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

Abstract 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 O 6 -methylguanine, O 6 MeG) 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 6 MeG levels were only quantifiable at ≥10 μM MNU; O 6 MeG 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.