Temporal and molecular characteristics of mutations induced by ethylnitrosourea in germ cells isolated from seminiferous tubules and in spermatozoa of lacZ transgenic mice.

Abstract

The lacZ transgenic mouse (Muta mouse) model was used to examine the timing of ethylnitrosourea (ENU)-induced mutations in germ cells. The spectrum of mutations was also determined. Animals received five daily treatments with ENU at 50 mg/kg and were sampled at times up to 55 days after treatment. In mixed germ-cell populations isolated from seminiferous tubules, there was little increase in the mutant frequency 5 days after treatment; subsequently, there was a continuous increase until the maximum (17.5-fold above background) was reached by -35 days. In the spermatozoa, an increase in mutant frequency was not seen until 20 days after treatment, with the maximum (4.3-fold above background) being achieved no sooner than -35 days. Based on the timing of sampling, these data demonstrate the detection of both spermatogonial and postspermatogonial mutations. The most prominent feature of the ENU-induced basepair mutations in testicular germ cells sampled 55 days after treatment is that 70%o are induced in AT base pairs, compared to only 16% in spontaneous mutations. These findings are consistent with comparable data from ENU studies using assays for inherited germ-cell mutations in mice. This study has demonstrated the utility and potential of the transgenic mouse lacZ model (Muta mouse) for the detection and study of germ-cell mutations and provides guidance in the selection of simplified treatment and sampling protocols.in bacteria, A-T base pairs are the predominate source of mutations in mammalian species in vivo (13). This change is related to the reduced capacity of DNA alkyltransferase to remove ethyl adducts other than 06-ethylguanine from mammalian cells (13). ENU induces both spermatogonial stem cell and postspermatogonial mutations in mice (14). The former are characterized by base-substitution mutations in A-T base pairs (15-19); the latter have been shown to result in relatively more multilocus lesions (20) and, if adducts are induced late in development, mosaic mutations in the progeny result (21).In this study we have used a lacZ transgenic mouse (Muta mouse) model to determine the timing of ENU-induced mutation induction in a mixed population of germ cells isolated from seminiferous tubules and in spermatozoa isolated from the vas deferens. The mutation spectrum for mutations from mixed germ cells was also determined. It is anticipated that such information will be influential in determining the acceptability of transgenic mouse models for the study of germ-cell mutagenicity, in suggesting appropriate experimental protocols for future studies, and in alleviating the need to conduct costly experiments on the offspring of exposed animals when it is not necessary. It is envisaged that such a transgenic mouse germ-cell assay, once validated, may become a companion to the dominant lethal test in terms of its role in hazard identification.The establishment of transgenic mouse models for the quantitative detection of gene mutations (1, 2) has provided unprecedented access to the study of mutagenesis ...

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

“…Of base alkylations, the most significant pre-mutagenic lesions induced by ENU are O 6 -ethylguanine, O 2 -ethylthymine, and O 4 -ethylthymine. The first of these adducts is, however, readily repaired by O 6 -alkylguanine transferase [38] and by nucleotide excision repair, while the other two types of adducts have been reported not to be removed by either mechanism [20]. This leads to the greater proportion of mutations in A-T than G-C base pairs that has been reported for ENU-induced SG mutations recovered in the specific-locus test [4,5,8] or in transgenic mice, both λlacZ [20] and λlacI [39].…”

“…Sequencing revealed 27 of them, at four loci, to be single base-pair changes, with no evidence for large lesions in the remaining 5 [5 (review),8]. In λlacZ transgenic mice, none of 31 mutations induced by ENU in spermatogonia was found to be a deletion or insertion, even though the test is capable of detecting such changes up to 550 bp [20]. The present experiment generated SG mutations induced by MNU.…”

“…When males are mated continuously, instead of sequentially, stage assignments become very uncertain, since, with the much smaller number of mating opportunities available, the possibility of sperm storage cannot be entirely dismissed (see frequency of fertile mating, discussed in Section 3.1). Similarly, histological identification of germ cells in mixed populations, as encountered in mutagenesis studies on transgenic mice, makes it more difficult to attribute mutants to specific stages [20].…”

Assessing human germ‐cell mutagenesis in the Postgenome Era: A celebration of the legacy of William Lawson (Bill) Russell

“…George R. Douglas (Health Canada) emphasized the need for parallel studies of mutational mechanisms in mouse models, and he presented data from ENU-treated lacZ transgenic mice (Muta mouse) that illustrated the utility of transgenic rodent models for studying the etiology and mechanisms of induced gene mutation in male germ cells [Douglas et al, 1995]. Although the lacZ model does not permit the detection of gene mutations in the ovum per se, Douglas described a method to detect lacZ mutations in granulosa cells, which provide the immediate environment for the ovum in female mice [Yauk et al, 2005].…”

Transgenic Rodent Assay for Quantifying Male Germ Cell Mutant Frequency

“…An example of this assay performed on seminiferous tubule germ cells can be found in Douglas et al 27 , where MF was determined in tubule germ cells at various time intervals following a 5 day repeat intraperitoneal injection of 50 mg/kg ENU. In that study, mutant frequencies in seminiferous cells increased up to 15 days after treatment and remained constant thereafter.…”