title: 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 5replicates/dose (4-h treatment) with MMS or MNU (0.0069-50muM), 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 10muM MMS and 0.69muM 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 >/=10muM MMS or 3.45muM MNU. O(6)MeG levels were only quantifiable at >/=10muM MNU; O(6)MeG was not quantifiable in control or MMS-treated cells at current detection limits. Thus, (1) cells treated with </=0.69muM MNU or </=10muM 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.
authors.affiliation: Toxicology and Environmental Research & Consulting, The Dow Chemical Company, Midland, MI 48674, USA. lpottenger@dow.com
authors.forename: Lynn H
authors.initials: LH
authors.lastname: Pottenger
authors.forename: Melissa R
authors.initials: MR
authors.lastname: Schisler
authors.forename: Fagen
authors.initials: F
authors.lastname: Zhang
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Bartels
authors.forename: Donald D
authors.initials: DD
authors.lastname: Fontaine
authors.forename: Lisa G
authors.initials: LG
authors.lastname: McFadden
authors.forename: B
authors.initials: B
authors.lastname: Bhaskar Gollapudi
mesh_terms: D000818:Animals; D018736:DNA Adducts; D004305:Dose-Response Relationship, Drug; D007940:Leukemia L5178; D008741:Methyl Methanesulfonate / Q000633:toxicity*; D008770:Methylnitrosourea / Q000633:toxicity*; D051379:Mice; D009152:Mutagenicity Tests; D009153:Mutagens / Q000633:toxicity*; D019055:No-Observed-Adverse-Effect Level; D020711:Threshold Limit Values
chemical_list: D018736:DNA Adducts; D009153:Mutagens; D008770:Methylnitrosourea; D008741:Methyl Methanesulfonate