Distinctive Toxicity of TiO₂ Rutile/Anatase Mixed Phase Nanoparticles on Caco-2 Cells

Titanium dioxide has a long-standing use as a food additive. Micrometric powders are, e.g., applied as whiteners in confectionary or dairy products. Possible hazards of ingested nanometric TiO₂ particles for humans and the potential influence of varying specific surface area (SSA) are currently under discussion. Five TiO₂-samples were analyzed for purity, crystallinity, primary particle size, SSA, ζ potential, and aggregation/agglomeration. Their potential to induce cytotoxicity, oxidative stress, and DNA damage was evaluated in human intestinal Caco-2 cells. Only anatase-rutile containing samples, in contrast to the pure anatase samples, induced significant LDH leakage or mild DNA damage (Fpg-comet assay). Evaluation of the metabolic competence of the cells (WST-1 assay) revealed a highly significant correlation between the SSA of the anatase samples and cytotoxicity. The anatase/rutile samples showed higher toxicity per unit surface area than the pure anatase powders. However, none of the samples affected cellular markers of oxidative stress. Our findings suggest that both SSA and crystallinity are critical determinants of TiO₂-toxicity toward intestinal cells

Fluorescent staining of RAW 264.7 macrophages after 4 h treatment with ZnO or staurosporine (STS) using TUNEL assay.

<p>Representative multichannel images are shown for apoptotic DNA fragmentation (green staining) and corresponding nuclei (blue staining). Original magnification 100×.</p

Effects of ZnO exposure in murine bone marrow-derived macrophages of p47^phox−/− versus wt animals.

<p>(A) Superoxide detection via lucigenin-amplified chemiluminescence depicted in arbitrary units (AU). (B) FACS analysis of sideward scatter related granularity to investigate particle uptake (n = 2). (C) Cell viability determined by WST-1 assay (n = 2). (D) Content of hypodiploid cells determined by FACS analysis after 7-AAD staining (n = 2). STS: staurosporine.</p

Cytotoxic effects of a panel of ZnO particles in RAW 264.7 macrophages.

<p>(A) Cell viability determined by WST-1 assay after 4 h treatment with four different ZnO particles at the indicated concentrations. Data are expressed as a percentage of the non-treated control cells, and represent three independent experiments (i.e. n = 3). As positive control, cells were treated for 4 h with 1 µM staurosporine (STS). (B) FACS analysis after 7-AAD staining revealing cells with hypodiploid DNA content after 4 h treatment with the panel of ZnO particles or staurosporine. Data are expressed as percentage of total cell events (n = 3).</p

Firoin Reduces MAPK Activation and Neutrophilic Lung Inflammation in vivo.

<p>Female Fischer 344 rats (n = 7) were exposed to 2.5 mg/kg CNP in the presence or absence of the indicated doses [mM] of firoin <i>(F)</i>. A: exposure scenario. B: Quantification of phospho-specific signals in lung homogenates in relation to total Erk1/2 and representative Western blots. Total cell counts (C), numbers of neutrophils (D) and macrophages (E), and cinc-1 concentrations (F) were determined in bronchoalveolar lavage. (G) Staining of neutrophil elastase in lungs of animals treated according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111485#pone-0111485-g003" target="_blank">figure 3A</a>. Light bars, control groups with PBS or 1 mM firoin exposure; dark bars, CNP exposed animals. * Significantly different from CNP alone treated animals (p<0.05, Mann-Whitney-U Test). Arrows indicate cells considered as positive for neutrophil elastase.</p

Firoin and Ectoine Prevent CNP-induced MAPK Activation and Subsequent Endpoints of Tissue Homeostasis.

<p>RLE cells were pre-treated with the indicated final concentrations [mM] of firoin <i>(F)</i> in (A), ectoine <i>(E)</i> in (B), or controls PBS or H₂O (Sol), 1 h prior to CNP exposure. Quantitative analysis of MAPK phosphorylation (n = 3–5) and representative Western-blots. Light bars, control treatment with PBS, dark bars, CNP-treatment [10 µg/cm²] for 8 h (Erk1/2) or 4 h (JNK1/2). C: BrdU incorporation and caspase-3 activity (each n = 3) are shown relative to PBS treated controls. Cells were treated as described above. BrdU incorporation was determined after 24 h of exposure, caspase activity after 8 h. * Significantly different from CNP alone exposed controls (p<0.05 ANOVA and Tukey-HSD post hoc testing).</p

Firoin Acts in a Preventive Manner.

<p>Female Fischer 344 (n = 7) rats were pre-treated with 1 mM firoin <i>(F)</i> and subsequently exposed to 2.5 mg/kg CNP as depicted in A. B: Quantification of phospho-specific signals in relation to total Erk1/2 and representative Western blots. Total cell counts (C), numbers of neutrophils (D) and macrophages (E), and cinc-1 concentrations (F) were determined. Light bars, control groups with PBS or firoin pre-treatment; dark bars, PBS or firoin pre-treated animals exposed to CNP. * Significantly different from PBS pre-treated CNP-exposed animals (p<0.05, Mann-Whitney-U Test).</p

Particle-induced Changes in Apoptosis are Restored by Firoin.

<p>Peripheral blood neutrophils from healthy donors (n = 7), 2 h pre-treated with indicated amounts of firoin <i>(F)</i> or 1 mM ectoine <i>(E)</i> before CNP exposition (33 µg/ml). A: After 16 h of CNP treatment, cells were stained with Annexin V and analysed flow cytometrically. Normalized values of % Annexin V-positive cells are shown. Natural apoptosis of untreated cells was considered as 1. Dark bars, CNP-treated, light bars, untreated. * Significantly different from CNP alone (Mann-Whitney U Test after Bonferroni correction for multiple testing p<0.05). B: 6 h post CNP-treatment the anti-apoptotic Mcl-1 expression was measured by Western blot analysis. GAPDH was used as a loading control. Two representative blots from different individuals are shown and irrelevant lanes were removed.</p

Cell viability (WST-1 assay) after 4 h of ZnO exposure observed in a mutant Jurkat T-cell line deficient in caspase-9 (JMR) versus their caspase-9-restored counterpart (JMR/C9).

<p>Data of treated cells are related to corresponding control cells (100% activity, n = 3).</p

Representative Scanning Electron Microscopy images of the four ZnO samples used in present study.

<p>Samples were analyzed by a LEO (Zeiss) 1530 Scanning Electron Microscope at a magnification of 10,000× as well as 50,000× (inserts).</p