Genotoxic evaluation of selective serotonin-reuptake inhibitors by use of the somatic mutation and recombination test in Drosophila melanogaster

This study evaluated different concentrations of selective serotonin-reuptake inhibitors (citalopram and sertraline) for genotoxicity by use of the somatic mutation and recombination test (SMART) in Drosophila melanogaster. Three-day-old larvae, trans-heterozygous for the multiple wing hairs (mwh) and flare (flr3) genes were treated with these two compounds. Two recessive markers were located on the left arm of chromosome 3, i.e. 'multiple wing hairs' (mwh) in map position 0.3 and 'flare-3' (flr3) at 38.8, while the centromere was located in position 47.7. SMART is based on the loss of heterozygosity, which may occur through various mechanisms, such as mitotic recombination, mutation, deletion, half-translocation, chromosome loss, and non-disjunction. Genetic changes occurring in somatic cells of the wing's imaginal discs, cause the formation of mutant clones on the wing blade. The results of this study show that citalopram had a genotoxic effect in the Drosophila SMART. Sertraline, however, did not show any genotoxic effect in balancer heterozygous wings. This study concluded that more information is needed to be certain regarding the mutagenic effects of sertraline.     

Inhalation of methyl bromide gas induces mitotic recombination in somatic cells of Drosophila melanogaster

The fumigant methyl bromide was evaluated for genotoxicity in the somatic wing-spot assay of Drosophila melanogaster. Third instar larvae trans-dihybrid for mwh and flr3 were exposed to varying concentrations (0-16 mg/l) of the gas for 1 h. Following this exposure via inhalation, the larvae were placed into vials containing Instant Medium. 7 days after the exposure, the adult flies in the vials were collected, and their wings were scored under 400X magnification for the presence of clones of cells possessing malformed wing-hairs. Such clones appeared as mwh-flr3 twin spots and single spots of either mwh or flr3 phenotype. Exposure to methyl bromide was found to result in the positive induction of both twin spots and large (greater than 2 cells) single spots. For each endpoint, a significant exponential association was obtained between concentration and frequency of spots per wing. Methyl bromide was found to be a negative inducer of small (1-2 cells) single spots at all concentrations except 16 mg/l where a positive effect was observed. Because twin spots arise exclusively from mitotic recombination, methyl bromide was identified as having recombinogenic activity in the somatic tissue of Drosophila larvae.     

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.     

The influence of heterochromatin, inversion-heterozygosity and somatic pairing on x-ray induced mitotic recombination in Drosophila melanogaster

Summary Mitotic recombination has been induced with X-rays in Drosophila melanogaster larvae and assayed later as twin mosaic spots in the adult eyes. When the X-chromosomes are marked with zeste and white and the third chromosomes with roughoid and sepia, the frequency of twin spots was about 20 times higher for the X-chromosome than for the third chromosome. The greater amount of heterochromatin in the X-chromosome was considered responsible for the difference.Experiments with different inversion heterozygotes support this interpretation. Euchromatic inversions of different lengths have, when heterozygous, little or no influence on the twin spot frequency. The shorter the heterochromatic segment between the kinetochore and the proxomal break point of the inversion the stronger is the reduction of the twin spot frequency.The heterozygotes for the long sc ⁸ and sc ^S1 inversions gave exceptionally low twin spot frequencies. It seems possible that potential twin spot daughter cells die after recombination because of genetic imbalance and/or lack of proper cell separation resulting from the persistence of the dikinetic chromosome elements.To test whether inaccurate somatic pairing in inversion heterozygotes could help explain the low twin spot frequencies in those of sc ⁸ and sc ^S1, neuroblast chromosomes were investigated. They show that chromosomal arrangement during metaphase is determined exclusively by the location of the kinetochore, which always points, irrespective of earlier somatic pairing, toward the center of the metaphase plate. It is possible that there is a lack of proper chromosome alignment at the X-ray sensitive stage for mitotic recombination.     

The genotoxicity of nitrilotriacetic acid (NTA) in a somatic mutation and recombination test in Drosophila melanogaster

The genotoxicity of a chelating agent, the trisodium salt of nitrilotriacetic acid (NTA), was assessed in a somatic mutation and recombination test (SMART) in Drosophila melanogaster employing the wing hair markers mwh and flr3. The experiments were performed in parallel in two different laboratories (Padua, Italy and Schwerzenbach, Switzerland). The effectively absorbed doses of NTA, which was administered by feeding to larvae, were determined by a sensitive method employing [3H]leucine which allowed individual consumption levels to be measured. The particular pattern of clone induction produced by this compound suggests that NTA is active in inducing mitotic recombination and possibly aneuploidy in somatic cells of Drosophila. This is discussed in relation to the data present in the literature regarding the genotoxicity of NTA in a variety of experimental systems.     

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.     

The effect of inversions on mitotic recombination in Drosophila melanogaster.

Summary The effect of inversions on mitotic recombination outside the inversion was studied in inversion-heterozygotes. Seven euchromatic inversions of the X-chromosome, with breakpoints within the interval between two cell markers, were chosen. The size of the inverted region and the distance from the proximal breakpoint to the proximal cell marker varied. Mitotic recombination was X-ray induced in larvae and clones scored in the tergites of emerged adults. The frequency of recombinants between both cell markers and the frequency of recombinants proximal to the proximal cell marker was used to estimate the effect of interference in pairing caused by the inversions. Such an effect only occurs in small chromosome intervals. This indicates that homologous sequences are tightly paired in the interphase nuclei of somatic cells. This conclusion is derived from data based on X-ray induced mitotic recombination. The possibility of extending this conclusion to non-irradiated cells is discussed.     

Chiral ruthenium(II) anthraquinone complexes as dual inhibitors of topoisomerases I and II

Abstract  

DNA topoisomerases (I and II) have been one of the excellent targets in anticancer drug development. Here two chiral ruthenium(II) anthraquinone complexes, Δ- and Λ-[Ru(bpy)₂(ipad)]²⁺, where bpy is 2,2′-bipyridine and ipad is 2-(anthracene-9,10-dione-2-yl)imidazo[4,5-f][1,10]phenanthroline, were synthesized and characterized. As expected, both of the Ru(II) complexes intercalate into DNA base pairs and possess an obviously greater affinity with DNA. Topoisomerase inhibition and DNA strand passage assay confirmed that the two complexes are efficient dual inhibitors of topoisomerases I and II by interference with the DNA religation. In MTT cytotoxicity studies, two Ru(II) complexes exhibited antitumor activity against HeLa, MCF-7, HepG2 and BEL-7402 tumor cell lines. Flow cytometry analysis shows an increase in the percentage of cells with apoptotic morphological features in the sub-G1 phase for Ru(II) complexes. Nuclear chromatin cleavage has also been observed from AO/EB staining assay and alkaline single-cell gel electrophoresis (comet assay). The results demonstrated that Δ- and Λ-[Ru(bpy)₂(ipad)]²⁺ act as dual inhibitors of topoisomerases I and II, and cause DNA damage that can lead to cell cycle arrest and/or cell death by apoptosis.     

Mechanistic and methodological aspects of chemically-induced somatic mutation and recombination in Drosophila melanogaster

This study presents the analysis of chemically-induced somatic mutations and chromosomal damage in the eye imaginal discs of Drosophila larvae, assayed later as twin (TS) and single light (LS) mosaic spots in the adult eyes. Regarding the question as to what kind of DNA alterations contribute to somatic cell mutagenicity, the approach followed here has been to investigate the possible differences in response between male (hemizygous for an X) and female (homozygous) larvae, rod-X/rod-X versus ring-X/rod-X genotypes and inversion-heterozygotes versus genotypes not carrying an inversion. The systems chosen for this analysis were the white-coral/white (wco/w) and the white+/white (w+/w) eye mosaic system. The principle findings with 12 mutagens of different modes of action are as follows: (1) At least 98% of all TS and LS induced by cisplatin (DDP) in wco/w female larvae and about 95% of those by formaldehyde (FA) appear as the result of recombinogenic activity between the two homologous X-chromosomes. The corresponding estimates for MMS, EMS and ENU are 81%, 73% and 61%, respectively. (2) The long scS1L sc8R inversion, which also contains In(1)dl-49, suppresses induction of TS to 83-93%. There was also a sharp decline in the frequency of LS in inversion heterozygotes for DDP (91%), FA (86%), MMS (52%) and EMS (47%). (3) Ethylnitrosourea (ENU) was the mutagen for which introduction of the inverted chromosome reduced only slightly (23%) the frequency of LS, indicating that the majority of them were somatic mutations (and deletions) at the white locus. (4) In w/RX females heterozygous for a ring-X chromosome, the frequency of LS was only approximately one tenth of that of the control (w+/w) group, after exposure to MMS or DDP. The explanation is that exchange processes involving the ring frequently lead to genetic imbalance with subsequent cell killing.     

Modulatory effects of Tabebuia impetiginosa (Lamiales, Bignoniaceae) on doxorubicin-induced somatic mutation and recombination in Drosophila melanogaster

The wing Somatic Mutation and Recombination Test (SMART) in D. melanogaster was used to study genotoxicity of the medicinal plant Tabebuia impetiginosa. Lapachol (naphthoquinone) and β-lapachone (quinone) are the two main chemical constituents of T. impetiginosa. These compounds have several biological properties. They induce apoptosis by generating oxygen-reactive species, thereby inhibiting topoisomerases (I and II) or inducing other enzymes dependent on NAD(P)H:quinone oxidoreductase 1, thus affecting cell cycle checkpoints. The SMART was used in the standard (ST) version, which has normal levels of cytochrome P450 (CYP) enzymes, to check the direct action of this compound, and in the high bioactivation (HB) version, which has a high constitutive level of CYP enzymes, to check for indirect action in three different T. impetiginosa concentrations (10%, 20% or 40% w/w). It was observed that T. impetiginosa alone did not modify the spontaneous frequencies of mutant spots in either cross. The negative results observed prompted us to study this phytotherapeuticum in association with the reference mutagen doxorubicin (DXR). In co-treated series, T. impetiginosa was toxic in both crosses at higher concentration, whereas in the HB cross, it induced a considerable potentiating effect (from ~24.0 to ~95.0%) on DXR genotoxity. Therefore, further research is needed to determine the possible risks associated with the exposure of living organisms to this complex mixture.     

Frequency of x-ray induced mitotic recombination in genotypes with different X-Y-combination-chromosomes in Drosophila melanogaster

Summary In first and second instar larvae of Drosophila melanogaster mitotic recombination has been induced by X-rays. Resulting mosaic spots were analysed in the eyes of adult females. The five examined genotypes varied in the combination of different X-chromosomes and in the presence or absence of the duplication Dp(1;3)N ^264-58. X-chromosomes used have been elongated by proximally or distally linked arms of the Y-chromosome. The portion of Y-heterochromatin in the genome is negatively correlated with the frequency of twin mosaic spots (Fig. 1). The frequency of these spots is lower in flies bearing the duplication than in those without the duplication. With respect to the X-chromosome combinations, there are no marked differences in the frequencies of single mosaic spots (Fig. 2); with respect to the duplication they are absent. In the genotype Y ^S X/X.Y ^S prophases of neuroblast mitoses (Fig. 4) show normal pairing of homologous chromosome sections. Reasons for the different spot frequencies are discussed.

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Vorgelegt von E. Hadorn