The alkylsilane-based product (NFP 2) had no effect at the concentrations studied. Experiments with different types of perfluorinated silanes and alkylsiloxanes showed that the toxic effects did not arise solely from the perfluorination. The number of free hydroxyl groups in the silanes/alkylsiloxanes was also critical for the toxicity

Modest effect on plaque progression and vasodilatory function in atherosclerosis-prone mice exposed to nanosized TiO2

<p>Abstract</p> <p>Background</p> <p>There is growing evidence that exposure to small size particulate matter increases the risk of developing cardiovascular disease.</p> <p>Methods</p> <p>We investigated plaque progression and vasodilatory function in apolipoprotein E knockout (<it>ApoE</it>^-/-) mice exposed to TiO₂. <it>ApoE</it>^-/-mice were intratracheally instilled (0.5 mg/kg bodyweight) with rutile fine TiO₂(fTiO₂, 288 nm), photocatalytic 92/8 anatase/rutile TiO₂(pTiO₂, 12 nm), or rutile nano TiO₂(nTiO₂, 21.6 nm) at 26 and 2 hours before measurement of vasodilatory function in aorta segments mounted in myographs. The progression of atherosclerotic plaques in aorta was assessed in mice exposed to nanosized TiO₂(0.5 mg/kg bodyweight) once a week for 4 weeks. We measured mRNA levels of <it>Mcp-1</it>, <it>Mip-2</it>, <it>Vcam-1</it>, <it>Icam-1 </it>and <it>Vegf </it>in lung tissue to assess pulmonary inflammation and vascular function. TiO₂-induced alterations in nitric oxide (NO) production were assessed in human umbilical vein endothelial cells (HUVECs).</p> <p>Results</p> <p>The exposure to nTiO₂was associated with a modest increase in plaque progression in aorta, whereas there were unaltered vasodilatory function and expression levels of <it>Mcp-1</it>, <it>Mip-2</it>, <it>Vcam-1</it>, <it>Icam-1 </it>and <it>Vegf </it>in lung tissue. The <it>ApoE^-/-</it>mice exposed to fine and photocatalytic TiO₂had unaltered vasodilatory function and lung tissue inflammatory gene expression. The unaltered NO-dependent vasodilatory function was supported by observations in HUVECs where the NO production was only increased by exposure to nTiO₂.</p> <p>Conclusion</p> <p>Repeated exposure to nanosized TiO₂particles was associated with modest plaque progression in <it>ApoE^-/-</it>mice. There were no associations between the pulmonary TiO₂exposure and inflammation or vasodilatory dysfunction.</p

Particle emission rates during electrostatic spray deposition of TiO2 nanoparticle-based photoactive coating

A new method for the covalent and specific labeling of fusion proteins of carrier proteins (CPs) with small organic molecules has been developed in this work. This technology combines the convenience of expressing genetically tagged reporter proteins with the versatility of synthetic organic molecules. Moreover it promises to overcome some of the limitations of the currently used approaches. The method is based on the posttranslational modification of CPs by phosphopantetheine transferase (PPTase). In this reaction, the 4'-phosphopantetheine group of coenzyme A (CoA) is transferred to a serine residue of CP by PPTase. The PPTase can also use as substrates CoA derivatives that are modified in the thiol moiety by fluorophores or affinity reporter groups that are transferred to CP by PPTase in a covalent and irreversible manner. In this work, several CoA derivatives were synthesized by coupling of CoA with reporter groups functionalized by maleimide. The labeling method using the acyl carrier protein (ACP) and the PPTase (AcpS) from E. coli was applied to the in vitro labeling of purified proteins or in E. coli and yeast lysates, but also to the labeling of proteins expressed on cell surfaces of yeast and mammalian cells. The labeling reaction is fast, specific and quantitative. Pulse-chase labeling experiments with different fluorophores allowed the visualization of different protein generations on yeast cell surfaces. Thus, the method was demonstrated to be attractive for fluorescence microscopy. The second objective was to create a system for the selective labeling of different CPs with different CoA derivatives in the same sample, which requires PPTases with different specificities. The labeling must be performed sequentially, in order that each CP is labeled with only one CoA derivative. The pair peptidyl carrier protein (PCP) from B. brevis and the PPTase from B. subtilis (Sfp) was chosen as counterpart of the pair ACP / AcpS from E. coli. AcpS that is specific towards ACP is used for the first labeling reaction, and after a washing step to remove excess of substrate, the second labeling is performed with Sfp which is promiscuous. The system was successfully tested in vitro in solution and with proteins immobilized on microarrays, and on the surface of yeast and mammalian cells. Finally, the last objective was to reduce the size of the carrier protein (∼ 80 amino acids) to a minimal motif that is efficiently recognized by the PPTase. ACP and PCP were truncated before and after helix II whose residues are involved in the recognition by AcpS and Sfp. The fragments of ACP (aa 27-50) and PCP (aa 37-59) were labeled by AcpS and Sfp respectively, but the kinetics of labeling was slow. Two libraries were created with randomization of the six amino acids around the modified serine. Selections were performed using a phage display system based on the phagemid technology. Mt1 (32 aa) was modified by AcpS at the same rate as wild type ACP. Additional truncations of mt1 sequence yielded mt1.4 (16 aa) that was efficiently recognized by AcpS and weakly by Sfp. In conclusion, this labeling method should become an important tool for studies of cell surface proteins as well as for in vitro applications

Carbon black nanoparticle instillation induces sustained inflammation and genotoxicity in mouse lung and liver

<p>Abstract</p> <p>Background</p> <p>Widespread occupational exposure to carbon black nanoparticles (CBNPs) raises concerns over their safety. CBNPs are genotoxic <it>in vitro </it>but less is known about their genotoxicity in various organs <it>in vivo</it>.</p> <p>Methods</p> <p>We investigated inflammatory and acute phase responses, DNA strand breaks (SB) and oxidatively damaged DNA in C57BL/6 mice 1, 3 and 28 days after a single instillation of 0.018, 0.054 or 0.162 mg Printex 90 CBNPs, alongside sham controls. Bronchoalveolar lavage (BAL) fluid was analyzed for cellular composition. SB in BAL cells, whole lung and liver were assessed using the alkaline comet assay. Formamidopyrimidine DNA glycosylase (FPG) sensitive sites were assessed as an indicator of oxidatively damaged DNA. Pulmonary and hepatic acute phase response was evaluated by <it>Saa3 </it>mRNA real-time quantitative PCR.</p> <p>Results</p> <p>Inflammation was strongest 1 and 3 days post-exposure, and remained elevated for the two highest doses (i.e., 0.054 and 0.162 mg) 28 days post-exposure (P < 0.001). SB were detected in lung at all doses on post-exposure day 1 (P < 0.001) and remained elevated at the two highest doses until day 28 (P < 0.05). BAL cell DNA SB were elevated relative to controls at least at the highest dose on all post-exposure days (P < 0.05). The level of FPG sensitive sites in lung was increased throughout with significant increases occurring on post-exposure days 1 and 3, in comparison to controls (P < 0.001-0.05). SB in liver were detected on post-exposure days 1 (P < 0.001) and 28 (P < 0.001). Polymorphonuclear (PMN) cell counts in BAL correlated strongly with FPG sensitive sites in lung (r = 0.88, P < 0.001), whereas no such correlation was observed with SB (r = 0.52, P = 0.08). CBNP increased the expression of <it>Saa3 </it>mRNA in lung tissue on day 1 (all doses), 3 (all doses) and 28 (0.054 and 0.162 mg), but not in liver.</p> <p>Conclusions</p> <p>Deposition of CBNPs in lung induces inflammatory and genotoxic effects in mouse lung that persist considerably after the initial exposure. Our results demonstrate that CBNPs may cause genotoxicity both in the primary exposed tissue, lung and BAL cells, and in a secondary tissue, the liver.</p

Effects of prenatal exposure to surface-coated nanosized titanium dioxide (UV-Titan). A study in mice

<p>Abstract</p> <p>Background</p> <p>Engineered nanoparticles are smaller than 100 nm and designed to improve or achieve new physico-chemical properties. Consequently, also toxicological properties may change compared to the parent compound. We examined developmental and neurobehavioral effects following maternal exposure to a nanoparticulate UV-filter (UV-titan L181).</p> <p>Methods</p> <p>Time-mated mice (C57BL/6BomTac) were exposed by inhalation 1h/day to 42 mg/m³aerosolized powder (1.7·10⁶n/cm³; peak-size: 97 nm) on gestation days 8-18. Endpoints included: maternal lung inflammation; gestational and litter parameters; offspring neurofunction and fertility. Physicochemical particle properties were determined to provide information on specific exposure and deposition.</p> <p>Results</p> <p>Particles consisted of mainly elongated rutile titanium dioxide (TiO₂) with an average crystallite size of 21 nm, modified with Al, Si and Zr, and coated with polyalcohols. In exposed adult mice, 38 mg Ti/kg was detected in the lungs on day 5 and differential cell counts of bronchoalveolar lavage fluid revealed lung inflammation 5 and 26-27 days following exposure termination, relative to control mice. As young adults, prenatally exposed offspring tended to avoid the central zone of the open field and exposed female offspring displayed enhanced prepulse inhibition. Cognitive function was unaffected (Morris water maze test).</p> <p>Conclusion</p> <p>Inhalation exposure to nano-sized UV Titan dusts induced long term lung inflammation in time-mated adult female mice. Gestationally exposed offspring displayed moderate neurobehavioral alterations. The results are discussed in the light of the observed particle size distribution in the exposure atmosphere and the potential pathways by which nanoparticles may impart changes in fetal development.</p