A study of ENU-induced mutagenesis in the mouse using the restriction site mutation (RSM) assay

We report here the application of the restriction site mutation (RSM) assay to study the induction of mutations by the alkylating agent ENU. Specifically, mutations were sought in the spleen and bone marrow of mice 3, 10, and 100 days after being treated with ENU; this was compared to data previously published from our laboratory on ENU-induced testes mutations. It was found that the ENU-induced mutations were all at GC bases implicating the O(6)-ethylguanosine adduct. The mutations detected reached a peak at day 10 in the spleen and were detectable to a lesser extent at 100 days, which is similar to the testes data. In the bone marrow, the mutation level rose until day 100, although the level remained below that of the spleen and testes. However, by studying the mutations detected in control animals, it was found that spontaneous mutational events were detectable at the day 100 time point in all three tissues. Hence the spleen, testes, and bone marrow mutations at day 100 in the ENU-treated samples were probably spontaneous mutational events with very few genuine ENU-induced mutations remaining in any of these tissues after 100 days. This paper also demonstrates the applicability of the inverse RSM methodology in the detection of ENU-induced mutations, whereby mutations can be detected by the conversion of one restriction site to another. The iRSM assay appears to be particularly suitable to studying alkylating agents due to their known sequence specific mutation induction. We also show a comparison of the bone marrow micronucleus data with the RSM assay and show that both assays are capable of detecting the genotoxicity of ENU to the mouse bone marrow in vivo.