Influence of experimental conditions and DNA repair ability on EMS-induced mutagenesis and DNA binding in Escherichia coli K12 : Comparison with mammalian cell mutagenesis

Abstract

In continuation of previous experiments aimed at quantitatively comparing the magnitude of ethyl methanesulfonate (EMS)-induced DNA binding and mutagenesis in bacterial and mammalian cells, the present studies were undertaken to determine the influence of various experimental conditions on EMS-induced changes in E. coli K12/343/113. The sensitivity to EMS mutagenesis in this strain was assayed for various genetic endpoints, namely gal +, MTR r, NAL r, VAL r and arg +. EMS mutagenesis and DNA binding in the repair-proficient wild-type was, furthermore, compared to that in derivatives deficient in excision repair (Δ uvrB101) and post-replication repair ( recA13). Attempts were, finally, undertaken to determine the shape of the dose-response curve for the most sensitive genetic systems found, namely NAL r and VAL r mutations, at relatively low EMS-exposure conditions of 0–7 mM for 120 min at 37°C. From the present experiments and from previous results obtained under identical EMS treatment conditions in E. coli 343/113 and in the mammalian cell lines V79 and L5178Y, the following conclusions can be drawn: (i) At EMS exposure concentrations ranging from 2.5 to 50 mM and treatment periods of 120 min at 37°C in buffer pH 7.2, the levels of DNA alkylation at termination of the treatment are similar in E. coli and mammalian cells and increase from 1.7 × 10 −4 to 1.3 × 10 −3 ethylations per nucleotide. (ii) The presence of nutrients such as l-arginine, D-glucose and fetal calf serum, during EMS treatment of stationary E. coli cells does not influence the level of DNA alkylation but greatly increases EMS-induced mutation yields, probably because of the accumulation of these nutrients into the thereby leading to increased capability of genetic fixation and/or phenotypic expression of the premutational DNA changes. (iii) The mammalian cells are more sensitive to the killing actions of EMS than E. coli repair-proficient cells (survival at 10 mM of 12 and 100%, respectively), which indicates that the genetic and/or physiologic target for inactivation of colony-forming ability is larger in the mammalian cells than in bacteria, or alternatively that these cells have lower ability to repair EMS-induced DNA lesions. (iv) The 2 new mutation systems tested in the present series of experiments, namely NAL r and VAL r mutations appear to be quite comparable to those tested in mammalian cells, such as thioguanine resistance (HGPRT −) and BUdR resistance (TK −/−), in the sense that at low doses, i.e. EMS-exposure concentrations of up to 7 mM for 120 min at 37°C in buffer, pH 7.2, the induction kinetics in all systems are compatible with a linear relationship between dose and mutation frequency, with no indication of a threshold. In the case of NAL r in E. coli and HGPRT − in V79 mammalian cells the Lowest Effective Concentrations of EMS are 1–2 mM for treatment periods of 120 min at 37°C. It remains to be determined if the same EMS-induced DNA adducts are responsible in both cell types for the genetic effects observed; further comparative experiments are also required to assess the general suitability of bacteria in quantifying mutagenic and tumor-initiating potency of chemicals.