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

Abstract

Mutagenic, reproductive, and toxicity effects of two closely related chemicals, ethylnitrosourea (ENU) and methylnitrosourea (MNU), were compared at equimolar and near-equimolar doses in the mouse specific-locus test in a screen of all stages of spermatogenesis and spermiogenesis. In stemcell spermatogonia (SG), ENU is more than an order of magnitude more mutagenic than MNU. During post-SG stages, both chemicals exhibit high peaks in mutation yield when differentiating spermatogonia (DG) and preleptotene spermatocytes are exposed. The mutation frequency induced by 75 mg MNU/kg during this peak interval is, to date, the highest induced by any single-exposure mutagenic treatment -chemical or radiation -that allows survival of the exposed animal and its germ cells, producing an estimated 10 new mutations per genome. There is thus a vast difference between stem cell and differentiating spermatogonia in their sensitivity to MNU, but little difference between these stages in their sensitivity to ENU.During stages following meiotic metaphase, the highest mutation yield is obtained from exposed spermatids, but for both chemicals, that yield is less than one-quarter that obtained from the peak interval. Large-lesion (LL) mutations were induced only in spermatids. Although only a few of the remaining mutations were analyzed molecularly, there is considerable evidence from recent molecular characterizations of the marker genes and their flanking chromosomal regions that most, if not all, mutations induced during the peak-sensitive period did not involve lesions outside the marked loci. Both ENU and MNU treatments of post-SG stages yielded significant numbers of mutants that were recovered as mosaics, with the proportion being higher for ENU than for MNU.Comparing the chemicals for the endpoints studied and additional ones (e.g., chromosome aberrations, toxicity to germ cells and to animals, teratogenicity) revealed that while MNU is generally more effective, the opposite is true when the target cells are SG.

Disruption of histone methylation in developing sperm impairs offspring health transgenerationally

“…These observations together with the high penetrance of phenotypes that was resolved by the fourth generation of non-transgenic offspring points to an epigenetic mechanism driving impaired development. It is unlikely that hKDM1A expression during spermatogenesis behaves comparable to a chemical mutagen or induces a mutator phenotype in germ cells or offspring given the very high frequency of abnormalities observed (44)(45)(46). Consistent with that notion, our assessments using multiple approaches revealed no DNA damage or chromatin instability.…”

In vitro responses to known in vivo genotoxic agents in mouse germ cells

“…This was not detected in other cell types, namely spermatocytes or spermatids; mRNA expression of bcl‐2 was also decreased in spermatogonia more than in spermatocytes and spermatids when treated with the lowest concentration of ENU. This result confirms that of a previous study in vivo , in which differentiating spermatogonial cells were observed to be the most sensitive testicular cells to ENU damage, due to their higher mitotic rate compared to other spermatogenic cells [Russell et al, ]. The data presented here show that the expression level of p53 is high in spermatogonial cells compared to levels in spermatocytes and spermatids; a potential consequence of actively dividing spermatogonia which are the most susceptible male germ cell.…”

“…Mutant frequencies in male rat germ cells were determined after exposure to ENU, MNU, 6MP, 5BrdU, MMS, and EMS and thus have been reported to cause specific DNA damage in isolated germ cells from adult rat testis [Ehling et al, ; Anderson et al, ; Russell et al, ; Levkoff et al, ; Kanemitsu et al, ; Habas et al, ]. To examine species differences in the DNA damage between rats and mice three male germ cell mutagens (ENU, 6‐MP and MMS) were selected for testing.…”

Enhanced Genetic Integrity in Mouse Germ Cells1

“…That there is indeed a low frequency of spontaneous mutations in mammalian germline cells was suggested by early studies of mice [2,3]. The specific locus test was used to examine spontaneous and induced germline mutation frequencies to evaluate the effects of suspected genotoxins [4][5][6][7][8]. These and related studies estimated the spontaneous mutation frequencies for female and male germ cells to be approximately 1.4 3 10 À6 and 6.6 3 10 À6 , respectively [9].…”