Improved high bioactivation cross for the wing somatic mutation and recombination test in Drosophila melanogaster.

The two tester strains of the high bioactivation (HB) cross for the wing somatic mutation and recombination test (SMART) in Drosophila melanogaster developed by Fr?lich and Würgler possess high metabolic capacity to activate promutagens. These strains contain chromosomes 1 and 2 of the DDT-resistant stock Oregon R(R) which exhibits a high constitutive level of cytochrome P450. However, they show several disadvantages for routine application, such as disturbed wing hair patterns in certain areas of the wing, making spot classification difficult, and a delay in development of the larvae. We have established and evaluated an improved HB cross (ORR; flr3 females and mwh males) producing ORR heterozygous individuals. These develop normally and have a normal, undisturbed wing hair pattern while exhibiting high bioactivation. The hybrid larvae of the improved HB cross show P450-dependent bioactivation capacity equal to or even slightly higher than those of the original HB cross. This was demonstrated by measuring the genotoxic activity of the promutagens diethylnitrosamine, 7,12-dimethylbenz[a]anthracene, N-nitrosopyrrolidine, and urethane. In addition, the improved HB cross has a sensitivity to the direct-acting alkylating agent ethyl nitrosourea equal to that of the standard cross. The main advantage of the improved HB cross is to combine the high bioactivation capacity with the ease of scoring the wings using the same criteria as for the standard cross.     

Molecular cloning of mei-41, a gene that influences both somatic and germline chromosome metabolism of Drosophila melanogaster

The mei-41 gene of Drosophila melanogaster plays an essential role in meiosis, in the maintenance of somatic chromosome stability, in postreplication repair and in DNA double-strand break repair. This gene has been cytogenetically localized to polytene chromosome bands 14C4-6 using available chromosomal aberrations. About 60 kb of DNA sequence has been isolated following a bidirectional chromosomal walk that extends over the cytogenetic interval 14C1-6. The breakpoints of chromosomal aberrations identified within that walk establish that the entire mei-41 gene has been cloned. Two independently derived mei-41 mutants have been shown to carry P insertions within a single 2.2 kb fragment of the walk. Since revertants of those mutants have lost the P element sequences, an essential region of the mei-41 gene is present in that fragment. A 10.5 kb genomic fragment that spans the P insertion sites has been found to restore methyl methanesulfonate resistance and female fertility of the mei-41 ^D3 mutants. The results demonstrate that all the sequences required for the proper expression of the mei-41 gene are present on this genomic fragment. This study provides the foundation for molecular analysis of a function that is essential for chromosome stability in both the germline and somatic cells.     

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 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.     

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 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.     

Evaluation of genotoxicity of captan,maneb and zineb in the wing spot test of Drosophila melanogaster: role of nitrosation

The wing spot test in Drosophila melanogaster is a suitable system for the analysis of genotoxic activity of compounds that need metabolic transformation to render them active. We have analysed the genotoxicity of three fungicides for which it was reported that the metabolic processes taking place in vivo may determine their activity. The compounds analysed are captan, maneb, zineb and ethylenethiourea (ETU) (a metabolic derivative of ethylenebisdithiocarbamates like maneb and zineb). We have also evaluated the ability of ETU to form genotoxic derivatives in vivo analysing this compound in combined treatments with sodium nitrite. Both standard and high bioactivation NORR strains have been used. Captan, usually considered a mutagen in vitro but a non-mutagen in vivo, gave negative results in the wing spot test with both crosses. Positive results were obtained for maneb in the standard cross and for ETU in both the standard and the high bioactivation cross. The genotoxicities of maneb and ETU were higher when treatments were made on media in which nitrosation is favoured. A low absorption of the fungicide and an inefficient availability of the compound in the target may explain negative results obtained with zineb in both crosses. The results obtained in this study with the wing spot test demonstrate once again the suitability of this in vivo assay, in which absorption, distribution and metabolism processes take place, for the evaluation of genotoxicity of compounds to which humans are exposed.     

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.     

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.     

The repair-deficient mei-9 alpha Drosophila female potentiates chromosome loss induced in the parenteral genome by diethylnitrosamine

Following matings of DEN-treated Xc2/BSYy+ males with repair-deficient mei-9 alpha females and ordinary females, significant increases in complete and partial sex chromosome loss as well as dramatic shifts in sex ratio were found with mei-9 alpha but not ordinary females. Accordingly, the mei-9 alpha female enhances the detection of chromosome lesions leading to chromosome loss induced in the male genome by DEN. To date, the 4 compounds tested in this way (DMN, DEN, MMS and procarbazine) exhibit strong potentiation of chromosome loss with mei-9 alpha females suggesting the possibility that a protocol involving treatment (or not) or Xc2/BSYy+ males mated with mei-9 alpha females may hold promise as an alternative to traditional tests for chromosome loss using repair-proficient females. Comparison with published translocation data on the 4 compounds indicated above suggests an overall greater sensitivity of the described mei-9 alpha chromosome-loss test compared with the traditional translocation test in the detection of chemically induced chromosome lesions.     

A negative test for mutagenic action of microwave radiation in Drosophila melanogaster.

Microwave radiation (2450 MHz CW) was tested for mutagenicity in Drosophila melanogaster. Embryos in water were exposed to the electromagnetic field with a mean specific absorption rate of 100 W/kg. A sensitive somatic test system was used, in which mutagenicity was measured as the frequency of somatic mutations for eye pigmentation. With the test system used, microwaves did not show any mutagenic activity.     

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.     

Induced chromosome loss following treatment of postmeiotic cells of the Drosophila melanogaster male with MMS and DMN and matings with repair-proficient females and the repair-deficient females mei-9a and st mus302

Drosophila melanogaster ring-X males carrying a double marked Y chromosome, BsYy+, were treated with MMS or DMN and mated with repair-proficient females or the repair-deficient females mei-9a and st. mus302. Frequencies of induced complete loss (principally the ring-X) and partial losses of the Y chromosome (loss of Bs or Y+) decreased in the sequence st must302 greater than mei-9a greater than repair-proficient females agreeing with the sequence obtained previously with procarbazine and DEN. With MMS and DMN, some 30-40% or more or partial Y chromosome losses are mosaics from mei-9a and only 0.4% from st mus302 females and a delay in mei-9a females. Similar findings with procarbazine and DEN are indicated. That the higher sensitivity of st mus302 relative to mei-9a results from impairments in both postreplication and excision repair in the former remains to be determined.     

Aneuploidy in Drosophila, I. Genetic test systems in the female Drosophila melanogaster for the rapid detection of chemically induced chromosome gain and chromosome loss

An account is provided of two genetic schemes in the Drosophila melanogaster female designed as rapid detectors of chemically induced aneuploidy, including both chromosome gain and chromosome loss. One scheme is referred to as FIX, in which the female carried free (heterozygously) inverted X (chromosomes) and the other, ZESTE, where females do not carry inversions and the X-linked sexually dimorphic zeste mutation plays the key role in the detection of aneuploid offspring. The principle attribute of the FIX system is that all euploid offspring are wild-type for body and eye color whereas aneuploid females have a yellow body and aneuploid males white eyes; int he ZESTE system all euploid individuals are wild-type for eye color, aneuploid females possess zeste-colored eyes and aneuploid males white eyes. In addition induced polyploidies (2X:2A gametes) appear as yellow and zeste male intersexes in the FIX and ZESTE systems, respectively. In this way all aneuploids are recognized immediately. Consequently, detection of compounds with weak effects requiring large sample sizes may be made in a fraction of the time associated with more traditional schemes for aneuploidy detection in Drosophila.     

Genotoxicity of Rogor studied in the sex-linked recessive lethal test and wing, eye and female germ-line mosaic assays in Drosophila melanogaster

The genotoxic potential of Rogor (dimethoate), an anticholinesterase organophosphate insecticide, has been studied in the sex-linked recessive lethal test and the wing, eye and female germ-line mosaic assays in Drosophila melanogaster. Larvae of different instars carrying suitable recessive genetic markers on their first and third chromosomes were exposed to the LD50 or half of this dose for the entire larval life. The Basc technique was followed for the detection of the induction of sex-linked recessive lethals. The wings and eyes of the adult flies and the eggs laid by the heterozygous females were checked for the induction of mosaicism. It is concluded that Rogor induces sex-linked recessive lethals in immature male germ cells and is recombinogenic and/or mutagenic in both the somatic and the germ-line cells of Drosophila.     

Genotoxicity of ziram established through wing, eye and female germ-line mosaic assays and the sex-linked recessive lethal test in Drosophila melanogaster

The genotoxicity of ziram (zinc-dimethyl dithiocarbamate, CAS No. 137-30-4), a carbamate fungicide, is studied in the wing, eye and female germ-line mosaic assays and the sex-linked recessive lethal test in Drosophila melanogaster. First-, second- and third-instar larvae, carrying suitable recessive genetic markers on their first and third chromosomes, were exposed to ziram. Wings and eyes of adults were screened for the induction of mosaic spots and the eggs laid by adult females for germ-line mosaicism. The Basc method was used to detect sex-linked recessive lethals. Ziram is genotoxic to the somatic and germ cells of Drosophila melanogaster.