Dose–response and operational thresholds/NOAELs for in vitro mutagenic effects from DNA-reactive mutagens, MMS and MNU

The dose–response relationships for in vitro mutagenicity induced by methylmethanesulfonate (MMS) or methylnitrosourea (MNU) in L5178Y mouse lymphoma (ML) cells were examined. DNA adducts (N7-methylguanine, N7MeG and O6-methylguanine, O6MeG) were quantified as biomarkers of exposure. Both endpoints were assessed using 5 replicates/dose (4-h treatment) with MMS or MNU (0.0069–50 μM), or vehicle (1% DMSO). Mutant frequency (MF) (thymidine kinase (TK) locus) was determined using the soft agar cloning methodology and a 2-day expression period; in addition, microwell and Sequester–Express–Select (SES) methods were used for MMS. Isolated DNA was acid-hydrolyzed, and adducts quantified by LC/ESI-MS/MS, using authentic and internal standards. MF dose–responses were analyzed using several statistical approaches, all of which confirmed that a threshold dose–response model provided the best fit. NOAELs for MF were 10 μM MMS and 0.69 μM MNU, based on ANOVA and Dunnett's test (p < 0.05). N7MeG adducts were present in all cell samples, including solvent-control cells, and were increased over control levels in cells treated with ≥10 μM MMS or 3.45 μM MNU. O6MeG levels were only quantifiable at ≥10 μM MNU; O6MeG was not quantifiable in control or MMS-treated cells at current detection limits. Thus, (1) cells treated with ≤0.69 μM MNU or ≤10 μM MMS did not demonstrate increases in TK− MF, but did demonstrate quantifiable levels of N7MeG adducts; and (2) the levels of N7MeG adducts did not correlate with induced MF, as MNU-treated cells had fewer N7MeG adducts but higher MF compared with MMS-treated cells, for quasi-equimolar doses. Taken together, these results demonstrate operational thresholds, defined as the highest dose for which the response is not significantly (statistically or biologically) distinguishable from the control/background values, for induction of mutations and N7MeG adducts in ML cells treated with MMS or MNU, and a lack of correlation between induced MF and levels of N7MeG adducts.