Establishment of a dose-response relationship for reverse mutation at the HPRT (hypoxanthine guanine phosphoribosyl transferase) locus L5178Y mouse lymphoma cells.

L5178Y mouse lymphoma cells were treated with the mismatching agent 6-hydroxy-aminopurine (HAP), a base analogue known to produce forward and reverse mutations in bacteria. Mutants with the phenotype deficient in hypoxanthine guanine phosphoribosyl transferase (HPRT) were selected in 6-thoiguanine(TG)-containing medium and isolated. Reverse mutations to the HPRT-proficient phenotype occurred both spontaneously and after treatment with ethyl nitrosourea (ENU), which suggested that the initial HAP treatment had induced point mutations at the HPRT locus. Reconstruction experiments, in which a small number of wild-type cells together with different numbers of mutant cells were seeded in HAT medium, indicated that densities up to 10(6) cells per ml can be used for the selection of revertants. Optimal expression of induced revertants was obtained two days after treatment. The dose-response relationship for induction of reverse mutations by ENU appears not to deviate from linearity. The highest revertant frequency observed was 3.3 x 10(-5) at an ENU concentration of 1 mM. The spontaneous reversion frequency per generation -- based on 3 spontaneous revertants -- was estimated to be 1.3 x 10(-9). All revertants were indistinguishable from the parental wild-type line with respect to the activity as well as the electrophoretic mobility of HPRT.