Airway exposure to silica-coated TiO2 nanoparticles induces pulmonary neutrophilia in mice.

The importance of nanotechnologies and engineered nanoparticles has grown rapidly. It is therefore crucial to acquire up-to-date knowledge of the possible harmful health effects of these materials. Since a multitude of different types of nanosized titanium dioxide (TiO(2)) particles are used in industry, we explored their inflammatory potential using mouse and cell models. BALB/c mice were exposed by inhalation for 2 h, 2 h on 4 consecutive days, or 2 h on 4 consecutive days for 4 weeks to several commercial TiO(2) nanoparticles, SiO(2) nanoparticles, and to nanosized TiO(2) generated in a gas-to-particle conversion process at 10 mg/m(3). In addition, effects of in vitro exposure of human macrophages and fibroblasts (MRC-9) to the different particles were assessed. SiO(2)-coated rutile TiO(2) nanoparticles (cnTiO(2)) was the only sample tested that elicited clear-cut pulmonary neutrophilia. Uncoated rutile and anatase as well as nanosized SiO(2) did not induce significant inflammation. Pulmonary neutrophilia was accompanied by increased expression of tumor necrosis factor-alpha (TNF-alpha) and neutrophil-attracting chemokine CXCL1 in the lung tissue. TiO(2) particles accumulated almost exclusively in the alveolar macrophages. In vitro exposure of murine and human macrophages to cnTiO(2) elicited significant induction of TNF-alpha and neutrophil-attracting chemokines. Stimulation of human fibroblasts with cnTiO(2)-activated macrophage supernatant induced high expression of neutrophil-attracting chemokines, CXCL1 and CXCL8. Interestingly, the level of lung inflammation could not be explained by the surface area of the particles, their primary or agglomerate particle size, or radical formation capacity but is rather explained by the surface coating. Our findings emphasize that it is vitally important to take into account in the risk assessment that alterations of nanoparticles, e.g., by surface coating, may drastically change their toxicological potential.

Respiratory and dermal exposure to organophosphorus flame retardants and tetrabromobisphenol A at five work environments.

Organophosphorus compounds (OPs) and tetrabromobisphenol A (TBBPA) are widely utilized as flame retardants (FRs) in plastics, textiles, rubbers, and building materials. Eight OPs and TBBPA were quantified by GC/MS from air samples collected from a furniture workshop, a circuit board factory, two electronics dismantling facilities, a computer classroom, and offices and social premises. In addition, dermal exposure was assessed with patch and hand wash samples at some workplaces. Triphenyl phosphate, tris(2-chloroethyl) phosphate, and tris(2-chloroisopropyl) phosphate were typical contaminants of the workplaces, whereas TBBPA, tricresyl phosphate, tri-n-butyl phosphate, and tris(2-ethylhexyl) phosphate were rather site-specific. The highest geometric mean of total FRs in the air samples was measured in personal samples atthe electronics dismantling facilities (2.9 and 3.8 microg/m3), whereas the stationary sample results from the other environments ranged between 90 and 720 ng/m3. Stationary samplings underestimated the personal exposure at three out of four work places where comparisons were made. Dermal exposure was shown for the first time at these occupational settings. The geometric mean of totalFR levels in patch samples ranged between 1.5 and 24 ng/cm2 and in hand wash samples between 3.5 and 34 microg/ two hands. The health effects of the measured FR levels remain unknown.

Fluorescence quenching-based assays for hydrolyzing enzymes. Application of time-resolved fluorometry in assays for caspase, helicase, and phosphatase.

We have developed assay technologies to measure hydrolyzing enzymes based on homogeneous time-resolved fluorescence quenching (TruPoint). High sensitivity was obtained using fluorescent europium chelates as labels, internally quenched by suitable quenchers and released upon enzymatic reaction. This approach allows robust and sensitive monitoring of low enzyme activities. The assay technology and the choice of donor-acceptor pairs were evaluated in three different enzymatic assays, a protease related to apoptosis, helicase involved in DNA unwinding, and phosphatase having an important role in cellular signaling cascades. All the assays produced an increasing signal, were sensitive, and had a good dynamic range. There were significant differences in optimized quenchers for each of the assays depending on the size, flexibility, and rigidity of the substrates. Also, clear differences in the energy-transfer reactions, their requirements for spectral overlapping, ionic interactions, and energy-transfer distances were found. Each of the enzymatic assays was briefly tested in a high-throughput screening environment by analyzing signal dynamics and statistical relevance as Z' factors.

Caspase multiplexing: simultaneous homogeneous time-resolved quenching assay (TruPoint) for caspases 1, 3, and 6.

Caspases are a group of cysteine proteases involved in apoptosis and inflammation. A multiparametric homogeneous assay capable of measuring activity of three different caspases in a single well of a microtiter plate is described. Different fluorescent europium, samarium, terbium, and dysprosium chelates were coupled to a caspase substrate peptide, their luminescence properties, were analyzed, and their function in a time-resolved fluorescence quenching-based caspase 3 assay was studied. Substrates for caspases 1, 2, 3, 6, and 8 and granzyme B were also synthesized and their specificities for different caspases were determined. By selecting suitable lanthanide chelates and substrates we developed a multiparametric homogeneous time-resolved fluorescence quenching-based assay for caspases 1, 3, and 6. The assay was capable of measuring the activity of both single caspases and a mixture of three caspases mixed in the same well.