A battery of reliable and validated in vitro assays is needed to test for genotoxic and cytotoxic effects of chemicals (as in the REACH programme) without resorting to animal experiments. The comet assay, a sensitive indicator of DNA damage, will be combined in this project with the Cell Array system, to establish and validate high capacity assays suitable for chemical testing. Up to 800 cell samples will be processed for comets on a single microscope slide. Arrays will use cells with different metabolic capabilities, and data on cytotoxicity will be obtained in parallel with DNA damage. A medium-throughput assay will also be developed. Comet analysis by differential staining of damaged/undamaged DNA using established and novel dyes combined with automated image analysis will be faster and more reliable than at present.
A crucial aspect of the cellular response to DNA damage is DNA repair; variations between people can affect cancer risk. Genotoxic chemicals can act by interfering with repair.
Two methods for measuring repair, one based on the comet assay, the other using a 'Repair Chip' approach, will be compared. In addition, fluorescent probes which lock onto the DNA after hybridization ('padlock probes') will be applied to study gene-specific DNA repair. The modified comet assay methods will be assessed for reproducibility and sensitivity in an inter-laboratory prevalidation trial, using a coded set of standard chemicals, to satisfy regulatory bodies as well as industrial users of the technology. The result will be robust, validated, high-throughput in vitro genotoxicity tests. This proposal brings together academic partners and SMEs, together with a large industrial concern, and the European Centre for the Validation of Alternative Methods (ECVAM). Dissemination through professional and trade publications, regulatory channels, and scientific conferences will lead to widespread adoption of the new methods.
Fields of science
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