Objectives

Screening of samples arising from WP1. Identifying the range of oxidant and inflammatory activity of particles obtained from across the network. Those with greatest (and least) oxidant and inflammatory activity will be further examined in animal studies. >From these screening activities samples will be identified for eventual use in mechanistic work (WP3) and for human instillation studies (WP5).

Methodology and study materials A) Particle samples of defined number, surface chemistry and area will be examined in an in vitro model of ELF. Particles (105/well) will be incubated with ELF for up to 2 hours. Samples will be drawn off at 30 min intervals for determination of ascorbic acid, uric acid and glutathione (GSH/GSSG) concentrations. Oxidant capacity will be related to extent of antioxidant depletion. (B) Toxic effects on epithelial primary cell cultures and epithelial cell line BEAS-2B. © The murine monocytic/macrophagic cell line RAW 264.7 will be used as an in-vitro model to evaluate the macrophagic inflammatory response after exposure to PM10. Using similar particle samples as described in (1) the release of IL-6 , IL-8 , AA and NO will be measured in the cell culture medium.

Deliverables - Approaches 1& 2 will be used to screen particles collected in WP1. These sensitive assays of oxidant and inflammatory activity will help identify those particles which should be taken forward for more intensive investigation such as animal exposure studies described in WP4. -Combined these screening procedures will identify particles, in an efficient manner, which are appropriate for mechanistic studies in-vitro in WP3 and in mechanistic and relevance confirming studies in humans in WP5.

Milestones and expected results The first milestone is to have particles screened in-vitro. The second to determine samples with contrasts in biological activity that will go on to animal studies in WP4. It is expected that PM from different locations in Europe with different traffic situations will vary in bioactivity, but also PM at different time from same location. Additional comparison will be made with pure diesel and gasoline exhaust particulates as references. Delivery of obtained information comes gradually over 7-18 months as fresh PM are investigated which follows the sampling period of ambient PM, rather than being done in one single batch. It is expected that considerable contrasts will be found and that these will relate to the physico-chemical characteristics determined in WP1