Culture of Calu-3 cells at the air interface provides a representative model of the airway epithelial barrier.

PURPOSE: The aim of this study was to compare the effect of liquid-covered culture (LCC) and air-interfaced culture (AIC) on Calu-3 cell layer morphology and permeability, thus assessing the fitness of these culture systems as models of airway epithelium barrier function. METHODS: Cell layers were grown on 0.33 cm2 Transwell polyester cell culture supports. Cell layers grown using LCC and AIC were evaluated by using light and electron microscopy, transepithelial electrical resistance (TER), and permeability to the transepithelial flux of fluorescein sodium (flu-Na), and by varying molecular weight dextrans labeled with fluorescein isothiocyanate (FITC-dex). The tight junction protein, zona occludens protein-1 (ZO-1), was visualized by confocal microscopy and apical glycoprotein secretions were identified by using alcian blue. RESULTS: Cells grown via AIC produced a more columnar epithelium with a more rugged apical topography and greater glycoprotein secretion compared to cells grown via LCC. Apical protrusions appearing to be cilia-like structures were observed on occasional cells using AIC, but typical airway ciliated cell phenotypes were not produced under either condition. Secretory granules were observed in cells cultured under both conditions. Cells cultured using LCC exhibited higher levels of ZO-1 protein than the AIC counterpart. The maximal TER of cells using LCC, 1,086 +/- 113 ohms cm2 at 11-16 days, was significantly greater than the TER of cells cultured using AIC, 306 +/- 53 ohms cm2 at 11-13 days. Apparent permeability (P(app)) values for the transport of flu-Na using LCC and AIC were 1.48 +/- 0.19x10(-7) and 3.36 +/- 0.47x10(-7) cm s(-1), respectively. Transport rates of flu-Na and FITC-dex were inversely proportional to molecular weight, and were significantly lower (p < 0.05) in cell layers grown using LCC than AIC. Renkin analysis fitted the data to single pore populations of radii 7.7 and 11.0 nm for LCC and AIC, respectively. CONCLUSION: Distinct differences in morphology and permeability result when Calu-3 cells are grown using AIC or LCC. Cells cultured using AIC generate a model more morphologically representative of the airway epithelium than cells cultured using LCC.

Variations in the source, metal content and bioreactivity of technogenic aerosols: a case study from Port Talbot, Wales, UK.

Atmospheric aerosol samples were collected during different prevailing wind directions from a site located close to a busy motorway, a major steelworks, and the town of Port Talbot (Wales, UK). A high-volume collector was used (1100 l/min), enabling relatively large amounts of particulate matter (PM(10-2.5) and PM(2.5)) samples to be obtained on a polyurethane foam [PUF, H(2)N-C(O)O-CH(2)CH(3)] substrate over periods of 2-7 days. Four samples were chosen to exemplify different particle mixtures: SE- and NE-derived samples for particles moving along and across the motorway, a NW-derived sample from the town, and a mixed SW/SE-derived sample containing a mixture of particles from both steelworks and motorway. The latter sample showed the highest average collection rate (0.9 mg/h, 13 microg/m(3)) and included a prominent pollution episode when rainy winds were blowing from the direction of the steelworks. Both NW and SE samples were collected under dry conditions and show the same collection rate (0.7 mg/h, 10 microg/m(3)), whereas the NE sample was collected during wetter weather and shows the lowest rate (0.3 mg/h, 5 microg/m(3)). Scanning electron microscopy (SEM) and energy-dispersive X-ray microanalysis system (EDX) analyses show all samples are dominated by elemental and organic carbon compounds (EOCC) and nitrates, with lesser amounts of sulphates, felsic silicates, chlorides and metals. ICP-MS analyses show the SW/SE sample to be richest in metals, especially Fe, Zn, Ni, and Mn, these being attributed to an origin from the steelworks. The SE sample, blown along the motorway corridor, shows enhanced levels of Pb, V, Ti, As, and Ce, these metals being interpreted as defining a traffic-related chemical fingerprint. The NW sample shows a very low metal content. DNA plasmid assay data on the samples show TM(50) values varying from 66 to 175 microg/ml for the adjusted whole sample and 89 to 203 microg/ml for the soluble fraction. The SW/SE-mixed metalliferous sample is the most bioreactive (both whole and soluble) and the soluble fraction of the metal-depleted NW sample is the least bioreactive. The metal content of the aerosol samples, especially soluble metals such as Zn, is suggested to be the primary component responsible for oxidative damage of the DNA, and therefore most implicated in any health effects arising from the inhalation of these particulate cocktails.

The collection of PM10 for toxicological investigation: comparisons between different collecting devices.

A positive correlation has been established between increased levels of airborne particulate pollution and adverse health effects, the toxicological mechanisms of which are poorly understood. For toxicologists to unambiguously determine these mechanisms, truly representative samples of ambient PM10 are required. This presents problems, as PM10 collecting equipment commonly employed, such as the Tapered Element Oscillating Microbalance (TEOM), heat the inflow to exclude moisture or use fibrous filters, resulting in a PM10 sample that may have undergone significant chemical change on the filter surface or is contaminated by filter fibres. Other systems (i.e. Negretti and Partisol) can successfully collect PM10 without chemical alteration or filter contamination. Comparative collections from Port Talbot, S. Wales suggest that TEOMs and Negretti/Partisol systems collect different PM10's; the principle difference arising from the TEOM's heating chamber, which precipitates water-soluble ions and volatilises some organic components. This results in both the mass and composition of the PM10's being altered. Particle size distributions for Negretti and Partisol collections highlighted differences mainly attributed to different flow rates. The results of this work demonstrate that simple correlations between PM10 mass and adverse health effects are problematic. Furthermore, elucidation of the complex fractionation and chemical changes in different collectors is necessary.

Particle-induced oxidative damage is ameliorated by pulmonary antioxidants.

This investigation focuses on the application of an in vitro assay in elucidating the role of lung lining fluid antioxidants in the protection against inhaled particles, and to investigate the source of bioreactivity in urban PM10 collections from South Wales. The Plasmid Assay is an in vitro method of assessing and comparing the oxidative bioreactivity of inhalable particles. This method has provided the basis of limited toxicological studies into various inhaled xenobiotics including asbestos, and more recently PM10. Carbon Black M120 and Diesel Exhaust Particles (DEP) were tested as PM10 surrogates, DEP displaying the greatest oxidative bioreactivity. Both urban PM2.5 (fine fraction) and PM2.5-10 (coarse fraction) (Cardiff, S. Wales, UK) caused significant damage, the coarse fraction displaying higher oxidative capacity. The soluble components were found to be responsible for most of the bioreactivity in both PM sizes. Low molecular components of fresh lung lavage were found to offer most antioxidant protection, and surrogate Epithelial Lining Fluid (sELF) showed significant amelioration of DNA damage by the coarse fraction but less effect against the fine. Overall, the coarse, soluble fraction of PM10 is a great source of oxidative bioreactivity, but natural pulmonary low molecular weight antioxidants can significantly ameliorate its effects.