A microplate version of the DNA-synthesis inhibition test for rapid detection of DNA-alteration potentials

A microplate version of the DNA-synthesis inhibition test (DIT) for fast detection of DNA-alteration potentials has been developed. The DIT is based on the concept that DNA damage causes inhibition of DNA synthesis that becomes detectable some time after replicating cells have been in contact with genotoxic agents. In this test procedure human tissue culture cells (HeLa S3), prelabeled with [14C]thymidine, arfe exposed for 90 min to the substances in question. After the cells are rinsed, they are allowed to recover for 2 1/2 h in fresh culture medium, thereby unspecific interactions interfering with DNA replication are practically eliminated. Next, [3H]thymidine is added for 30 min, and then the cells are harvested and thoroughly rinsed. Finally, incorporated radioactivity is determined by liquid scintillation counting for measurement of the 3H/14C ratio. This allows for the evaluation of DNA synthesis during the 3H-labeling period and of the extent of genotoxic damage. This microplate version of the DIT can be carried out fully automated in a laboratory workstation. The test is compared to other tests for genotoxicity. Its advantages are discussed.