Effect of pH shifts on the mutant frequency at the thymidine kinase locus in mouse lymphoma L5178Y TK+/- cells.
Evidence has been accumulating that conditions of nonphysiological pH may affect the results of in vitro genetic tests by mechanisms unrelated to the chemical being tested. Medium was pH-adjusted with HCl, NaOH or with organic buffers (Good's zwitterions). In the absence of S9 mix, no changes in mutant frequency were observed over a pH range of 6.4-9.2; a small, 1.9-fold increase was observed for a moderately toxic treatment (24% relative growth) at pH 6.3. However, in the presence of S9 mix, the mutant frequency increased sharply for pH values below 6.8. At pH 6.4, a 4-fold increase was induced, and pH 6.0 resulted in a 10-fold increase in mutant frequency. Basic pH shifts in the presence of S9 mix caused no changes in mutant frequency up to pH 8.0; treatment with pH 8.8 was highly toxic (5.3% relative growth) and caused a 3-fold increase in mutant frequency. Thirteen mutant clones induced at pH 6.0 with S9 mix were challenged with trifluorothymidine after their expansion in nonselective medium and all retained their resistance; another 14 clones were tested for thymidine utilization and all incorporated only 0.1-5.5% of the 14C-labeled thymidine used by the parental line. The induced mutants were primarily of the small-colony phenotype, which indicated clastogenic activity. This was confirmed with chromosome studies which showed a large increase in cells with aberrations consisting of chromatid breaks and complex rearrangements. The results show that the combination of weak acidity (pH 6-6.8) and S9 mix is mutagenic and clastogenic to L5178Y TK+/- cells.
D000818:Animals; D002021:Buffers; D002460:Cell Line; D002470:Cell Survival; D002869:Chromosome Aberrations; D004351:Drug Resistance; D006863:Hydrogen-Ion Concentration; D007940:Leukemia L5178 / Q000235:genetics*; D007942:Leukemia, Experimental / Q000235:genetics*; D051379:Mice; D008862:Microsomes, Liver / Q000378:metabolism; D009154:Mutation; D013937:Thymidine Kinase / Q000235:genetics*; D014271:Trifluridine
D002021:Buffers; D013937:Thymidine Kinase; D014271:Trifluridine