Dose-repetition increases the mutagenic effectiveness of N-ethyl-N-nitrosourea in mouse spermatogonia.

Abstract

In order to maximize the mutagenic effectiveness of N-ethyl-N-nitrosourea in mouse stem-cell spermatogonia, advantage was taken of the fact that these cells can accumulate mutations from repeated doses given over relatively long time periods. Repeated doses (100 mg/kg) of ethylnitrosourea injected intraperitoneally into male mice at weekly intervals were found to allow adequate survival and fertility with total dosages of 300 and 400 mg/kg. The specificlocus mutation frequencies at these dosages were, respectively, 1.8 and 2.2 times that obtained with the maximal practicable single dose of 250 mg/kg. The mutation frequency induced by a 400 mg/kg dosage of ethylnitrosourea is 12 times the maximal mutation frequency achievable with a single exposure to x-rays and 36 times that reported for procarbazine, the most effective chemical mutagen previously known for mouse stem-cell spermatogonia. Ethylnitrosourea is already the mutagen of choice in deliberate attempts to create mouse models for human disease and in any experiments in which a maximal mutation rate is desired. Repeated-dose regimens similar to the ones reported here should increase the efficiency of such studies.One advantage of determining induced mutation rates in stem-cell spermatogonia is that these cells can accumulate mutational lesions throughout the reproductive life span of the animal; therefore, repeated doses of mutagens can be given over relatively long time periods to amass a total dosage higher than that tolerated in a single exposure. Ethylnitrosourea (EtNU) has been shown to be the most effective mutagen for induction of specific-locus mutations in mouse spermatogonia (1,2). This has led rapidly to its being the mutagen of choice by many investigators (3,4). Therefore, it seemed useful to test whether repeated doses could be given to yield higher specific-locus mutation rates than those obtained by single exposures. This paper presents data from specific-locus tests with repeated doses of EtNU and from survival and fertility tests that were conducted prior to the specific-locus tests in order to choose feasible dosage regimens. A brief abstract of the preliminary results on survival and fertility has appeared (5). MATERIALS AND METHODSSurvival and Fertility Tests. EtNU, synthesized in our laboratory by D. G. Doherty, was dissolved in phosphate buffer (6) adjusted to pH 6. The dose injected intraperitoneally was matched to the body weight of the animals by adjusting the volume of solution injected, which never exceeded 1 ml. Male (101 x C3H)F1 mice were injected with dosages of 300 or 400 mg of EtNU/kg, given in 100 mg/kg doses. In four separate experiments, the individual doses were spaced 1 day, 2 days, 4 days, or 7 days apart, respectively. Six mice, 12 weeks old at the first injection, were in each dosage group in each experiment. All injections were completed within 1 hr after the EtNU was dissolved. Seven weeks after the final injection, these males were mated to females of our standard specific-locus test strain, T, which is h...

Random mutagenesis of proximal mouse chromosome 5 uncovers predominantly embryonic lethal mutations

“…An effective way to induce germ-line mutations in mice is with the chemical N-ethyl-N-nitrosourea (ENU) (Shedlovsky et al 1986(Shedlovsky et al , 1988. ENU induces mutations in the male germ line at a high frequency, enabling efficient screening of mutagenized pedigrees for aberrant phenotypes (Hitotsumachi et al 1985). As a point mutagen, it produces not only null alleles, but also hypomorphs and gain-of-function mutations.…”

Uncovering the uncharacterized and unexpected: Unbiased phenotype‐driven screens in the mouse

“…The vast majority of these mutations lie in non-coding DNA and do not affect gene function. When seven loci with visible phenotypes were examined, ENU generated one gene-inactivating mutation per locus in 700 F1 progeny (the specific locus test; Hitotsumachi et al, 1985). More recent large-scale screens have not uncovered multiple allele of single genes as the 1/700 rate would predict (Kile et al, 2003).…”

Cancer of mice and men: old twists and new tails

“…A chemical mutagen that is still in use today is N -ethyl-N -nitrosourea (ENU). ENU, once used as a chemotherapeutic drug, is an alkylating agent that is a powerful mutagen of mouse spermatogonial stem cells producing single locus mutations (typically point mutations that when translated into a protein product predominantly result in missense mutations) [15] at rates up to 1.5 × 10 −3 [16]. Large-scale ENU mutagenesis programmes have resulted in an explosion in the number of mutants, with phenotypic screening of these mice leading to the identification of new disease candidates [17][18][19].…”