Chlorambucil effectively induces deletion mutations in mouse germ cells.

Abstract

The chemotherapeutic agent chlorambucil was found to be more effective than x-rays or any chemical investigated to date in inducing high yields of mouse germ-line mutations that appear to be deletions or other structural changes. Induction of mutations involving seven specific loci was studied after exposures of various male germ-cell stages to chlorambucil at 10-25 mg/kg. A total of 60,750 offspring was scored. Mutation rates in spermatogonial stem cells were not significantly increased over control values; this negative result is not attributable to selective elimination of mutant cells. Mutations were, however, clearly induced in treated poststem-cell stages, among which marked variations in mutational response were found. Maximum yield occurred after exposure of early spermatids, with -1% of all offspring carrying a specific-locus mutation in the 10 mg/kg group. The stageresponse pattern for chlorambucil differs from that of all other chemicals investigated to date in the specific-locus test. Thus far, all but one of the tested mutations induced by chlorambucil in post-stem-cell stages have been proved deletions or other structural changes by genetic, cytogenetic, and/or molecular criteria. Deletion mutations have recently been useful for molecular mapping and for structure-function correlations of genomic regions. For generating presumed large-lesion germline mutations at highest frequencies, chlorambucil may be the mutagen of choice.Chromosomal deletions have been valuable in the molecular mapping of genetic loci in humans (1, 2). In the mouse, the existence of large arrays of radiation-induced deletion mutations involving marked loci has been an important factor in recent advances in structurally and functionally characterizing regions of the genome that surround these loci (for review, see ref.3). Multilocus deletions constitute a relatively high proportion of mutations induced by certain radiation treatments (4). Although the total yield of mutations produced by N-ethyl-N-nitrosourea (EtNU) considerably exceeds the yield of radiation-induced mutations (5), EtNU induces chiefly intragenic lesions, including base-pair changes (4, 6). This paper reports the finding that a chemical mutagen, chlorambucil, is considerably more effective than radiation or any chemical investigated to date in inducing high yields of germ-line mutations that appear to be multilocus deletions or other structural changes.Chlorambucil (7). Chlorambucil is a germ-cell mutagen in Drosophila, where it induces very high frequencies of sex-linked recessive lethals as well as translocations in meiotic and postmeiotic stages (8). Evidence that chlorambucil reaches the testis of mice comes from the results of histological studies (9), in which exposure to this chemical at 20 mg/kg was moderately to strongly cytotoxic to differentiating spermatogonia, and slightly toxic to spermatogonial stem cells. In this study, mutagenicity of chlorambucil was tested in male germ-cell stages of the mouse, ranging from spermatogonial stem cells t...

Further flow cytometric studies of the effects of triethylenemelamine on somatic and testicular tissues of the rat

“…This is consistent with the hypothesis that increased variation in the G, peak is due to chromosomal breaks that result in deletions and duplications (25). In particular, visible chromosomal deletions have been observed in mice exposed to chemical mutagens (29) and smaller deletions have been documented to be common by Southern blot analysis (27). But, binding of mutagens to DNA, resulting in interference with dye binding, could also play a role.…”

Development of a possible nonmammalian test system for radiation-induced germ-cell mutagenesis using a fish, the Japanese medaka (Oryzias latipes).

“…Indeed, our preliminary experiments using a potent chemical mutagen, ethylnitrosourea, demonstrated that 15 out of 17 total mutants obtained from gametes treated at the spermatogonial stage became viable mutants, giving VMF/TMF ratios very close to 1 (unpublished data). Also interesting should be the use ofthe mouse supermutagen chlorambucil (31,32).…”

N -Ethyl- N -Nitrosourea Mutagenesis: Boarding the Mouse Mutant Express

“…Whereas the frequency of X-ray mutagenesis (13 ϫ 10 Ϫ5 to 50 ϫ 10 Ϫ5 per locus) is 20 to 100 times that of spontaneous mutants, X rays cause a wide variety of chromosomal rearrangements, such as inversion, deletions, and translocations, which usually affect multiple genes. Similarly, the chemical chlorambucil also causes a range of chromosomal rearrangements, but with greater frequency (127 ϫ 10 Ϫ5 per locus) (49). In the 1970s, investigators at Oak Ridge National laboratory under the guidance of Bill Russell began to systematically explore the use of chemical mutagenesis with N-ethyl-N-nitrosourea (ENU) to produce new mouse mutations.…”