Objectives

To delineate the mechanisms though which ambient and vehicle PM cause cellular injury and inflammation. We will investigate the hypothesis that free radical activity is a pivotal component of PM. That exposure to PM leads to antioxidant depletion and generation of oxidised proteins and lipids in ELF. That these oxidation products enter pulmonary target cells and activate NFkB, resulting in a cytokine-mediated inflammatory response in the lung.

Methodology and study materials Particles shown to have strong oxidant capacity under WP3 will be examined in a range of assays designed to identify the nature and strength of free radical activity (A,B), the signalling mechanism(s) between particles and pulmonary target cells ©, the signalling pathway within cells target pulmonary cells (D), and the mediators of the inflammatory response (E). Each of these assays are outlined below. Antioxidant depletion capacity of PM will be examined as described previously: The oxidant potential of PM will be assessed as its ability to deplete plasmid DNA and the possible involvement of metals in the adverse effects of PM will be assessed using specific metal chelators such as desferroxamine. Secondary oxidation products, such as proteins and lipids, will be identified following co-incubation of PM with human ELF. ©NFk B activation will be assessed in epithelial cells using an immunoflorescent antibody to the p65 component of NFk B by the electrophoretic gel mobility shift assay and by a luciferase reporter system for NFk B transfected into epithelial cells. The murine monocytic/macrophagic cell line RAW 264.7 will be exposed to different concentrations of PM and various mediators of inflammation determined including: arachidonic acid, NO and the cytokines IL-6 and IL-8.

Deliverables This work package will deliver a report containing a comprehensive assessment of the nature of the free radical activity of PM from a range of different sites in Europe. It will also provide a basis for which to assess the most effective way to intervene in the toxic pathway of PM.

Milestones and expected results The first milestone will involve a comprehensive overview of the oxidant capacity of PM obtained from the sampling network. This will be reached after 24 months into the project. The second milestone will cover the extra- and intracellular signalling mechanisms and the characterisation of the mediator response to PM. This will be reached 36 months into the project.