The genotoxicities of N-nitrosamines in Drosophila melanogaster in vivo: the correlation of mutagenicity in the wing spot test with the DNA damages detected by the DNA-repair test

The genotoxicities of a series of N-nitrosamines were assayed in the wing spot test and a new short-term test of Drosophila melanogaster. In the spot test, larval flies trans-heterozygous for the somatic cell markers mwh and flr3 were fed the test reagents and the wing hairs in adults were inspected for clones expressing the phenotypes of the markers. In the other test, larval stock consisting of meiotic recombination-deficient (Rec-) double mutant mei-9a and mei-41D5 males and repair-proficient Rec+ females were grown on feed containing the reagents and the DNA damages were detected with the preferential killing of the Rec- larvae as an endpoint. The carcinogenic nitrosamines tested, N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), N-nitrosodi-n-butylamine (NDBA), N-nitrosomorpholine (NMOR), N-nitro-sopiperidine (NPIP) and N-nitrosopyrrolidine (NPYR), all showed clearly positive activities in both tests. The activities in the wing spot test were ranked in a sequence of NDMA much greater than NMOR greater than NPIP greater than NDEA greater than NPYR greater than NDBA. A similar ranking was obtained in the repair assay. The genotoxicity of N-nitrosodiphenylamine (NDPhA), carcinogenicity studies of which are inconclusive, was marginal in the spot test. The non-carcinogenic N-nitrosoproline (NPRO) and the non-mutagenic N-nitrosothioproline (NTPRO) were negative in the spot test. NDPhA and NPRO were negative in the repair test as well. The DNA-repair test is thus a convenient technique for estimating the mutagenicity of compounds because of its simplicity compared with the wing spot test. These Drosophila tests may be useful in predicting carcinogenic potentials of compounds.     

Genetic toxicity of six carcinogens and six non-carcinogens in the Drosophila wing spot test

Six rodent carcinogens, 5 of which are also human carcinogens, and 6 compounds recognized as non-carcinogens were tested for their genotoxic activity in the Drosophila melanogaster wing spot test. 72-h-old larvae trans-heterozygous for the recessive wing cell markers 'multiple wing hairs' (mwh) and 'flare' (flr3) were fed various concentrations of the test compounds for a period of 48 h. With amitrole and 4-aminobiphenyl, larvae of the same age were also given an acute treatment of 6 h with higher concentrations, and, in addition, 48-h-old larvae were fed for a longer period of 72 h. Repeats of all experiments document the good reproducibility of the results in the wing spot test. Amitrole and 4-aminobiphenyl were genotoxic after both 48-h and 72-h treatments, but their activity could not be detected following acute exposure of only 6 h. Chlorambucil and melphalan were clearly genotoxic. The carcinogens sodium arsenite and sodium arsenate, however, which are highly toxic to Drosophila, could only be tested at low exposure levels and were negative under these treatment conditions. The 6 non-carcinogens (ascorbic acid, 2-aminobiphenyl, mannitol, piperonyl butoxide, stannous chloride and titanium dioxide) were all definitely non-genotoxic in the Drosophila wing spot test. The data for the non-carcinogens demonstrate that non-genotoxic compounds can be identified in the wing spot test with a reasonable experimental effort.     

Genotoxic activity in vivo of the naturally occurring glucoside,cycasin, in the Drosophila wing spot test

Cycasin, methylazoxymethanol-β-glucoside, is a naturally occurring carcinogenic compound. The genotoxicity of cycasin was assayed in the Drosophila wing spot test. Cycasin induced small single and large single spots on feeding at 10 μmol/g medium. The presence of these spots indicates that cycasin is genotoxic in Drosophila melanogaster. Microorganisms which showed β-glucosidase activity for cleaving cycasin to toxic aglycon were isolated from gut flora of the Drosophila larvae. Consequently, the Drosophila wing spot test would be useful for mutagenicity screening of other naturally occurring glucosides.     

Genotoxicity of four herbicides in the Drosophila wing spot test.

The herbicides alachlor, atrazine, maleic hydrazide and paraquat were evaluated for genotoxicity in the Drosophila melanogaster wing spot test. Third-instar larvae trans-heterozygous for two recessive mutations of wing trichomes, multiple wing hairs (mwh) and flare (flr3), were treated by chronic feeding with different concentrations of the four herbicides. Feeding ended with pupation of the surviving larvae. The genotoxic effects were determined from the appearance of clones of cells with mwh, flr3 or mwh-flr3 phenotypes. Exposure to maleic hydrazide resulted in a significant increase in the frequency of the three categories of spots recorded (small single, large single and twin spots) in a dose-related fashion. Exposure to alachlor induced significant increases in both small and total spots at the four concentrations assayed and in the frequency of twin spots at the highest concentration tested (10 mM). Atrazine and paraquat also induced significant increases in both small and total spots at three of the four concentrations tested, without indication of a direct dose-effect relationship.     

Genotoxicity testing of five herbicides in the Drosophila wing spot test

Four triazine herbicides: amitrole, metribuzin, prometryn and terbutryn, and the bipyridal compound diquat dibromide have been evaluated for genotoxicity in the wing somatic mutation and recombination test of Drosophila melanogaster, following standard procedures. Third-instar larvae trans-heterozygous for the third chromosome recessive markers multiple wing hairs (mwh) and flare-3 (flr(3)) were chronically fed with different concentrations of the test compounds. Feeding ended with pupation of the surviving larvae. Genetic changes induced in somatic cells of the wing's imaginal discs lead to the formation of mutant clones on the wing blade. Point mutation, chromosome breakage and mitotic recombination produce single spots; while twin spots are produced only by mitotic recombination. Exposure to 0.5 mM and 1 mM of amitrole clearly increased the frequency of small single, large single and total spots. Terbutryn, at the concentration of 5 mM, induced a slight increase in the frequency of small single and total spots, but this result could be false positive. The other three herbicides tested did not show any genotoxic effect. When heterozygous larvae for mwh and the multiple inverted TM3 balancer chromosomes were treated, significant increases in the frequency of mutant spots were only detected for amitrole. The observed spot frequencies were lower than those found in mwh/flr(3)50%) of the total spot induction was due to mitotic recombination.     

Evaluation in Drosophila melanogaster of the mutagenic potential of furfural in the mei-9a test for chromosome loss in germ-line cells and the wing spot test for mutational activity in somatic cells.

The mutagenic potential of furfural was evaluated by means of the chromosome loss test in germ cells and the wing spot test in somatic cells of Drosophila melanogaster. The chromosome loss test was carried out employing repair-proficient as well as repair-deficient females. Males carried the compound Y chromosome, BSYy+. Two routes of administration were used: injection and feeding of adult males. Genetic damage was demonstrable after matings of treated males with females carrying the excision repair-deficient mutant mei-9a. The somatic mutation and recombination test was carried out treating 72-h transheterozygous mwh+/+flr3 larvae. Acute treatment of larvae was chosen as the method of exposure. Evidence indicates that furfural induces somatic damage as measured in the wing spot test.     

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.     

Evaluation of the genotoxicity of four herbicides in the wing spot test of Drosophila melanogaster using two different strains

In the present study, the herbicides bentazone, molinate, thiobencarb and trifluralin were evaluated for mutagenic and recombinagenic effects using the wing spot test of Drosophila melanogaster (somatic mutation and recombination test, SMART). Both standard (ST) and high-bioactivation (HB) fly crosses were used, the latter cross is characterised by a high sensitivity to promutagens and procarcinogens. Three-day-old larvae, transheterozygous for the multiple wing hairs (mwh, 3-0.3) and flare-3 (flr(3), 3-38.8) genes, were chronically fed with six different concentrations of each herbicide. Feeding ended with pupation of the surviving larvae and the genetic changes induced in somatic cells of the wing's imaginal discs lead to the formation of mutant clones on the wing blade. Point mutation, chromosome breakage and mitotic recombination produce single spots; while twin spots are produced only by mitotic recombination. Bentazone, usually considered as a non-mutagen, gave positive results in the wing spot test with the high-bioactivation cross. Molinate, about which information on mutagenic effects is inconclusive, gave positive responses in both the standard and the high-bioactivation crosses, while the other thiocarbamate, thiobencarb, gave positive results only in the standard cross and at the highest concentration tested (10 mM). Finally, trifluralin, one of the most widely studied herbicides for genotoxic effects, gave positive results in the wing spot test with both crosses. Apart from the interest of the results found in the genotoxic evaluation of the four selected herbicides, our results also contribute to extend the existing database on the Drosophila wing spot test, and corroborate the utility of the use of high-bioactivation strains for the genotoxic evaluation of xenobiotics.     

The inhibitory effect of the fungicides captan and captafol on eukaryotic topoisomerases in vitro and lack of recombinagenic activity in the wing spot test of Drosophila melanogaster

In studies on the mechanisms of mutagenic and carcinogenic action of captan and captafol-related chloroalkylthiocarboximide fungicides, two effects were tested: (i) the effect of both compounds on the activity of eukaryotic topoisomerases I and II in vitro, and (ii) their mutagenic and recombinagenic activity in the somatic mutation and recombination test (SMART) in wing cells of Drosophila melanogaster. Only captafol inhibited the activity of topoisomerase I (10-20% inhibition of activity in the range of 10-100microM). In contrast, both chemicals decreased the activity of topoisomerase II already at 1microM concentration (50 and 20% inhibition of activity by captafol and captan, respectively).Genotoxicity was tested in vivo by administrating both compounds by acute (3h) and chronic feeding (48h) of 3-day-old larvae. In acute feeding, captan and captafol demonstrated positive results only for small single and total spots in 10-100mM exposure concentration range. Both chemicals were inconclusive for large single spots, as well as for twin spots. In chronic treatment, captan showed positive results only for small single and total spots at 2.5 and 5mM concentrations. Captafol gave inconclusive results over all concentrations tested. The results of the acute treatment experiments which have been performed at very high doses (50% toxicity at higher doses) indicate very weak overall mutagenic activity of both test fungicides.     

Involvement of eddy currents in the mutagenicity of ELF magnetic fields

Possible carcinogenic and/or mutagenic activity of extremely low frequency magnetic fields was examined using somatic mutation and recombination test system of Drosophila melanogaster. An X-linked semi-dominant DNA repair defective mutation mei-41(D5) was introduced into the conventional mwh/flr test system to enhance mutant spot frequency. Virgin females of w mei-41(D5)/FM6; flr/TM6 were crossed with w mei-41(D5)/Y; mwh jv; spa(pol) males. The F(1) third instar larvae were exposed to a 50Hz, 20mT sinusoidal AC magnetic field for 24h. After moulting from pupal cases, their wings were examined under a bright field microscope to detect hair spots with mwh or flr mutant morphology. The exposure caused a statistically significant enhancement in somatic recombination spot frequency. Mutant spots arising due to chromosomal non-disjunction or terminal deletion also increased but the frequency of spots resulting from point mutation was not altered. The enhancement in the recombination spot frequency was suppressed to the control level when a culture medium without electrolytes was used during exposure. When larvae were exposed to a magnetic field in an annular dish, flies from the outer ring showed more mutant spots compared to those from the inner ring. These results suggest that the detected mutagenic activity was that of the induced eddy current, rather than that of the magnetic field itself.     

Evidence that chlorophyllin (CHLN) may behave as an inhibitor or a promoter of radiation-induced genetic damage in somatic cells of drosophila

Irradiation of 96h old Drosophila following a 24h pretreatment with 5% chlorophyllin (CHLN) was delayed 0-4 days. The antimutagenic effect of CHLN in somatic cells monitored by the wing spot test persisted for 3 days after completion of the pretreatment and appeared to terminate at a time corresponding to the cessation of mitotic divisions of wing anlagen cells. Within the same population of cells, CHLN demonstrated both an inhibitory effect as measured in mwh single spot classes, and contrarily, a promoting effect in the class of mwh/flr twin spots and to an extent in the class of large flr spots. The reason for the contrasting effects of CHLN remains to be determined.     

Genotoxicity of lapachol evaluated by wing spot test of Drosophila melanogaster

This study investigated the genotoxicity of Lapachol (LAP) evaluated by wing spot test of Drosophila melanogaster in the descendants from standard (ST) and high bioactivation (HB) crosses. This assay detects the loss of heterozygosity of marker genes expressed phenotypically on the fly's wings. Drosophila has extensive genetic homology to mammals, which makes it a suitable model organism for genotoxic investigations. Three-day-old larvae from ST crosses (females flr(3)/TM3, Bd(s) x males mwh/mwh), with basal levels of the cytochrome P450 and larvae of high metabolic bioactivity capacity (HB cross) (females ORR; flr(3)/TM3, Bd(s) x males mwh/mwh), were used. The results showed that LAP is a promutagen, exhibiting genotoxic activity in larvae from the HB cross. In other words, an increase in the frequency of spots is exclusive of individuals with a high level of the cytochrome P450. The results also indicate that recombinogenicity is the main genotoxic event induced by LAP.     

Genotoxicity of triasulfuron in the wing spot test of Drosophila melanogaster is modulated by winter wheat seedlings

Triasulfuron (TS) is a widely used sulfonylurea herbicide which inhibits the acetolactate synthase in broad-leaf weeds and in some wheat crop grasses (Triticum aestivum L.). Residues can be found in soil and superficial water with high toxicity to primary producers. In cereals, TS metabolism depends on cytochromes P450 (CYPs), the age of seedlings and the interaction with compounds. The genotoxicity of TS was demonstrated in the wing spot test of Drosophila melanogaster, an in vivo assay based on the loss of heterozygosity of the mwh and flr markers in the wing imaginal disk cells of larvae fed with chemical agents. Chronic treatments with analytical grade TS, commercial formulation TS (Amber) 75WG) (0.5mg/mL) and commercial formulation bentazon (Basagran) 480) (0.24mg/mL) were performed with three-day-old larvae of the standard (ST) and the high bioactivation (HB) crosses with regulated and high constitutive levels of CYPs, respectively. To demonstrate the effect of winter wheat metabolism on TS genotoxicity, T. aestivum L. seedlings were immersed for 4h in these herbicides, and aqueous extracts (AEs) of the roots were prepared to expose the larvae. TS and Amber 75WG produced similar genotoxic effects in both crosses. Wheat metabolism modulated the genotoxicity because the AEs yielded statistically significant lower spot frequencies in the HB cross than in the ST cross. Differences between the two crosses of the wing spot test in D. melanogaster must be related to CYPs levels. Basagran 480 was genotoxic only in the HB cross, and wheat metabolism did not modulate its genotoxicity.     

Evaluation of genotoxicity of the indenoisoquinoline analogues of fagaronine and nitidine in Drosophila melanogaster

The wing spot test of Drosophila melanogaster was done to evaluate the genotoxicity of the antitumor indenoisoquinoline analogues of nitidine chloride and fagaronien chloride in larvae. Both compounds have toxic effects but no statistically significant increase in the frequency of spots was detected with the analogue of nitidine chloride. This strongly suggests that this compounds is not mutagenic to Drosophila larvae at the concentrations tested. Results with the analogue of fagaronine chloride were ambiguous. Low mutagenicity was detected in only one of two experiments and in the pooled results at 2 mM but not at 5 mM or 10 mM. These results suggest at best a very weak genotoxic effect but its biological significance needs confirmation by results from other assays.     

The genotoxicity of the anti-cancer drug mitoxantrone in somatic and germ cells of Drosophila melanogaster.

The novel antineoplastic drug mitoxantrone was studied for its genotoxic effects in Drosophila melanogaster. In male germ cells, the clinical preparation Novantrone, the dihydrochloride salt of mitoxantrone, did not induce sex-linked recessive lethal mutations in feeding and injection experiments with adult flies, although statistically the results were inconclusive rather than truly negative. However, the free base mitoxantrone was weakly, but significantly genotoxic in this test (0.14% lethals/mM exposure concentration); this is most probably the result of prolonged exposure. On the other hand, both forms of mitoxantrone assayed were clearly genotoxic in the somatic mutation and recombination test of the wing. This test assays the cells of the proliferating imaginal wing discs of larvae. Depending on the feeding method used, the overall clone induction frequency was in the range of about 2-6 x 10(-5) per cell and cell generation and per mM exposure dose. Correction of these frequencies according to mean clone size led to slightly higher estimates (by about 5-25% higher). Although the majority of the clone induction events are due to mitotic recombination, a significant proportion can be attributed to mutational events (gene and chromosome mutations). The genotoxicity of mitoxantrone seems to depend mainly on impaired DNA synthesis in cycling cells owing to the compound's ability to inhibit topoisomerase II by intercalation into DNA.     

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.     

Thirty compounds tested in the Drosophila wing spot test

The Drosophila wing somatic mutation and recombination test (SMART) was evaluated for its suitability in genotoxicity screening by testing 30 chemicals. Of the 2 crosses used, the mwh-flr3 cross turned out to be more convenient than the previously used mwh-flr cross. Based on the experience gained with both acute exposures and chronic exposures of different duration, we suggest that the optimal strategy in genotoxicity screening is to start with chronic exposure of 3-day-old larvae for 48 h (that is, until pupation). Only for unstable compounds and very volatile compounds and gases are acute treatments, including inhalation, recommended. In general, a qualitative evaluation of the genotoxicity of a compound in the wing assay is possible with as few as 1-2 different exposure concentrations. A more quantitative evaluation of genotoxicity, based upon dose-response data, can often be achieved with as few as 3-4 concentrations. The results reported here were obtained in 2 different laboratories, demonstrating that the wing spot test is easily transferable to other laboratories. The experience gained indicates that the assay has now been developed to an extent that a coordinated international comparative validation study is desirable.     

Evidence that chlorophyllin (CHLN) may behave as an inhibitor or a promoter of radiation-induced genetic damage in somatic cells of Drosophila

Irradiation of 96 h old Drosophila following a 24 h pretreatment with 5% chlorophyllin (CHLN) was delayed 0–4 days. The antimutagenic effect of CHLN in somatic cells monitored by the wing spot test persisted for 3 days after completion of the pretreatment and appeared to terminate at a time corresponding to the cessation of mitotic divisions of wing anlagen cells. Within the same population of cells, CHLN demonstrated both an inhibitory effect as measured in mwh single spot classes, and contrarily, a promoting effect in the class of mwh/flr twin spots and to an extent in the class of large flr spots. The reason for the contrasting effects of CHLN remains to be determined.     

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).     

Genotoxicity of neutrons in Drosophila melanogaster. Somatic mutation and recombination induced by reactor neutrons

This paper describes the observation of a direct relationship between the absorbed doses of neutrons and the frequencies of somatic mutation and recombination using the wing somatic mutation and recombination test (SMART) of Drosophila melanogaster. This test was used for evaluating the biological effects induced by neutrons from the Triga Mark III reactor of Mexico. Two different reactor power levels were used, 300 and 1000 kW, and two absorbed doses were tested for each power level: 1.6 and 3.2 Gy for 300 kW and 0.84 and 1.7 Gy for 1000 kW. A linear relationship was observed between the absorbed dose and the somatic mutation and recombination frequencies. Furthermore, these frequencies were dependent on larval age: In 96-h-old larvae, the frequencies were increased considerably but the sizes of the spots were smaller than in 72-h-old larvae. The analysis of the balancer-heterozygous progeny showed a linear absorbed dose- response relationship, although the responses were clearly lower than found in the marker-trans-heterozygous flies. Approximately 65% of the genotoxicity observed is due to recombinational events. The results of the study indicate that thermal and fast neutrons are both mutagenic and recombinagenic in the D. melanogaster wing SMART, and that the frequencies are dependent on neutron dose, reactor power, and the age of the treated larvae.