Dose--response curve for ethylnitrosourea-induced specific-locus mutations in mouse spermatogonia.

Abstract

The extreme mutagenic effectiveness ofN-ethyl-N-nitrosourea in the mouse has permitted the accumulation of the most extensive dose-response data yet obtained for chemical induction of specific-locus mutations in spermatogonia. In the lower portion of the curve, below a dose of 100 mg/kg, the data fall statistically significantly below a maximum likelihood fit to a straight line. Independent evidence indicates that, over this dose range, ethylnitrosourea reaches the testis in amounts directly proportional to the injected dose. It is concluded that, despite the mutagenic effectiveness of ethylnitrosourea, the spermatogonia are apparently capable of repairing at least a major part of the mutational damage when the repair process is not swamped by a high dose. This finding is important both in basic studies on the mutagenic action of chemicals in mammals and in risk estimation.In 1979, it was shown (1) with the specific-locus method that N-ethyl-N-nitrosourea (ENU) is, by more than an order ofmagnitude, a more effective mutagen in mouse spermatogonia than any other compound tested. It was pointed out that ENU could, therefore, serve as a model chemical in exploring, with relative ease, the various factors-such as dose, dose fractionation, sex, and cell stage-that might affect mutagenic action. Preliminary findings on the effects of these and other factors have been reported (2-7). A more detailed dose-response curve than that obtained for any other compound tested for transmitted mutations in the mouse is now available and is presented here.MATERIALS AND METHODS ENU (purchased from Bio-Clinical Laboratories, Bohemia, NY) was dissolved in phosphate buffer (8) adjusted to pH 6. The dose injected intraperitoneally was matched to the body weight of the animal by adjusting the volume of solution injected, which approximated 1 ml for all doses. Wild-type (101 x C3H)F1 male mice 11.5-14.5 wk old were injected with a single dose. The experiment was done in two series, the first with doses of 100, 150, 200, and 250 mg/kg, and the second, 11 wk later, with doses of 25, 50, 75, and 100 mg/kg. The same crystalline batch of ENU, kept in a dessicator under refrigeration, was used throughout but, to check for deterioration of the chemical or other change in the conditions, the 100-mg/kg dose was included in both series. All injections were completed within 2 hr after the chemical was dissolved. The injected males were mated to females of our standard specific-locus test strain (T), which is homozygous for seven marker genes (9). Each male was mated to a group of either two or four females and moved to a new group of females each week. After each 7-wk period, the males were rotated back to the original group offemales to start the cycle over again. All the offspring reported here came from conceptions occurring more than 7 wk after injection, thus ensuring that they were derived from cells that had been exposed to the chemical in spermatogonial stem-cell stage. The offspring were scored for mutations at the seven loci. RES...

Comparison of the genetic effects of equimolar doses of ENU and MNU: While the chemicals differ dramatically in their mutagenicity in stem-cell spermatogonia, both elicit very high mutation rates in differentiating spermatogonia

“…During the first four mating weeks, the chemicals can be compared both at the equimolar doses (50 mg ENU/kg versus 44 mg MNU/ kg) and at the roughly equimolar doses (100 mg ENU/kg versus 75 mg MNU/kg). (Note that calculations to extrapolate from 100 to the equimolar dose of 85.2 mg ENU/kg are probably legitimate since these doses lie within the straight portion of the dose curve [2].) Neither chemical yields overall higher frequencies than the other during this time, except for week-3 matings (sampling early spermatids), when MNU rates are higher than ENU rates (though not significantly so) in each of four possible comparisons (two scenarios for each of two doses).…”

“…Almost three decades ago, N-ethyl-N-nitrosourea (ENU) was demonstrated to be the most effective germline mutagen in the mouse when administered to stem-cell spermatogonia [1,2]. Productive dosing regimens that were subsequently developed [3] yield mutation rates 12 times higher than those obtainable with X-rays.…”

“…The mutagenic effects of ENU on stem-cell spermatogonia have been studied and reported extensively [1][2][3]13,16]. This paper reports on large-scale ENU experiments on the subsequent germ-cell stages, as well as on MNU mutagenesis studies on all male germ-cell stages.…”

Next-generation gene targeting in the mouse for functional genomics

“…The phenotype-driven approach with a potent alkylating mutagen ENU was taken in Phase I mouse mutagenesis because 1) ENU had been well known to effectively induce point mutations in a genomewide manner (21)(22)(23)(24)(25)(26), which resemble most of the cause of human genetic diseases, and 2) the phenotype-driven approach enable to identify yet-unknown genes responsible for human diseases. On the other hand, the ENU mutagenesis has been conducted mostly for dominant phenotype screenings but not for recessives (27).…”

ENU-Based Gene-Driven Mutagenesis in the Mouse: A Next-Generation Gene-Targeting System

“…Their major objectives were to develop a new infrastructure for mammalian functional genomics by foreseeing the completion of whole sequencing of the human genome by the Human Genome Project. ENU is known to be the most potent chemical mutagen in the mouse, inducing heritable base substitutions in spermatogonia [24,27,28]. By inducing point mutations genomewide, mutant mice can be used to explore biological functions of genomic DNA sequences as well as to provide human disease models.…”