The influence of serum components on the growth and mutation of Chinese hamster cells in medium containing 8-azaguanine

Abstract

Low concentrations (less than or equal 20 mug/ml) of 8-azaguanine are 1000 fold more toxic to V79 Chinese hamster cells in medium containing 10% dialyzed fetal calf serum than in medium containing 10% undialyzed serum. Serum enzyme activity that converts AG to nontoxic 8-azaxanthine degrades AG at the same rate, whether or not the serum is dialyzed. However, cytotoxicity results similar to those obtained with US were produced in medium containing DS and 2.5 mug of hypoxanthine (HX)/ml (DSH). Therefore, serum HX is considered to be responsible for the relatively low cytotoxicity of AG in medium containing US. Colonies that arose in medium containing AG were isolated and characterized. Those that remained resistant to AG (40 mug/ml) and sensitive to aminopterin in the presence of HX and thymidine (HAT) were considered mutants; non-mutants were sensitive to AG and resistant to HAT. Colonies isolated from medium containing DSH or US and low concentrations of AG were not mutants, but those from medium containing high concentrations (greater than or equal 30 mug/ml) of AG were mutants. Spontaneous and N-methyl-N-nitro-N-nitrosoguanidine induced mutants were detectable in medium containing DSH without replating the cells prior to adding AG (greater than or equal 30 mug/ml), but in order to detect MNNG induced mutations in medium containing DS replating was essential. In DS, the mutation frequency increased as an exponential function of the toxicity of MNNG, but remained two orders of magnitude lower than the induced mutation frequencies that occurred in DSH. HX, in DSH or US, produced profound effects, other than interference with AG toxicity, that distort the results of mutagenesis assays. To study mutation using AG resistance as the endpoint, it is essential to use dialyzed serum.