Genotoxicity modulation by cadmium treatment: studies in the Drosophila wing spot test

The genotoxic activity of cadmium chloride (CC) has been evaluated in the somatic mutation and recombination test (SMART) in Drosophila melanogaster. In addition, its possible modulating effect on the genotoxicity of two known mutagenic agents, potassium dichromate (PDC) and ethyl methanesulfonate (EMS), was investigated. Three different types of combined treatments of CC with the two genotoxins were performed: pretreatment, cotreatment, and posttreatment. The SMART assay is based on the principle that loss of heterozygosity for the recessive markers, multiple wing hairs (mwh) and flare-3 (flr(3)), leads to the formation of mutant clones in the imaginal disks of larvae, which are expressed as mutant spots on the wings of adult flies. Thus, after adult emergence, the wings of the adult flies were scored for the presence of single and/or twin spots. Our results show that CC alone was not effective in increasing the frequency of any of the three categories of spots (small, large, and twin). In the cotreatment experiments, CC increased the genotoxicity of PDC but it decreased the genotoxicity of EMS. No effects of CC were observed in the pretreatment or posttreatment experiments; however, only low concentrations of CC, PDC, and EMS were tested in the pretreatment assays due to the high toxicity of the treatment. Although our results with PDC are consistent with the hypothesis that cadmium can interfere with repair mechanisms, the EMS data suggest that other modulating mechanisms are also involved in the genotoxicity of this metal.     

Genotoxicity of vegetable cooking oils in the Drosophila wing spot test

Seven vegetable oils consumed by humans were tested for genotoxic activity in the Drosophila somatic mutation and recombination test. The oils included five seed oils (sesame, sunflower, wheat germ, flax, and soy oil) and both first-class extra-virgin and low-grade (refined) olive oil. Larvae of the standard (STD) and highly bioactive (NORR) crosses of Drosophila melanogaster were fed medium containing 6% and 12% of each of the oils. Flax oil produced the strongest response, while sesame, wheat germ, and soy oil showed some genotoxic activity. Sunflower and the low-grade olive oil gave inconclusive results, and extra-virgin olive oil was clearly nongenotoxic. It is argued that the genotoxicity is probably due to the fatty acid composition of the oils, which after peroxidation can form specific DNA-adducts.     

The genotoxicity of N4-aminocytidine in the Drosophila wing spot test

The nucleoside analogue N⁴-aminocytidine is known to inducemutations in bacteria, and was shown to induce somatic mutationsin Drosophila melanogaster after larval administration. Theassay system employed was a wing-hair mutation spot test developedby Würgler and co-workers. The potency of N⁴-aminocytidineto induce somatic mutation is comparable to those of severalfood-pyrolysate mutagens previously reported. The occurrenceof twin spots, i.e. two types of recessive mutant-hair clonesin adjacent positions, suggests that N⁴-aminocytidine inducessomatic recombination in Drosophila. Another feature of themutagenicity of N⁴-aminocytidine is that both the acute andthe chronic larval feedings gave rise to mutant hair formationof similar patterns with respect to the spot-size distributions:small single spots were formed predominantly and the largerthe spotsize, the lower their frequency.     

Genotoxicity studies on the phenoxyacetates 2,4-D and 4-CPA in the Drosophila wing spot test.

The phenoxyacetates 2,4-D and 4-CPA were evaluated for genotoxicity using the Drosophila melanogaster wing spot test, which assesses for somatic mutation and recombination events. Third-instar larvae trans-heterozygous for two recessive mutations affecting the expression of wing trichomes, multiple wing hairs (mwh), and flare (flr) were treated by chronic feeding with different concentrations of the two chemicals. Feeding lasted until pupation of the surviving larvae and the genotoxic effects induced were evaluated in adults for the appearance of wing-blade cell clones with the mwh, flr, or mwh-flr phenotypes. Exposure to 2,4-D, at the highest concentration evaluated (10 mM), induced a weak but significant increase in the frequency of two of the categories of recorded spots: large single and total spots; in contrast, the 4-CPA treatments failed to induce any significant increase in the frequency of evaluated spots. When the heterozygous larvae for mwh and the multiple inverted TM3 balancer chromosome were treated with the chemicals, no increases were detected, either after the 2,4-D nor the 4-CPA treatments.     

Antigenotoxic effects of Citrus aurentium L. fruit peel oil on mutagenicity of two alkylating agents and two metals in the Drosophila wing spot test

Antigenotoxic effects of Citrus aurentium L. (Rutaceae) fruit peel oil (CPO) in combination with mutagenic metals and alkylating agents were studied using the wing spot test of D. melanogaster. The four reference mutagens, potassium dichromate (K₂Cr₂O₇), cobalt chloride (CoCl₂), ethylmethanesulfonate (EMS), and N‐ethyl‐N‐nitrosourea (ENU) were clearly genotoxic. CPO alone at doses from 0.1 to 0.5% in Tween 80 was not mutagenic and did not enhance the mutagenic effect of the reference mutagens. However, antigenotoxic effects of CPO were clearly demonstrated in chronic cotreatments with mutagens and oil, by a significant decrease in wing spots induced by all four mutagens. The D. melanogaster wing spot test was found to be a suitable assay for detecting antigenotoxic effects in vivo. Environ. Mol. Mutagen., 2009. © 2009 Wiley‐Liss, Inc.     

Use of the Drosophila wing spot test in the genotoxicity testing of different herbicides

Four herbicides, namely propanil, maleic hydrazide, glyphosate, and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), were investigated for genotoxicity in the wing spot test of Drosophila melanogaster. The herbicides were administered by chronic feeding to 3-day-old larvae. Two different crosses, a standard (ST) and a high-bioactivation (HB) cross, involving the flare-3 (flr(3)) and the multiple wing hairs (mwh) markers, were used. The HB cross uses flies characterized by an increased cytochrome P-450-dependent bioactivation capacity, which permits a more efficient biotransformation of promutagens and procarcinogens. In both crosses, the wings of the two types of progeny, which are inversion-free marker heterozygotes and balancer heterozygotes, were analyzed. Maleic hydrazide and glyphosate proved to be more genotoxic in the ST cross, whereas propanil appeared to be slightly more genotoxic in the HB cross. On the other hand, the herbicide 2,4,5-T increased the mutation frequency for only the small single spots in the ST cross.     

Metabolism of arsenic in Drosophila melanogaster and the genotoxicity of dimethylarsinic acid in the Drosophila wing spot test

Inorganic arsenic is nongenotoxic in the Drosophila melanogaster wing somatic mutation and recombination test (SMART). Recent evidence in mammalian systems indicates that methylated metabolites of arsenic are more genotoxic than inorganic arsenic. Thus, we hypothesized that inorganic arsenic is nongenotoxic in Drosophila because they are unable to biotransform arsenic to methylated forms. In the present study, we fed trivalent and pentavalent inorganic arsenic to Drosophila larvae and adults and measured the production of methylated derivatives. No biomethylated arsenic species were found in the organisms or in the growth medium, which suggests that Drosophila are unable to biomethylate inorganic arsenic. Exposure of Drosophila to the methylated arsenic derivative dimethylarsinic acid (DMA(V)) resulted in incorporation of this organoarsenic compound without demethylation. In addition, we used the SMART wing spot assay, which measures loss of heterozygosity (LOH) resulting from gene mutation, chromosomal rearrangement, chromosome breakage, and chromosome loss, to evaluate the genotoxicity of DMA. DMA by itself induced significant increases in the frequency of total spots, small spots, and large single spots. These results are consistent with the important role of arsenic biomethylation as a determinant of the genotoxicity of arsenic compounds. The absence of biomethylation in Drosophila could explain the lack of genotoxicity for inorganic arsenic and the genotoxicity of methylated arsenic species in the SMART wing spot assay.     

Influence of sodium arsenite on the genotoxicity of potassium dichromate and ethyl methanesulfonate: studies with the wing spot test in Drosophila

The wing spot test in Drosophila melanogaster was used to investigate the genotoxicity of arsenic and its effects on the action of two clearly genotoxic agents: potassium dichromate (PDC) and ethyl methanesulfonate (EMS). This assay is based on the principle that the loss of heterozygosity of the suitable recessive markers multiple wing hairs (mwh) and flare-3 (flr(3)) can lead to the formation of mutant clones of larval cells, which are then expressed as spots on the wings of adult flies. These spots can be attributed to different genotoxic events: either mitotic recombination or mutation (deletion, point mutation, and specific types of translocation). Pretreatments and chronic cotreatments were comparatively used for combined treatments. From the results obtained it is evident that sodium arsenite (SA) does not increase the frequency of any of the three categories of spots recorded (small, large, and twin spots) at the concentrations tested. The effects of SA in combination with PDC, in both cotreatments and pretreatments, indicate that SA almost suppressed the clones induced by PDC. Nevertheless, no effects of arsenic were observed with respect to the pre- and cotreatments with EMS. Thus, SA does not modify the frequencies of mutant clones induced by EMS.     

Assessing the impact of pollution on the Japaratuba river in Brazil using the Drosophila wing spot test

The Drosophila melanogaster somatic mutation and recombination test (SMART) was used to assess the genotoxicity of surface (S) and bottom (B) water and sediment samples collected from Sites 1 and 2 on the Japaratuba River (Sergipe, Brazil), an area impacted by a petrochemical industrial complex that indirectly discharges treated effluent (produced water) into the river. The genotoxicity tests were performed in standard (ST) cross and high bioactivation (HB) cross flies and were conducted on samples taken in March (dry season) and in July (rainy season) of 2003. Mutant spot frequencies found in treatments with unprocessed water and sediment samples from the test sites were compared with the frequencies observed for similar samples taken from a clean reference site (the Jacarecica River in Sergipe, Brazil) and those of negative (ultrapure water) controls. While samples from the Japaratuba River generally produced greater responses than those from the Jacarecica River, positive responses were detected for both the test and reference site samples. All the water samples collected in March 2003 were genotoxic. In July 2003, the positive responses were restricted to water samples collected from Sites 1 B and 2 S in the ST cross. The genotoxicity of the water samples was due to mitotic recombination, and the samples produced similar genotoxic responses in ST and HB flies. The spot frequencies found in the July water samples were considerably lower than those for the March water samples, suggesting a seasonal effect. The only sediment samples that were genotoxic were from Site 1 (March and July) and from the Jacarecica River (March). The genotoxins in these samples produced both somatic mutation (limited to the Site 1 sample in HB flies) and recombination. The results of this study indicate that samples from both the Japaratuba and Jacarecica Rivers were genotoxic, with the most consistently positive responses detected with Site 1 samples, the site closest to the putative pollution source.     

Genotoxicity of the anti-juvenile hormone agent precocene II as revealed by the Drosophila wing spot test

The anti-juvenile hormone agent, precocene II, designated as a prototype of potential fourth-generation insecticides, was subjected to genotoxicity screening by means of the somatic mutation and recombination test in Drosophila melanogaster. Larvae heterozygous for recessive wing trichome mutations, mwh and flr3, were exposed to sublethal concentrations of precocene II, and wings of emerged adult females were inspected for the presence of phenotypically mutant mosaic spots. The compound significantly increased the frequency of mosaic spots in mwh/flr3 wings, but revealed only a slight effect in mwh/TM2 wings. The results suggest that the main sources of genotoxic activity of precocene II are due to chromosome-breakage phenomena resulting from mitotic recombination. The possible mechanism of this effect is discussed.     

Genotoxicity of tamoxifen citrate and 4-nitroquinoline-1-oxide in the wing spot test of Drosophila melanogaster

Tamoxifen (TAM) is an anti-oestrogen used for treatment and prevention of human breast cancer, but it is also related to human endometrial and uterine cancer. The wing spot test in Drosophila melanogaster was employed to determine the genotoxic effects of TAM and 4-nitroquinoline-1-oxide (4-NQO), a carcinogen that produces adducts similar to TAM-DNA adducts detected in rodent liver and human liver microsomes. As Drosophila spp. have no oestrogen receptor, no effects can result in binding of TAM to a receptor. Chronic treatments with TAM citrate were performed with 3-day-old larvae of the standard (ST) and high bioactivation (HB) crosses of the wing spot test at concentrations of 0.66, 1.66 and 3.33 mM. In addition, the carcinogen 4-NQO was administered at 2.5 and 5.0 mM. Somatic spots on normal wings from marker-heterozygous flies and on serrate wings from balancer-heterozygous flies were scored to determine mutation and recombination events in somatic cells for each compound. The results showed genotoxic effects of TAM at 1.66 and 3.33 mM in the ST cross only and without a clear dose-response effect. This suggests a weak genotoxicity of this anti-oestrogen. The negative results obtained with TAM in the HB cross may indicate efficient detoxification of the compound by the increased xenobiotic metabolism present in this cross. As reported before, 4-NQO showed genotoxic effects in the ST cross with a clear dose-response effect. For the first time, we report enhanced effects of this compound in the HB cross. It is concluded that the genotoxicity of TAM in the Drosophila wing spot test is different from that of 4-NQO.     

Recombinagenic activity of four compounds in the standard and high bioactivation crosses of Drosophila melanogaster in the wing spot test

The wing somatic mutation and recombination test (SMART) using Drosophila melanogaster was employed to determine the recombinagenic and mutagenic activity of four chemicals in an in vivo eukaryotic system. Two different crosses involving the wing cell markers mwh and flr(3) were used: the standard cross and a high bioactivation cross. The high bioactivation cross is characterized by a high constitutive level of cytochromes P450 which leads to an increased sensitivity to a number of promutagens and procarcinogens. Three-day-old larvae derived from both crosses were treated chronically with the oxidizing agent potassium chromate and with the three procarcinogens cyclophosphamide, p-dimethylaminoazobenzene and 9,10-dimethylanthracene. From both crosses two types of progeny were obtained: marker-heterozygous and balancer-heterozygous. The wings of both genotypes were analysed for the occurrence of single and twin spots expressing the mwh and/or flr(3) mutant phenotypes. In the marker-heterozygous genotype the spots can be due either to mitotic recombination or to mutation. In contrast, in the balancer-heterozygous genotype only mutational events lead to spot formation, all recombination events being eliminated. The oxidizing agent potassium chromate was equally and highly genotoxic in both crosses. Surprisingly, the promutagen cyclophosphamide also showed equal genotoxicity in both crosses, whereas p-dimethylaminoazobenzene was negative in the standard cross, but clearly genotoxic in the high bioactivation cross. 9,10-Dimethylanthracene showed a rather weak genotoxicity in the high bioactivation cross. Analyses of the dose-response relationships for mwh clones recorded in the two wing genotypes demonstrated that all four compounds are recombinagenic. The fraction of all genotoxic events which are due to mitotic recombination ranged from 83% (9,10-dimethylanthracene) to 99% (p-dimethylaminoazobenzene). These results demonstrate that the wing spot test in Drosophila is most suited to the detection of recombinagenic activity of genotoxic chemicals.     

Genotoxic activity of different chromium compounds in larval cells of Drosophila melanogaster, as measured in the wing spot test.

Two chromium(VI) compounds (potassium chromate and potassium dichromate) and one chromium(III) compound, chromium chloride, were evaluated for genotoxic effects in the wing spot test of Drosophila melanogaster following standard procedures. This assay detects both somatic recombination and mutational events. The genotoxic effects were determined from the appearance of wing spots in flies transheterozygous for the third chromosome recessive markers multiple wing hairs (mwh) and flare-3 (flr(3)), as well as in flies heterozygous formwh and the multiply inverted TM3 balancer chromosome. Genetic changes induced in somatic cells of the wing's imaginal discs lead to the formation of mutant clones on the wingblade. Single spots are due to different genotoxic mechanisms: point mutation, deletion, chromosome breakage, and mitotic recombination; while twin spots are produced only by mitotic recombination. From our results it appears that both chromium(VI) compounds clearly increase the incidence of mutant clones by inducing high increases in the frequency of all types of clones recorded. On the contrary, chromium(III) did not increase the frequency of mutant clones. A high proportion of the total spot induction was due to mitotic recombination, confirming previously reported data on the strong recombinogenic activity of chromium(VI) compounds.     

Induction of somatic mutation and recombination by four inhibitors of eukaryotic topoisomerases assayed in the wing spot test of Drosophila melanogaster

Four inhibitors of eukaryotic topoisomerases were investigatedfor genotoxic effects in the wing spot test of Drosophila melanogaster.As a somatic mutation and recombination test (SMART) this assayassesses mitotic recombination and mutational events of variouskinds. We studied camptothecin as a topoisomerase I inhibitor,as well as ellipticine as an intercalating inhibitor and teniposideand etoposide as two non-intercalating inhibitors of topoisomeraseII. Wing spots were induced in flies trans-heterozygous forthe recessive wing cell markers multiple wing hairs (mwh) andflare (flr³) as well as in flies heterozygous for mwh and themultiply inverted TM3 balancer chromosome. All four compoundsproved significantly genotoxic in this test The spot inductionfrequencies formally standardized to the millimolar unit ofexposure dose decreased in the order camptothecin > teniposide> ellipticine     

The somatic white-ivory eye spot test does not detect the same spectrum of genotoxic events as the wing somatic mutation and recombination test in Drosophila melanogaster

A group of six chemical compounds was tested in parallel in two different somatic genotoxicity assays in Drosophila melanogaster, the wing somatic mutation and recombination test (SMART) and the white-ivory eye spot test. The wing spot test makes use of the wing cell markers multiple wing hairs (mwh) and flare (flr) and detects both mitotic recombination and various types of mutational events. The white-ivory eye spot test makes use of the white-ivory (w′) quadruplication and detects the somatic reversion of the recessive eye color mutation w′ to the wild-type (w⁺). Three- or two-day-old larvae were fed chronically with the compounds ethylnitrosourea (ENU), N-nitrosopyrrolidine (NNP), caffeine (CAF), chromium (VI) oxide (CRO), potassium chromate (POC), and 2,4-dichlorophenoxyacetic acid (2,4-D). All six compounds are genotoxic to various degrees in the wing spot test. The percentage of the genotoxic activity that is due to mitotic recombination was between 84% and 91% for the hexavalent chromium compounds CRO and POC and about 68% for 2,4-D. In contrast, ENU and NNP showed only 46% and 25% recombinagenic activity, respectively. In the white-ivory eye spot test, the three compounds [CRO, POC, and 2,4-D] with high recombinagenic activity and CAF were clearly nongenotoxic, whereas only ENU and NNP gave a positive response. From these results, it is concluded that the spectrum of genotoxic events detected by the two assays is different. In particular, the white-ivory eye spot test appears not to detect mitotic recombination the way the wing spot test does. © 1996 Wiley-Liss, Inc.     

Genotoxicity testing of antiparasitic nitrofurans in the Drosophila wing somatic mutation and recombination test

Nifurtimox and eight structurally related 5-nitrofuran compounds active against Trypanosoma cruzi were tested for genotoxicity in the wing somatic mutation and recombination test in Drosophila melanogaster. Nifurtimox, compound ada and compound 1B were clearly mutagenic and recombinogenic whereas the remaining six compounds were negative. In contrast to the situation in bacterial mutagenicity tests, nitroreductase activity is probably not decisive for the genotoxicity of these compounds in Drosophila. The three non-genotoxic nitrofurans with high antiparasitic activity are promising candidates for the replacement of nifurtimox. However, these compounds require further genotoxicity testing in eukaryotic assay systems for a final evaluation.     

Antigenotoxic properties of Terminalia arjuna bark extracts.

Compounds possessing antimutagenic properties (polyphenols, tannins, vitamins, etc.) have been identified in fruits, vegetables, spices, and medicinal plants. Terminalia arjuna (Combretaceae), a tropical woody tree occurring throughout India and known locally as Kumbuk, is a medicinal plant rich in tannins and triterpenes that is used extensively in Ayurvedic medicine as a cardiac tonic. The aim of the present collaborative work was to test six solvent extracts from the bark of Terminalia arjuna for antigenotoxic activity using in vitro short-term tests. Terminalia arjuna extracts were obtained by sequential extraction using acetone, methanol, methanol + HCl, chloroform, ethyl acetate, and ethyl ether. The antigenotoxic properties of these extracts were investigated by assessing the inhibition of genotoxicity of the directacting mutagen 4-nitroquinoline-N-oxide (4NQO) using the "comet" assay and the micronucleus (MN) test. Human peripheral blood leukocytes were incubated with different concentrations of the six extracts (from 5 to 100 microg/ mL) and with 4NQO (1 and 2 microg/mL, for the "comet" assay and MN test, respectively). Each extract/4NQO combination was tested twice; in each experiment, positive control (4NQO alone) and negative control (1% DMSO) were set. "Comet" assay results showed that acetone and methanol extracts were highly effective in reducing the DNA damage caused by 4NQO, whereas the acidic methanol, chloroform, ethyl acetate, and ethyl ether extracts showed less marked or no antigenotoxic activity. In the MN test, a decrease in 4NQO genotoxicity was observed by testing this mutagen in the presence of acetone, methanol, chloroform, and ethyl acetate extracts, even though the extent of inhibition was not always statistically significant.     

On the use of excision repair defective cells in the wing somatic mutation and recombination test in Drosophila melanogaster

Ten chemical mutagens were tested in the wing somatic mutation and recombination test in Drosophila melanogaster. This assay makes use of genetic markers expressed on the wing of adult flies. Larvae which are trans-heterozygous for mwh (multiple wing hairs) and flr (flare) were fed with the compounds either acutely (2, 4, or 6 hr) or chronically (48 or 72 hr), or were treated by inhalation (1 hr). Genetic changes induced in the somatic cells of the wing imaginal discs lead to the formation of mutant clones on the wing (mwh and/or flr). Single spots are produced by point mutation, chromosome breakage, and mitotic recombination; twin spots are produced exclusively by mitotic recombination. All 10 mutagens belonging to different chemical classes were clearly positive in this assay. However, the choice of the optimal treatment conditions (concentration of compound, duration of treatment, age of larvae at treatment) is essential. Eight of the compounds were also tested in excision repair defective cells by introducing the mei-9L1 mutation into the test system. This seems not to improve the detection capacity of the assay, mainly because only small spots are found in excision repair defective wings. In addition, the frequencies of spots in these wings are enhanced four to five times, which makes the scoring more tedious. For these and other practical reasons the use of this specific cross is not recommended in the wing spot test for routine screening purposes.     

Over-expression of DREF in the Drosophila wing imaginal disc induces apoptosis and a notching wing phenotype

Background DNA replication‐related element binding factor (DREF) has been suggested to be involved in regulation of DNA replication‐ and proliferation‐related genes in Drosophila. While the effects on the mutation in the DNA replication‐related element (DRE) in cultured cells have been studied extensively, the consequences of elevating wild‐type DREF activity in developing tissues have hitherto remained unclear. Results We over‐expressed DREF in the wing imaginal disc using a GAL4‐UAS targeted expression system in Drosophila. Over‐expression of DREF induced a notching wing phenotype, which was associated with ectopic apoptosis. A half reduction of the reaper, head involution defective and grim gene dose suppressed this DREF‐induced notching wing phenotype. Furthermore, this was also the case with co‐expression of baculovirus P35, a caspase inhibitor. In addition, over‐expression of the 32 kDa boundary element‐associated factor (BEAF‐32), thought to compete against DREF for common binding sites in genomic regions, rescued the DREF‐induced notching wing phenotype, while a half reduction of the genomic region, including the BEAF‐32 gene, exerted enhancing effects. To our knowledge, this is the first evidence for a genetic interaction between DREF and BEAF‐32. Conclusion The DREF‐induced notching wing phenotype is caused by induction of apoptosis in the Drosophila wing imaginal disc.