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Mutants of a diploid human lymphoblast line resistant to 6-thioguanine (6TG) appear 6–16 generations after treatment with any of a diverse group of mutagens: methylnitrosourea (MNU), methylnitrosoguanidine (MNNG), ICR-191 5-bromodeoxyuridine (BUdR). A hypothesis is advanced that expression of the 6-thioguanine-resistant state may require the removal of essentially all pre-existing hypoxanthine—guanine phosphoribosyl transferase (HGPRT) molecules via division, dilution, and protein turnover. Design of protocols for quantitative mutation assays requires attention to this phenomenon.

The lag in phenotype expression of methylnitrosourea(MNU)-induced mutation to 6-thioguanine (6TG) resistance has been studied in a diploid human lymphoblastoid cell line. We find that a considerable period (8-12 days) elapses before new mutants appear in treated cultures; after 2 weeks, however, a stable maximum fraction is attained, as would be expected for a genetic mutation. We present preliminary data linking this phenotypic lag to the slow degradation rate of hypoxanthine-guanine phosphoribosyl transferase (HGPRT) and to an apparent requirement for very low (less than 0.2% normal) cellular HGPRT content in order for cells to be resistant to 10 mug 6TG/ml. A series of reconstruction experiments are presented, the results of which support the conclusion that selective pressures in the assay procedure do not bias the quantitative estimates of induced mutant fraction.

The toxic and mutagenic effects of the alkylating agents N-methyl-N-nitrosourethane (MNUT) and ..beta..-propioactone (BPL) were quantitatively measured in human lymphoblasts and Salmonella typhimurium. Forward mutation to 6-thioguanine resistance was measured in the human lymphoblasts, and forward mutation to 8-azaguanine resistance was measured in the bacterial cells after equigenerational (1.5 doubling times) exposures. In both systems, the induced mutant fraction rose linearly as a function of concentration for BPL and was biphasic for MNUT. The responses of the two assay systems to eight alkylating agents were compared. The exposure of the cells to each alkylating agent was calculated as exposure concentration multiplied by the time of exposure, and allowance was made for the decomposition of the alkylating agents during exposure (integral exposure). Human cells were 2.5 to 13 times more sensitive than was S. typhimurium to the alkylating agents methyl methanesulfonate, ethyl methanesulfonate, propyl methanesulfonate, N-methyl-N'-nitro-N-nitrosoguanidine, methylnitrosourea, and MNUT. S. typhimurium cells were three times more sensitive to butyl methanesulfonate and 25 times more sensitive to BPL than were human cells.

Our primary reasons for using diploid human lymphoblast lines in studies of mutagenesis are that they can be grown in free suspension; they appear to be genetically stable for chromosome number, as well as immortal; and they are readily derived from humans of different genetic backgrounds and possibly different sensitivities to mutagenic agents. Of these properties, the most important in assessing the mutagenicity of a large number of suspect chemicals is the lymphoblasts' ability to grow in suspension culture. This characteristic offers the potential for preprogrammed, automatic handling, which probably could not be achieved with anchorage-dependent cells. In this chapter, we will introduce the basics of lymphoblast husbandry and some protocols (tricks) that we have used to facilitate their use in studying genetic change.