This study presents the first application of an in vivo alkaline comet assay using haemocytes of Drosophila melanogaster larvae. These cells, which play a role similar to that of mammalian blood, can be easily obtained and represent an overall exposure of the treated larvae. To validate the assay, we evaluated the response of these cells to three well-known mutagenic agents: ethyl methanesulfonate (EMS), potassium dichromate (PD), and gamma radiation (γ-irradiation). Third-instar Drosophila larvae were exposed to different concentrations of EMS (1, 2, and 4 mM) and PD (0.5, 1, and 2.5 mM) and to different doses of γ-irradiation (2, 4, and 8 Gγ). Subsequently, haemolymph was extracted from the larvae, and haemocytes were isolated by centrifugation and used in the comet assay. Haemocytes exhibited a significant dose-related increase in DNA damage, indicating that these cells are clearly sensitive to the treatments. These results suggest that the proposed in vivo comet test, using larvae haemocytes of D. melanogaster, may be a useful in vivo assay for genotoxicity assessment.
Doxorubicin (DOX) is an important tumor chemotherapeutic agent, acting mainly by genotoxic action. This work focus on cell processes that help cell survival, after DOX-induced DNA damage. In fact, cells deficient for XPA or DNA polymerase eta (pol eta, XPV) proteins (involved in distinct DNA repair pathways) are highly DOX-sensitive. Moreover, LY294002, an inhibitor of PIKK kinases, showed a synergistic killing effect in cells deficient in these proteins, with a strong induction of G2/M cell cycle arrest. Taken together, these results indicate that XPA and pol eta proteins participate in cell resistance to DOX-treatment, and kinase inhibitors can selectively enhance its killing effects, probably reducing the cell ability to recover from breaks induced in DNA.
Diphenyl ditelluride (DPDT) is a potential prototype for the development of novel biologically active molecules. Thus, it is important to evaluate the toxic effects of this compound. In the present study, we evaluated the cytotoxic, genotoxic and mutagenic properties of DPDT in Chinese hamster fibroblast (V79) cells, in strains of the yeast Saccharomyces cerevisiae both proficient and deficient in several DNA repair pathways and in Salmonella typhimurium. DPDT induced frameshift mutations in both S.typhimurium and a haploid wild-type strain of S.cerevisiae. Mutants of S.cerevisiae defective in base excision repair and recombinational repair were more sensitive to DPDT. The results of a lactate dehydrogenase leakage assay suggest that DPDT is cytotoxic to V79 cells. At cytotoxic concentrations, this compound increased thiobarbituric reactive species levels and decreased the glutathione:GSSH ratio in yeast and V79 cells. DPDT generated single- and double-strand DNA breaks in V79 cells, both with and without metabolic activation, as revealed by alkaline and neutral comet assays. Moreover, an induction of oxidative DNA base damage was indicated by a modified comet assay using formamidopyrimidine DNA glycosylase and endonuclease III. Treatment with DPDT also induced micronucleus formation in V79 cells. Pre-incubation with N-acetylcysteine reduced DPDT's oxidative, genotoxic and mutagenic effects in yeast and V79 cells. Our results suggest that the toxic and mutagenic properties of DPDT may stem from its ability to disturb the redox balance of the cell, which leads to oxidative stress and the induction of DNA damage.
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