A study of ENU-induced mutagenesis in the mouse using the restriction sitemutation (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. Specifica lly, 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 previousl y published from our laboratory on ENU-induced testes mutations. It was fou nd 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 t he spleen and were detectable to a lesser extent at 100 days, which is simi lar to the testes data. In the bone marrow, the mutation level rose until d ay 100, although the level remained below that of the spleen and testes. Ho wever, by studying the mutations detected in control animals, it was found that spontaneous mutational events were detectable at the day 100 time poin t 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 the se tissues after 100 days. This paper also demonstrates the applicability o f the inverse RSM methodology in the detection of ENU-induced mutations, wh ereby mutations can be detected by the conversion of one restriction site t o another. The iRSM assay appears to be particularly suitable to studying a lkylating agents due to their known sequence specific mutation induction. W e 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 o f ENU to the mouse bone marrow in vivo. (C) 1999 Wiley-Liss, Inc.