Relationships between cisplatin-induced adducts and DNA strand-breaks, mutation and recombination in vivo in somatic cells of Drosophila melanogaster, under different conditions of nucleotide excision repair.

Cisplatin is a chemotherapeutic drug widely used in the treatment of several tumours, but this chemotherapy presents problems in terms of side-effects and patient resistance. The detection and determination of cisplatin-induced adducts and the relationship with the physiological or clinical effects of this drug under different repair conditions could be a good measure to assess patient's response to such chemotherapy. A new methodological approach to detect and quantify cisplatin adducts by use of high-performance liquid chromatography with inductively coupled plasma mass-spectrometric detection (HPLC-ICP-MS) and isotope-dilution analysis (IDA), is evaluated for its application in vivo, under different repair conditions. This analysis is combined with the use of the Comet assay, which detects DNA strand-breaks, and the w/w(+) SMART assay, which monitors induction of somatic mutation and recombination in Drosophila melanogaster in vivo under different conditions of nucleotide-excision repair. Results show that (i) cisplatin induces in Drosophila several adducts not detected in mammals. The two most abundant cisplatin-induced adducts, identified by electrospray-mass spectrometry as G monoadduct and G-G intrastrand cross-links, were quantified individually; (ii) cisplatin induces higher levels of G monoadducts and G-G cross-links in NER-proficient than in NER-deficient cells; (iii) the level of adducts correlates with their biological consequences, both in terms of DNA strand-breaks (tail-moment values), and of somatic mutation and recombination (frequency of mosaic eyes and clones in 10(4) cells), when the repair status is considered. This work demonstrates the validity and potential of the adduct detection and quantification methodology in vivo, and its use to correlate adducts with their genetic consequences.

Mus308 processes oxygen and nitrogen ethylation DNA damage in germ cells of Drosophila.

The D. melanogaster mus308 gene, highly conserved among higher eukaryotes, is implicated in the repair of cross-links and of O-ethylpyrimidine DNA damage, working in a DNA damage tolerance mechanism. However, despite its relevance, its possible role on the processing of different DNA ethylation damages is not clear. To obtain data on mutation frequency and on mutation spectra in mus308 deficient (mus308(-)) conditions, the ethylating agent diethyl sulfate (DES) was analysed in postmeiotic male germ cells. These data were compared with those corresponding to mus308 efficient conditions. Our results indicate that Mus308 is necessary for the processing of oxygen and N-ethylation damage, for the survival of fertilized eggs depending on the level of induced DNA damage, and for an influence of the DNA damage neighbouring sequence. These results support the role of mus308 in a tolerance mechanism linked to a translesion synthesis pathway and also to the alternative end-joinig system.

Female germ cell mutagenicity of model chemicals in Drosophila melanogaster: mechanistic information and analysis of repair systems.

In spite of differences between female and male germ cells, and although both of them contribute to the gene pool of future generations, most germ cell mutagenicity studies in higher eukaryotes have been carried out on males. To study the response of female germ cells to mutagen/carcinogen exposure, the mutagenicity of two model chemicals like diethyl sulfate (DES) and hexamethylphosphoramide (HMPA), and the monofunctional methylating chemotherapeutic drug streptozotocin (STZ), has been analysed on repair efficient females of Drosophila melanogaster. Results previously obtained with N-ethyl-N-nitrosourea (ENU), another model chemical, have also been included in the analysis. The activity of bypass tolerance mechanism (BTM; represented by the mus308 locus) and nucleotide excision repair (NER) on the removal of oxygen and nitrogen ethylations was studied by determining DES mutagenicity in NER deficient females, comparing it with existing results for ENU, and by analysing both chemicals on BTM deficient females. Results indicate that (1) all chemicals are mutagenic on repair efficient females; (2) a measure of mutagenic activity ranked from the lowest DES to STZ, HMPA, and ENU as the highest. This order correlates with the repair of the respectively induced DNA damages, and with the mutagenic and carcinogenic potency of these compounds, considering the toxicity of cross-linking agents; (3) NER efficiently repairs nitrogen ethylation damage and seems to contribute to the processing of oxygen damage in female germ cells; and (4) BTM is involved on the processing of oxygen ethylation damage, whereas the results on nitrogen ethylation are not clear. Finally, these results indicate that differences between male and female germ cells affect the response to chemical exposure, and therefore demonstrate the necessity of analysing also female cells in germinal mutagenicity studies. In addition, these studies can provide important mechanistic information about germ cell chemical mutagenesis, and even when the analysis of oogonia is not possible, since all female germ cells are pre-meiotic, studies of oocytes could be a model for pre-meiotic cells.

Influence of mus201 and mus308 mutations of Drosophila melanogaster on the genotoxicity of model chemicals in somatic cells in vivo measured with the comet assay.

To check the possibilities of the recently developed comet assay, to be used in mechanistic studies in Drosophila melanogaster, neuroblast cells of third instar larvae are used to analyse in vivo, the effect of two repair deficient mutations: mus201, deficient on nucleotide excision repair, and mus308, deficient in a mechanism of damage bypass, on the genotoxicity of methyl methanesulphonate (MMS), ethyl methanesulphonate (EMS) and N-ethyl-N-nitrosourea (ENU). The obtained results reveal: (1) MMS-induced breaks are most probably consequence of N-alkylation damage mediated abasic (AP) site breakage; (2) MMS and at least part of the EMS induced damage leading to DNA strand breaks are efficiently repaired by the nucleotide excision repair mechanism; (3) ENU and part of EMS induced damage need a functional Mus308 protein to be processed, otherwise they can lead to DNA strand breaks. In addition, the results of this work confirm the validity of neuroblast cells to conduct the comet assay, and the usefulness of this assay in in vivo mechanistic studies related to DNA repair in D. melanogaster.