Cytotoxicity of monofunctional alkylating agents methyl methanesulfonate and methyl- N′-nitro- N-nitrosoguanidine have different mechanisms of toxicity for [formula omitted] cells

Abstract

N-methyl- N′-nitro- N-nitrosoguanidine (MNNG) and methyl methanesulfonate (MMS) are directly active alkylating agents that methylate cellular macromolecules by SN 1 and SN 2 mechanisms, respectively. These two chemicals produce similar types of alkylation products in DNA and a similar level of total alkylations on a molar basis, but strikingly different proportions of alkylations of ring oxygen atoms of purines and pyrimidines. Because of this attribute, they have been used in combination to attempt to determine which types of alkylation products are responsible for mutation, transformation, and toxicity. Studies have suggested that the mutation rates produced by these and similar chemicals in cells surviving toxicity correlate well with the number of methyl adducts at the O 6 position of guanine, but that cytotoxicity (reduced colony-forming efficiency) does not correlate with any single adduct or with the total level of alkylation of DNA. In this study we have investigated the cytotoxic mechanisms of MNNG and MMS in synchronized 10 T 1 2 cells, using colony-forming ability as a measure of toxicity. Both MNNG and MMS cause dose-dependent reduction in the ability of 10 T 1 2 cells to produce colonies of more than 50 cells after 2 weeks in culture. MNNG is about 100-fold more toxic than MMS on a molar basis. As indicated by the inability of cells to exclude trypan blue, MMS kills a fraction of the population of treated 10 T 1 2 cells after a 30-min exposure; the fraction of cells that excludes trypan blue is correlated with dose of MMS and with colony-forming efficiency. Neither the fraction of cells that is permeable to trypan blue nor the relative colony-forming efficiency is affected by the phase of the cycle when 10 T 1 2 cells are treated with MMS. Furthermore, MMS toxicity for 10 T 1 2 cells is not potentiated by caffeine, MMS treatment does not delay progress of S phase, and cells that survive acute membrane toxicity complete the cell cycle without significant delay. In contrast, MNNG treatment produces toxicity that is maximal when 10 T 1 2 cells are exposed during the S phase and the effect of potentiated by caffeine. MNNG treatment delays DNA replication and this delay is reversed by caffeine. In sharp contrast to 10 T 1 2 cells treated with MMS. MNNG-treated cells are not made permeable to trypan blue, but are blocked in their ability to proliferate. These observations indicate that MNNG and MMS kill 10 T 1 2 cells by drastically different mechanisms, MNNG producing toxicity mainly by preventing chromosome replication and MMS producing toxicity mainly by damaging cell membranes.