Project 1 (PLUS)
Project 1: New cellular and cell-free models for assessing NP-induced oxidative stress
Fellow: Paul Schlinkert (ESR1), 36 months
Tutors: Prof. Albert Duschl (PLUS)
PLUS has developed a reporter gene cell based screening method to detect immunotoxic effects of environmental pollutants. A panel of cell lines was stably transfected with cytokine- promoter sequences linked to the luciferase gene. These reporter cells were used to follow immune activation or suppression for on-line exposure to diesel exhaust (FP5 MAAPHRI), to chemicals and mixtures thereof in the absence and presence of cellular stress (FP6 IP NOMIRACLE), and to analyse immunomodulatory effects of NP within the FP6 STREP DIPNA. Many reports suggest that oxidative stress associated with production of reactive oxygen species (ROS) is induced by NP. An ESR will perform a study to screen for particularly relevant gene regulation events related to cellular stress, and will go on to produce several new stable cell lines with relevant promoters, including hemoxygenase-1. The effect of silver NP known to be immunotoxic as well as of other NP will be tested using real time PCR, Western blot and ELISA or intracellular staining of protein prior to the final selection of cell stress parameters. Using GFP as a reporter protein instead of luciferase has already been established, and allows the investigation of single cell methods together with partner 3, as cells do not need to be lysed for analysis. However, since the sensitivity of the luciferase assay is higher, both reporter systems have to be considered. The system will be applied to NP mixed with chemicals likely to occur in working environments. In addition, cell-free tests developed by partner 7 will be used to analyse the ability of NP to trigger the formation of ROS in the absence of cells. The balance between cellular antioxidants and ROS (generated both on the surface of particles and by cells following exposure to NP) will determine the induction of cellular stress. The project will thus investigate both intrinsic and cell-mediated mechanisms leading to cellular stress responses.
Main skills acquired are molecular and cell-based assays, and their application to investigating nanomaterials. Secondments to partners 3 (reporter assays in chip-format), 4 (validation of tests in ongoing screening projects), 7 (cell-free DTT-assay for ROS), 11 (airborne NP delivery to cultured cells). In WP1 (tasks 1.1 and 1.5), WP2 (tasks 2.1, 2.4 and 2.5), WP3 (task 3.2) and WP4 (tasks 4.1, 4.2, 4.3 and 4.5).