O‐ethylthymidine adducts are the most relevant damages for mutation induced by N‐ethyl‐N‐nitrosourea in female germ cells of Drosophila melanogaster

Abstract

Responses to genotoxic agents vary not only among organisms, test systems, and cellular stages, but also between sexes; little, however, is known about the mutagenic consequences of chemical exposures to female germ cells. In this study, the mutagenicity of N-ethyl-N-nitrosourea (ENU) was analyzed in female germ cells of Drosophila melanogaster using the recessive-lethal test and the vermilion system, which simultaneously generates information on induced mutation frequency and mutation spectrum. ENU was mutagenic in all stages of oogenesis, although there were differences among the stages. In mature and immature oocytes, ENU-induced mutations in the vermilion locus were 43.5% A:T-->G:C transitions, 39.1% A:T-->T:A transversions, 8.7% G:C-->A:T transitions, and 8.7% A:T-->C:G transversions, indicating that the most important premutagenic lesions induced by this chemical are O(4)-ethylthymine and O(2)-ethylthymine. The low frequency of mutation involving O(6)-ethylguanine (i.e., G:C-->A:T transitions) could be a consequence of the repair of these lesions by O(6)-methylguanine DNA methyltransferase. Comparison of these results with those previously obtained in male germ cells stresses the importance of the repair activity of the analyzed cells, because the mutation spectrum in female germ cells was similar to the spectrum obtained with repair-proficient spermatogonial cells and different from repair-deficient postmeiotic cells. The results also indicate that studies with female germ cells could be an alternative to the use of premeiotic male germ cells, especially when the analysis of these cells is difficult or almost impossible and when studies of in vivo DNA repair in premeiotic germ cells are performed.

Mus308 Processes Oxygen and Nitrogen Ethylation DNA Damage in Germ Cells of Drosophila

“…In D8-9 no mutation was found, and the same mutation was present in the independent mutants D8-21 and D8-30, as previously reported for other vermilion spectra [14, 18, 21, 31, 32]. In D8-26 no mutation was detected in the coding region nor in the proximal part of the promoter, but the distal part of the promoter could not be amplified, suggesting the presence of a mutation.…”

“…Although it is not considered a lethal lesion [33, 36], it is able to block DNA replication in mammalian cells in a NER deficient background [46]. NER is apparently implicated in its repair in Drosophila [27, 32], although rather inefficiently, because AT-GC transitions are one of the most frequently ENU-induced damages in the repair-active premeiotic germ cells of this organism [21, 31]. Therefore, this adduct fits with the proposed requirements for the substrates of Mus308 [13, 14].…”

“…The isolation of DNA and PCR amplifications were as described [21]. Mutant vermilion genes were cloned in the M13mp19 vector or in a pUC18 plasmid [22].…”

“…Sequencing reactions for the coding region were carried out using the dideoxy method, with a set of 10 internal primers. A fragment of about 1.8 Kb, localized upstream of the coding region, was analyzed as described before [21] in those mutants which did not show changes in the coding sequence. In order to exclude Taq polymerase-introduced errors, at least two plaques or colonies from independent PCR reactions were sequenced for each mutant.…”