Professor Richard Walmsley
The response to Genome Damage in Human cells: genotoxicity assessment GFP reporter assays.
DNA damage, chromosome breakage and chromosome mis-segregation can result from direct damage to chromosomes or chemical interference with the proteins that ensure the proper replication and repair of DNA or the mitotic machinery. In human and other mammalian cells, genome damage is sensed by a variety of mechanisms, and the result is an appropriate response. This ranges from a delayed mitosis, and induction of repair systems, to apoptotic cell death. Proper regulation of a gene called GADD45a is essential to the control of these processes. My group has developed a standardised assay to monitor the induction of GADD45a, by including sequences encoding the Green Fluorescent Protein into a modified GADD45a gene. The new GADD45a-GFP reporter is maintained in a human cell line called TK6. Testing of many many chemicals has shown that induction of the reporter is a property of genotoxic chemical carcinogens.
The assay is carried out in 96 well microplates, and can be carried out quickly and reproducibly and as a consequence the GADD45a-GFP reporter is now widely used to assess the potential of chemicals to cause genome damage. It compliments the better known Ames reverse mutation assay, providing eukaryotic targets and processes not present in bacteria, and together the 2 tests identify the majority of rodent carcinogens.
Our most recent work addresses an important extra factor that has to be considered when trying to compounds that damage the genome, metabolism. Chemicals we eat, inhale or apply to our skin are often modified, partly as a consequence of our evolved defenses. The liver, which can be characterised as a chemical engineering facility for the body, is particularly active in the neutralisation of harmfil 'xenobiotics', but in some cases it can increase the toxicity of a substance. An example of this is the poison aflatoxinB1, produced by fungal spoilage, particularly in the tropics. This poison can kill. At low doses where you may not even feel ill, you are exposed to a metabolite of the toxin which is a carcinogen. Potentially useful new drugs are often metabolised, so regulatory safety tests have been adapted to try and predict such effects before going to animal tests or human therapeutic use. For our own test we are currently assessing the use of liver extracts, or even liver derived cell lines to produce the metabolites for assessment in our test.