SHORT TERM MUTAGEN TESTING WITH HUMAN AND MURINE CELLS

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Short-term mutagen test systems will be established using mutations at the APRT locus in mouse TK+/-L5178Y cells and in human cell line HL-60. The L5178Y system is extensively used in short-term mutagen testing, but some doubt has been expressed as to the reliability of the thymidine kinase gene as a test marker. This strain has now been made heterozygotic for APRT and APRT- mutants derived from it. Heterozygotes can be distinguished for homozygotes on the basis of colony size in soft agar. Preliminary analysis indicates that this system can be used to differentiate a deletion mutant from other types of mutant events. Since the protein is easy to purify and the gene cloned, this system will be used for molecular analysis. This will be carried out using DNA hybridization studies with the appropriate probes to characterize possible alterations in DNA sequences. A series of well-characterized mutants will then be used in reversion studies. A comparison will be made between the rate of mutation to TK- and APRT- from the double heterozygotes. Human cell lines have been screened for high cloning efficiency. Hl-60, a human myelocytic leukemic cell line, clones efficiently in soft agar. Over 100 APRT- mutants have been isolated in HL-60 following mutagenesis. Selected mutants will be characterized at the level of APRT protein and DNA. Suitable mutants will be tested for reverse mutation in order to establish a mutagen test system. A molecular analysis of the mutants will be done to characterize the genetic lesion. The above two test systems will be compared with the Ames Salmonella system using a series of pesticides of known mutagenic spectrum.