Effects of ambient air particles on nitric oxide production in macrophage cell lines

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We assessed the in vitro toxicity of various particles on three murine macrophage cell lines, (J774A.1, WR19M.1, RAW264.7). The cells were exposed to aqueous suspensions (0–100 μg/30 mm2 well) of urban particulate matter (SRM-1648, SRM-1649, EHC-93), fine particulate matter (PM2.5), titanium dioxide (SRM-154b), and respirable cristobalite (SRM-1879) for 2 h and were then stimulated with lipopolysaccharide (LPS, 100 ng/ml) and recombinant interferon-gamma (IFN, 100 U/ml). After overnight incubation with the particles and LPS/IFN, nitric oxide production was estimated from culture supernatant nitrite. Cell viability was determined by monitoring the rate of AlamarBlue™ reduction. The dose-effect relationships for nitrite and viability were modeled as a power function (Fold change = [Dose + 1]β), where β represents the slope of the dose-response curve. Potency was defined as the rate of change in nitrite production corrected for cell viability (βPOTENCY = βNITRITE – βVIABILITY). Overall, the urban particles decreased nitric oxide production (βPOTENCY <0), while exposure of the cells to fine particulate matter or cristobalite increased the production of nitric oxide (βPOTENCY >0). Titanium dioxide (TiO2) was essentially inactive (βPOTENCY≈0). The decrease in nitric oxide production seen in cells exposed to the urban particles was directly correlated to a decrease in the expression of inducible nitric oxide (iNOS) as determined by Western blot analysis. The results indicate that particles are modulators of nitric oxide production in murine macrophages and may directly disrupt expression of iNOS during concomitant pathogen exposure. Pathways leading to enhanced NO production causing cell injury, and to decreased NO release resulting in lower bacterial clearance, may both be relevant to the health effects of ambient particles.

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