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.     

Genotoxic evaluation of bupivacaine and levobupivacaine in the Drosophila wing spot test

Bupivacaine and levobupivacaine are amino amide local anesthetics commonly used in medical practice. Although bupivacaine consists of a racemic mixture of S (–)-bupivacaine and R (+)-bupivacaine enantiomers, levobupivacaine is comprised of pure S (–)-bupivacaine. It has been known that levobupivacaine is preferable to bupivacaine since it may cause cardiovascular and nervous system toxicity. For determining genotoxicity of these anesthetics, we used the wing somatic mutation and recombination test in Drosophilamelanogaster. Three-day-old trans-heterozygous larvae were treated with bupivacaine and levobupivacaine. Analysis of the standard crosses indicated that bupivacaine and levobupivacaine did not exhibit mutagenic or recombinogenic activity until toxic doses have been reached at the larval stage. When we examined bupivacaine and levobupivacaine in the HB cross, bupivacaine did not exhibit any genotoxicity at high concentrations (500 µg/mL), but levobupivacaine did exert genotoxicity at high concentrations (1000 µg/mL)—depending on the substantial recombinogenic effect.     

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.     

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.     

Genotoxicity testing of six insecticides in two crosses of the Drosophila wing spot test

Among the great variety of genotoxicity assays available, the wing spot test in Drosophila melanogaster has some characteristics that make it very suited for the screening of genotoxic activity, i.e., it is an easy and inexpensive assay using a eukaryotic organism in vivo. One of the most interesting characteristics of the assay is its capacity to detect genotoxic activity of promutagens without the necessity of an exogenous metabolic activation system. In this paper we present results obtained with a recently developed high bioactivation cross of the wing spot test (NORR cross). The positive results obtained with the five well-known procarcinogens 7, 12-dimethylbenz[a]anthracene, N-nitrosopyrrolidine, p-dimethylaminoazobenzene, diethylnitrosamine and urethane clearly show that the NORR strains are similar to the other high bioactivation strains previously described, but they lack their methodological disadvantages. We have tested six insecticides, which are characterised by having contradictory results in other genotoxicity tests, using both the standard and the high bioactivation (NORR) cross. The six insecticides analysed are the pyrethroid allethrin, the methylenedioxyphenolic compound piperonyl butoxide, the chlorinated hydrocarbons dieldrin and endrin, and the organophosphates dimethoate and malathion. We obtained negative results for all six compounds. Our results show the suitability of the wing spot test for the evaluation of compounds at the first level of genotoxicity testing.     

Antimutagenic profile of three antioxidants in the Ames assay and the Drosophila wing spot test

Antimutagens and anticarcinogens are known to play an important role in combating the action of factors involved in the etiology of cancer. It is expected that inhibitors of mutagenesis also act as inhibitors of carcinogenesis. In the present study, two short-term genotoxicity assays, namely the Ames assay and the Drosophila wing spot test, have been selected for examining the antimutagenic potential of three antioxidants. For this purpose, a promutagen aflatoxin B(1) (AFB(1)) was chosen as a positive mutagen against which antimutagenic potential of alpha-tocopherol (Vit. E), caffeic acid (CA) and glutathione (GSH) was assessed. Vit. E did not exert any antimutagenic response while CA and GSH were effective in reducing the mutational events induced by AFB(1).     

Genotoxicity of ethyl carbamate in the Drosophila wing spot test: dependence on genotype-controlled metabolic capacity

Ethylcarbamate has a clear genotoxic potential in Drosophila melanogaster. In the somatic mutation and recombination test (SMART) it induces, in a dose-dependent manner, single spots as well as twin spots. The twin spots indicate a recombinogenic activity of ethyl carbamate. A strong strain difference demonstrates that the metabolic activation of ethyl carbamate to DNA binding metabolites most probably also in Drosophila involves cytochrome P450-dependent enzyme activities.     

Genotoxic effects of eugenol, isoeugenol and safrole in the wing spot test of Drosophila melanogaster

In the present study, the phenolic compounds eugenol, isoeugenol and safrole were investigated for genotoxicity in the wing spot test of Drosophila melanogaster. The Drosophila wing somatic mutation and recombination test (SMART) provides a rapid means to evaluate agents able to induce gene mutations and chromosome aberrations, as well as rearrangements related to mitotic recombination. We applied the SMART in its standard version with normal bioactivation and in its variant with increased cytochrome P450-dependent biotransformation capacity. Eugenol and safrole produced a positive recombinagenic response only in the improved assay, which was related to a high CYP450-dependent activation capacity. This suggests, as previously reported, the involvement of this family of enzymes in the activation of eugenol and safrole rather than in its detoxification. On the contrary, isoeugenol was clearly non-genotoxic at the same millimolar concentrations as used for eugenol in both the crosses. The responsiveness of SMART assays to recombinagenic compounds, as well as the reactive metabolites from eugenol and safrole were considered responsible for the genotoxicity observed.     

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.     

High recombinagenic activities of three antiviral agents, adenine derivatives, in the Drosophila wing spot test

Three adenine derivatives (R,S)-9-(2,3-dihydroxypropyl)adenin (DHPA), D-eritadine (EA), and 9-(2-phosphonylmethoxyethyl)adenine (PMEA), prospective antiviral drugs, were subjected to genotoxicity analysis using the somatic mutation and recombinatino test in Drosophila melanogaster. All three compounds were found to be very potent inducers of mosaic spots on Drosophila wings in a dose-related fashion. Data obtained in inversion-free flies revealed that the compounds, in particualr DHPA and EA (nucleoside analogues), are highly effective in the induction of mitotic recombination. PMEA, a nucleotide, exhibited a rather different genotoxic profile from those of DHPA and EA, indicating a different mechanism of genetic action of this compound. Of somatic mutations, chromosome aberrations, rather than point mutations seem to play a major role in the genotoxicity of PMEA. In flies carrying an inversion chromosome, which eliminates most products of mitotic recombination, reduced spot frequencies were obtained, which, however, were still unexpectedly high for compounds with strong recombinagenic activities. Most probably, in additino to structural mutations of chromosomes, double mitotic crossing-over and non-reciprocal recombinatino events similar to unequal sister-strand recombination of gene conversion significantly contributed to spot induction in the inversion heterozygous flies. Concerning the mechanism of genotoxic action, we suggest that these adenine derivatives can be incorporated into DNa chains during replication. This would result, via breaks and DNa repair mechanisms, either in various recombination events or in chromosome aberrations.     

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.     

Genotoxicity and antigenotoxicity of some essential oils evaluated by wing spot test of Drosophila melanogaster

Essential oils extracted from the three medicinal plants; Helichrysum italicum, Ledum groenlandicum and Ravensara aromatica, together with their mixture were tested for their genotoxic and antigenotoxic activities against urethane, a well-known promutagen. We have adopted the somatic mutations and recombination test (SMART) in the wings of Drosophila melanogaster. Three days old larvae, trans-heterozygous for two genetic markers mwh and flr, were treated by essential oil and/or urethane. A negative control corresponding to solvent was also used. Our results do not show any significant effect of the oils tested but they reduce the mutation ratio resulting from urethane. The mixture of the three oils at equal volume seems to be the most effective. The antimutagenic effect of these oils could be explained by the interaction of their constituents with cytochrome P-450 activation system leading to a reduction of the formation of the active metabolite. The effect could also be attributed to certain molecules that are involved in these oils.     

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.     

Genotoxic activities in vivo of cobaltous chloride and other metal chlorides as assayed in the Drosophila wing spot test

A series of metal chlorides were subjected to the wing spot test of Drosophila melanogaster. In the test, larvae trans-heterozygous for the wing-hair mutations mwh and flr were orally treated at the third instar stage with a test compound and the wings were inspected at the adult stage for spots expressing phenotypes of the markers. CoCl₂, MnCl₂, MoCl₃, NiCl₂ and ZnCl₂ were clearly effective in inducing spots with one or two mutant hairs (small spots). CoCl₂ was clearly effective in inducing spots with three or more mutant hairs (large spots) as well. CrCl₃, FeCl₂ and FeCl₃ were negative under the conditions used. Based on estimated frequencies of small spots induced at the LD₅₀, the genotoxic effectiveness of the positive metal salts were ranked in a sequence of CoCl₂ > ZnCl₂ > MoCl₃ > (MnCl₂, NiCl₂). Since CoCl₂ did not induce large spots in the wings of the mwh/TM3 flies with a suppressed ability of mitotic crossing-over, the large spots induced by this compound in the mwh/flr system were ascertained as mutant clones due to mitotic crossing-overs.     

Mutagenicity testing of the juvenoid methoprene (ZR-515) by means of the Drosophila wing spot test

The juvenile hormone analogue methoprene, which is used in insect pest control, was subjected to mutagenicity testing by means of the Drosophila wing spot test. Larvae heterozygous for recessive wing trichome mutations were exposed to a sublethal dose of methoprene. Wings of emerged adult females were inspected for the presence of phenotypically mutant mosaic spots. Methoprene exhibited a weak mutagenic effect. The fact that only small mosaic clones were induced is discussed.