Genotoxicity testing with the somatic white-ivory system in the eye of Drosophila melanogaster

The white-ivory test in Drosophila melanogaster is designed to detect chemically induced reversions of the sex-linked, recessive unstable eye-color mutation white-ivory to the wild-type form. After exposure of larvae reversions are detectable as clones of red facets in the eye of newly enclosed adult flies. Tester strains containing a quadruplication of the white-ivory gene on the X-chromosome(s) were used. In a strain with males carrying 4 copies of the gene and females carrying 8 copies of the gene, spontaneous reversions occurred proportional to the gene copy number. In contrast to this, chemically induced reversions occurred only 1.36 times more frequently in females (carrying 8 copies of the gene) than in males (carrying 4 copies). Since chemicals inducing different lesions in DNA (bleomycin, cyclophosphamide, daunomycin, diethyl sulfate and 7,12-dimethylbenz[a]anthracene) did induce statistically significant frequencies of reversions the test appears to be capable of detecting a wide variety of genotoxic chemicals with different modes of action. The recombinogen strychnine did not induce reversions.     

Genotoxicity of p-aminophenol in somatic and germ line cells of Drosophila melanogaster

p-Aminophenol (PAP; as a component of, e.g., hair dyes, photographic developers, as adsorbent in gas filters, as a metabolite of various fungicides, pesticides and drugs) has been tested for genotoxicity in Drosophila by means of the sex-linked recessive lethal test (SLRLT) and the somatic mutation and recombination test (SMART) of the wing. While the SLRLT was not significant, the SMART clearly indicated that the compound has genotoxic properties in this in vivo test in agreement with a majority of mammalian short-term tests in vitro and in vivo. The reducing agent dithiothreitol enhanced the genotoxic effects of PAP in the SMART; the reasons for this interaction remain to be elucidated.     

Genotoxicity of 5-azacytidine in somatic cells of Drosophila

The newly developed somatic mutation and recombination test, utilizing the wing-hair mutations mwh and flr3, was used to evaluate the genotoxicity of the base analog 5-azacytidine in larvae of Drosophila melanogaster. Third instar larvae were fed media wetted with various concentrations of the compound, and wings of surviving adults were removed and scored for the presence of clones of cells possessing malformed hairs. Wings of exposed flies trans-dihybrid for mwh and flr3 had significantly increased frequencies of twin spots, small single spots and large single spots. Significant linear regression of twin-spot frequencies upon concentration was also obtained. Induction of twin spots by 5-azacytidine unambiguously demonstrates its recombinogenic activity in somatic cells of Drosophila. Significantly increased frequencies of large single spots on wings of inversion-heterozygous flies were also observed and suggest that 5-azacytidine may also be inducing somatic gene mutations (or deletions).     

Induction of somatic and male crossing-over by bleomycin in Drosophila melanogaster

Bleomycin (BLM) is well known as an antibiotic as well as for its antineoplastic activity. A clinical preparation of BLM was tested for its recombinogenicity in a higher eukaryotic organism, Drosophila melanogaster. Feeding of the F1 larvae on a medium with BLM increased somatic crossing-over spots on female tergites and induced recombination in male germ cells. However, nonlinear dose-response curves were obtained. Malformed tergites were also observed in females treated with BLM.     

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).     

Interactions between germ cells and somatic cells in Drosophila melanogaster

Interactions between germ cells and somatic cells are important at several stages of Drosophila development. The types of interactions that will be discussed include: (1) molecules physically transferred from one cell to another; (2) long range interactions by hormones; and (3) local interactions between germ cells and somatic cells when they are in close proximity. These interactions have been mostly characterized during oogenesis.     

Modifications of the effect of bleomycin in the somatic mutation and recombination test in Drosophila melanogaster

Exposure to oxygen has been implicated as an important mechanism of mutations, cancer and aging. Most data supporting this notion have been obtained in vitro, but the elaborate defense systems against oxygen stress in aerobic organisms make it difficult to extrapolate in vitro data to in vivo conditions. In the present investigation the somatic mutation and recombination test (SMART) in Drosophila with the wing spot system (Graf et al., 1984) has been used as an in vivo system to study the effect of oxygen radicals generated by bleomycin (BLM). BLM causes a dose-related increase of wing spots and this effect drastically increases by increasing oxygen in the atmosphere to 70%. Data from treatment of larvae of different ages, as well as post-treatment with oxygen, indicate that BLM can persist, presumably intercalated in DNA, and subsequently be activated by oxygen to generate free radicals. By the use of inversion heterozygosity, which eliminates somatic recombination, it was shown that the majority of wing spots induced by BLM emanate from somatic recombination. A small number of flies deviated from the rest by an abnormally high frequency of BLM-induced wing spots. Preliminary results from a selection of such flies indicate that this extreme response to BLM is genetically determined. Treatment with BLM was also combined with agents known to interfere with the defense mechanisms against radicals or function as radical scavengers. Only ascorbic acid cotreatment had a modifying effect on BLM mutagenicity. The other agents did not alter or at most had a marginal effect on BLM mutagenicity. These data indicate that the defense mechanisms do not constitute a limiting factor in this case. BLM intercalates between DNA bases, presumably giving little time and opportunity for modifying agents to react with radicals generated in direct contact with the gene targets. No effect of BLM was observed on male germ cells by measuring loss and non-disjunction of ring-X/Y, neither in air nor in a 70% oxygen atmosphere.     

Genotoxicity evaluation of five tricyclic antidepressants in the wing somatic mutation and recombination test in Drosophila melanogaster

Five tricyclic antidepressants were tested for genotoxicity using the somatic mutation and recombination test (SMART) in wing cells of Drosophila melanogaster. Three-day-old larvae trans-heterozygous for 2 linked recessive wing hair mutants (multiple wing hairs and flare) were fed the test compounds in water mixed with a standard dry food for 48 h. Wings of the emerging adult flies were scored for the presence of spots of mutant cells which can be the consequence of either somatic mutation or mitotic recombination. Desipramine and imipramine were clearly genotoxic at concentrations above 1 mM whereas amitriptyline, nortriptyline and protriptyline were not genotoxic at concentrations up to 100 mM. This seems to implicate the nitrogen atom at position 5 in the 7-membered ring of the tricyclic molecule as being responsible for the genotoxic property of the compounds in Drosophila.     

Transposition of the hobo element in Drosophila melanogaster somatic cells

Somatic mutation and recombination test on wing cells of Drosophila melanogaster showed that the recombination frequency in the somatic tissues of strains studied correlated with the presence of a full-length copy of the hobo transposable element in the genome. Transposition of hobo in somatic tissue cells at a frequency 3.5 x 10-2 per site per X chromosome was shown by fluorescence in situ hybridization with salivary gland polytene chromosomes of larvae of one of the D. melanogaster strains having a full-length hobo copy.     

Transposition of the hobo element in Drosophila melanogaster somatic cells

Somatic mutation and recombination test on wing cells of Drosophila melanogaster showed that the recombination frequency in the somatic tissues of strains studied correlated with the presence of a full-length copy of the hobo transposable element in the genome. Transposition of hobo in somatic tissue cells at a frequency 3.5 × 10^?2 per site per X chromosome was shown by fluorescence in situ hybridization with salivary gland polytene chromosomes of larvae of one of the D. melanogaster strains having a full-length hobo copy.     

Genotoxic effects of two nickel-compounds in somatic cells of Drosophila melanogaster

In view of the scarcely available information on the in vivo mutagenic and co-mutagenic activity of nickel, the genotoxic potential of two nickel-compounds, nickel chloride (NiCl(2)) and nickel sulphate (NiSO(4)), was assessed in Drosophila melanogaster by measuring two different genetic endpoints. On the one hand, we used the wing-spot assay, which is based on the principle that the loss of heterozygosity of two suitable recessive markers, multiple wing hairs (mwh) and flare-3 (flr(3)), can lead to the formation of mutant clones in the imaginal disks of larval cells. On the other hand, the in vivo comet assay, which detects single- and double-strand DNA breaks, was also used with larval haemocytes. These cells offer several advantages: they are highly sensitive to genotoxic agents, the sampling and processing methodologies are quite simple and the level of basal DNA damage is relatively low. No significant increases in the frequencies of the three categories of mutant spots (i.e. small single spots, large single spots, and twin spots) were observed in the wing-spot assay; however, NiSO(4) induced significant dose-dependent increases in DNA damage in the comet assay. In addition, the combined treatments with gamma-radiation and NiCl(2) and NiSO(4) showed a slight but significant increase in the frequency of the three categories of mutant spots compared with the frequency induced by gamma-radiation alone, indicating that both nickel compounds have a synergistic interaction. These results support the assumption that both nickel compounds could act as co-mutagens interfering with DNA-repair processes and that the in vivo comet assay is a sensitive and effective method for detecting the DNA damage induced by NiSO(4) in haemocytes of D. melanogaster.     

Biochemical and genetic basis of the response to 5-fluorouracil in Drosophila melanogaster

Mutant strains sensitive and resistant to the drug 5-fluorouracil (FU) have been isolated from the wild-type Pac strain of Drosophila melanogaster. The resistant strain, termed flu^r, is resistant to at least 0.0035%FU (2.7 × 10^–4 m) in the food media and exhibits cross-resistance to 5-fluorodeoxyuridine (FUdR) but not to 5-fluorouridine (FUR). The sensitive strain termed flu ^S , exhibits over 90% mortality on 0.0008% FU (6 × 10^–5 m). Genetic analysis indicates that the flu gene is located on the third chromosome, which agrees with results of previous workers. An analysis of the enzyme thymidylate synthetase from the selected sensitive and resistant strains indicates that the resistant strain enzyme possesses an elevated specific activity. Levels 4 times that of the sensitive strain were observed when the enzymes were assayed at 20 C. This increase is apparently not due to induction by FU in the food media. It is suggested that the enzyme thymidylate synthetase may be involved in the resistance process.     

Distinct P-element excision products in somatic and germline cells of Drosophila melanogaster

The footprints remaining following somatic P-element excision from the Drosophila white locus were recovered and characterized. Two different types of footprints were observed. Over 75% of the footprints were short, composed of 4 or 7 nucleotides of the P-element inverted terminal repeat, and were similar to those found in a previously described plasmid excision assay. The remaining footprints were composed of 14-18 nucleotides of both inverted terminal repeats. These large footprints were indistinguishable from those recovered following germline P-element excision. Enhanced expression of the Drosophila homologue of the Ku70 protein did not affect the structure of the somatic footprints. Therefore, this protein is not a limiting factor for double-strand break repair by nonhomologous end-joining in Drosophila somatic cells.     

Comparative genotoxic effect of vincristine,vinblastine, and vinorelbine in somatic cells of Drosophila melanogaster

In this study, the vinca alkaloids vincristine (VCR), vinblastine (VBL) and vinorelbine (VNR) were investigated for genotoxicity in the wing Somatic Mutation and Recombination Test (SMART) of Drosophila. Our in vivo experiments demonstrated that all drugs assessed induced genetic toxicity, causing increments in the incidence of mutational events, as well as in somatic recombination. Another point to be considered is the fact that VNR was able to induce, respectively, approximately 13.0 and 1.7 times more mutant clones per millimolar exposure unit as their analogues VCR and VBL. The replacement of a CH(3) attached to vindoline group in VBL by a CHO in VCR seems to be responsible for the approximately seven times higher potency of the former. In contrast, the structural modifications on VNR's catharantine group could be related to its higher genotoxic potency, as well as its similar mutagenic and recombinagenic action.     

Genotoxicity of zineb detected through the somatic and germ-line mosaic assays and the sex-linked recessive-lethal test in Drosophila melanogaster

The genotoxicity of zineb, a carbamate fungicide, has been tested through eye, wing and female germ line mosaic assays and the sex-linked recessive-lethal test in Drosophila melanogaster. Larvae of different instars, heterozygous for appropriate recessive genetic markers, were exposed to the fungicide in food for different durations of time. The adult eyes and wings were screened for induction of mosaic spots and the eggs laid by the females were checked for induction of female germ-line mosaicism. It is concluded that zineb is genotoxic to both somatic and germ-line cells of Drosophila.     

Genotoxic damage induced by isopropanol in germinal and somatic cells of Drosophila melanogaster

Isopropanol (isopropyl alcohol, 2-propanol, IPA) is a volatile solvent widely used in domestic or industrial environments and reported as innocuous in various test systems. The aim of this work was to search for in vivo genotoxic effects of IPA in Drosophila melanogaster, studying its ability to induce nondisjunction (ND) in females, sex linked recessive lethals (SLRL) in males, and somatic mutation and/or recombination (SMART) in larvae. Treatments were acute (60min) and were administered via inhalation. IPA had low toxicity in adult flies (75% IPA mortality index, MI=12.7% (females) and 2.6% (males)) and larvae (MI=14.3%, 75% IPA). Female fertility was severely affected during the first 24h (brood I, BI) after treatment, but, afterwards, control values were recovered. IPA induced a 50-fold increase of ND (%) in 24h old females, and a six-fold rise in 4-5 d old BI offspring. Nondisjunction frequencies (%) in the offspring of broods II to V (24h in each case) were similar to control values. IPA doses of 25% and 50% (v/v), tested in 24h old females, showed a significant dose-dependent increase of ND(%)in BI only, with control values in subsequent broods. Flies gave normal offspring when kept in regular media for 24h before mating. The eye spot test (SMART) showed a significant increase at 50% IPA (p<0.05, m=2), but the response was not dose-dependent. IPA failed to induce SLRL in any of the spermatogenesis stages tested. These findings suggest that the main effect of IPA is to induce chromosomal malsegregation; IPA must be present at the resumption of M-phase I after fertilization, to exert these effects. The alcohol does not affect DNA directly, but perturbations of the nuclear membrane may be responsible for induction of ND.