Frequency Of Mutant Clones Research Articles

A comprehensive study of the harmful effects of ZnO nanoparticles using Drosophila melanogaster as an in vivo model

This study planned to determine the range of biological effects associated with ZnO-NP exposure using Drosophila melanogaster as an in vivo model. In addition, ZnCl2 was used to determine the potential role of Zn ions alone. Toxicity, internalization through the intestinal barrier, gene expression changes, ROS production, and genotoxicity were the end-points evaluated. No toxicity or oxidative stress induction was observed in D. melanogaster larvae, whether using ZnO-NPs or ZnCl2. Internalization of ZnO-NPs through the intestinal barrier was observed. No significant changes in the frequency of mutant clones (wing-spot test) or percentage of DNA in tail (comet assay) were observed although significant changes in Hsp70 and p53 gene expression were detected. Our study shows that ZnO-NPs do not induce toxicity or genotoxicity in D. melanogaster, although uptake occurs and altered gene expression is observed.

Genotoxicity of cobalt nanoparticles and ions in Drosophila

Nanogenotoxicology is an emergent area of research, relevant for estimating the potential carcinogenic risk of nanomaterials. Since most of the approaches use in vitro studies, and neglecting the whole organism limits the accuracy of the obtained results, we have used Drosophila melanogaster to study the possible genotoxic potential of cobalt nanoparticles (Co NPs). The wing somatic mutation and recombination test has been the test of choice. This test is based on the principle that the loss of heterozygosis and the corresponding expression of the suitable recessive markers, multiple wing hairs and flare-3 can lead to the formation of mutant clone cells in growing up larvae, which are expressed as mutant spots on the wings of adult flies. Co NPs, as well as the ionic form cobalt chloride, were given to third instar larvae through the food, at concentrations ranging from 0.1 to 10 mM. The results obtained indicate that both cobalt forms are able to induce significant increases in the frequency of mutant clones. Although at low concentrations only Co NPs were genotoxic, the level of genetic damage obtained at the highest dose tested of cobalt chloride (10 mM) showed a significant higher increase in the frequency of total spots than those observed after the treatment with cobalt nanoparticles. As conclusion, our results indicate that Co NPs were able to induce genotoxic activity in the wing-spot assay of D. melanogaster, mainly via the induction of somatic recombination. The differences observed in the behaviour of the two selected cobalt forms may result from differences in the uptake.

Induction of an adaptive response in Drosophila imaginal disc cells exposed in vivo to low doses of alkylating agents.

The adaptive response of Drosophila larvae to three alkylating agents (ethyl methanesulfonate, methyl methanesulfonate and N-nitroso-N-ethylurea) has been studied in the wing spot test. The experimental procedure included a 24 h pretreatment of 2-day-old larvae with two different adaptive doses followed by a challenge dose applied until the end of development. The genotoxic effects were analysed in trans-heterozygous larvae for the third chromosome recessive markers multiple wing hairs (mwh) and flare (flr(3)). Genetic changes induced in somatic cells of the wing imaginal discs led to loss of heterozygosity, expressed as mutant clones of the genetic markers used. From our results it appears that the adaptive doses clearly reduce the frequency of mutant clones induced by the challenge dose. As far as we know, this is the first time that the existence of an adaptive response to alkylating agents after Drosophila larval treatment has been reported using the wing spot assay.

Accumulation of deletions and point mutations in mitochondrial genome in degenerative diseases.

Accumulation of various mutations in the mitochondrial genome is proposed as an important contributor to aging and degenerative diseases. Extensive fragmentation of mtDNA was detected in association with increased 8-hydroxydeoxyguanosine content in the heart mitochondrial DNA (mtDNA) from a patient with premature aging and mitochondrial cardiomyopathy, who carried a mutation within the mitochondrial tRNA(Asp) gene. This result suggests that damage to mtDNA by hydroxyl radical and accumulation of deleted mtDNA can be accelerated by a specific mitochondrial genotype. Similarly, extensive fragmentation of mtDNA was also detected in cultured cells exposed to a high oxygen concentration atmosphere, implying that mtDNA is vulnerable to reactive oxygen species. To clarify the role of point mutations accumulated in mtDNA, we examined the sequence heterogeneity of mtDNA in the skeletal muscle of a MELAS patient who carried a mutation within the mitochondrial tRNA(leu)(UUR) gene. The analysis revealed that the frequency of mutant clones in the MELAS muscle was significantly higher than those in an age-matched control muscle and a control placenta. Some of these nucleotide substitutions were missense and nonsense mutations, which potentially have deleterious effects on the mitochondrial function. The frequency of nucleotide substitutions in the striatum of three patients with Parkinson's disease was also significantly higher than that in control tissues. We also observed increased protein modification by 4-hydroxy-2-nonenal, a lipid peroxidation by-product, in Parkinson's disease. These results suggests that a vicious cycle contributes to the progression of degenerative process. In this cycle, first a primary mitochondrial mutation(s) induces a mitochondrial respiratory defect, which increases the leakage of reactive oxygen species (ROS) from the respiratory chain. Then the ROS would trigger accumulation of secondary mtDNA mutations in postmitotic cells, leading to further aggravation of mitochondrial respiratory defects and increased production of ROS and lipid peroxides from mitochondria, and thus resulting in degeneration of cellular components.

Accumulation of Somatic Nucleotide Substitutions in Mitochondrial DNA Associated with the 3243 A-to-G tRNALeu(UUR)Mutation in Encephalomyopathy and Cardiomyopathy

To understand the pathogenesis of mitochondrial encephalomyopathy and cardiomyopathy, we analyzed the sequence heterogeneity of the skeletal muscle mitochondrial DNA from a patient with Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-like episodes (MELAS). A mtDNA segment of 347 bp amplified from the total DNA was cloned into a vector. Analysis of 60 independent clones (20,800 bp in total) revealed the 3243 A→G transition in all the sequenced clones and additional nucleotide substitutions at 5 sites in 10 clones. The frequency of mutant clones (10/60) in the MELAS patient was significantly higher [χ2= 10.909,P< 0.05] than that in an age-matched skeletal muscle control (0/60) as well as in a normal placenta (2/60). These results supports our hypothesis that secondary somatic mtDNA mutations can be initiated by the 3243 A→G mutation and that the accumulation of somatic mutation in individuals with deleterious inherited mitochondrial genotype can contribute to the progressive mitochondrial dysfunction in MELAS.

Genotoxicity testing of five compounds in three Drosophila short-term somatic assays

To provide further background data for the somatic mutation and/or recombination tests in Drosophila melanogaster, we have evaluated the responses in 3 assyas ( zeste-white, white-ivory and wing spot) of 5 chemicals classified by the U.S. National Toxicology Program (NTP) as genotoxic non-carcinogens (or ambiguous). The selected compounds were 2-chloromethylpyridine, 1-nitronaphthalene, 4-nitro- o-phenylenediamine, 3-nitropropionic acid and p-phenylenediamine. Our results show that all the compounds tested produce significant increases in the frequency of mutant clones, in at least one of the assays, p-phenylenediamine being the compound which present a clearer mutagenic activity, and the wing spot test, the assay the detects more genotoxic compounds (4/5).