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 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.     

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.     

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.     

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.     

On the persistence of the radioprotective effect of chlorophyllin (CHLN) in somatic cells of Drosophila

By delaying the time of gamma irradiation of 72 h larvae, pretreated at 48 h with 5% chlorophyllin (CHLN), it was established that the overall inhibiting effect of CHLN in somatic cells of Drosophila, as measured in the wing spot test, persists for about 4 days or until the time of cessation of the proliferation of wing anlagen. In the same population of cells, some spot classes gave evidence of an inhibitory effect whereas others did not arguing against the suggestion that the radioprotective effect of CHLN is a consequence of an induced delay in development, shrinking of the potential radiation target and lowering the probability of induced events. Other observations of interest are described.     

Evidence suggesting that chlorophyllin (CHLN) may act as an inhibitor or a promoter of genetic damage induced by chromium(VI) oxide (CrO3) in somatic cells of Drosophila

In Drosophila, 48h-old larvae were pretreated for 24h with chlorophyllin (CHLN) or sucrose and then treated with chromium(VI) oxide (CrO(3)) immediately following completion of the pretreatment period (0-day delay) or delayed 1, 2 or 3 days. The effects were scored in the wing spot test. After delays of 0 and 1 day, clear evidence of a protective effect of CHLN was found. Contrarily, after delays of 2 and 3 days, the results showed a reversal, i.e. CHLN-related events appeared more frequently than those in the sucrose control suggesting a promoting effect. It would appear prudent that CHLN be tested in a variety of situations in any given organism before decisions are reached regarding its inhibitor/promoter effects.     

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.     

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

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

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).     

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.     

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.     

Induced Rates of Mitotic Crossing over and Possible Mitotic Gene Conversion per Wing Anlage Cell in Drosophila Melanogaster by X Rays and Fission Neutrons

As a model for chromosome aberrations, radiation-induced mitotic recombination of mwh and flr genes in Drosophila melanogaster strain (mwh +/+ flr) was quantitatively studied. Fission neutrons were five to six times more effective than X rays per unit dose in producing either crossover-mwh/flr twins and mwh singles-or flr singles, indicating that common processes are involved in the production of crossover and flr singles. The X-ray-induced rate/wing anlage cell/Gy for flr singles was 1 X 10(-5), whereas that of crossover was 2 x 10(-4); the former and the latter rate are of the same order of magnitude as those of gene conversion and crossover in yeast, respectively. Thus, we conclude that proximal-marker "flr" singles induced in the transheterozygote are gene convertants. Using the model based on yeast that recombination events result from repair of double-strand breaks or gaps, we propose that mitotic recombination in the fly is a secondary result of recombinational DNA repair. Evidence for recombinational misrepair in the fly is given. The relative ratio of radiation-induced mitotic crossover to spontaneous meiotic crossover is one order of magnitude higher in the fly than in yeast and humans.     

Modulation of genotoxicity in Drosophila.

The extensive knowledge of the genetics of Drosophila melanogaster and the long experimental experience with this organism have made it of unique usefulness in mutation research and genetic toxicology. The development of somatic mutation and recombination tests (SMART) has provided sensitive, rapid and cheap assays for investigations of mutagenic and recombinogenic properties of chemicals. The present paper deals with the SMART wing spot assay, developed by Graf et al. (1984). The use of two genetic markers, multiple wing hair (mwh) and flare (flr) in the third chromosome, makes it possible to discern localized recombinogenic effects on the two intervals--the major, euchromatic, part of the chromosome, and the mostly heterochromatic centromere region. The distribution of induced mitotic recombination varied between test chemicals. Ethylene oxide caused a specific increase of twin spots, indicating a localized induction of somatic recombination in the centromere region. The wing spot assay has turned out to be suitable for combined treatment with chemicals in order to study antimutagenic and other modulating effects by mutagenic and recombinogenic chemicals. Examples of the use of this assay for such a purpose are presented in this paper. The inhibitor of poly ADP-ribosylation, 3-aminobenzamide (3AB), caused a pronounced increase of wing spots, induced by alkylating agents. The data indicate that this interaction between alkylating agents and 3AB is solely due to an effect on somatic recombination but not on point mutations. The inhibitor of topoisomerases, novobiocin, which presumably acts on the chromatin configuration, had different modulating effects on spots induced by methyl methanesulfonate (MMS) and ethylnitrosourea (ENU). Novobiocin essentially acted as an antirecombinogenic agent in cotreatment experiments with MMS and as antimutagenic agent with ENU. Attempts to interfere with mutagenic and recombinogenic effects of the radical-generating agents bleomycin, menadione and paraquat, by agents acting on the defence mechanisms against oxygen radicals, were essentially unsuccessful.     

Somatic mutation in the wings of Drosophila melanogaster females dysgenic due to P elements when reared at 29 degrees C.

The possibility of somatic mobilisation of P elements in Drosophila melanogaster was investigated. Flies, trans-heterozygous for the genetic markers mwh and flr3, were obtained by crossing males containing transposition-competent P elements with females having M cytotype. The hybrid dysgenic flies were reared at 29 degrees C and their wings examined for mutant clones. The frequency of mutant spots found on the wings of the female flies was significantly higher than that of female control flies. We postulate that this increase in frequency may be due to P element mobilisation at high temperature in the wing cells of dysgenic hybrids. This is in direct contrast to the large body of research which indicates that P-transposition-mediated mutation is restricted to the germline.     

Chlorophyllin [CHLN] and the mutagenicity of monofunctional alkylating agents in Drosophila: the action of CHLN need not include an influence on metabolic activation

The effect of chlorophyllin (CHLN) on the mutagenicity of four monofunctional alkylating agents (MFAAs) was evaluated in the wing spot test in Drosophila. Three of the compounds are direct-acting (ethylnitrosamine (ENU), methylnitrosourea (MNU), and methylmethanesulfonate (MMS)) and one indirect-acting (diethylnitrosamine, DEN). Results indicate that the mutagenicity of all four compounds is strongly inhibited by CHLN. The findings are not in agreement with the conclusion of Romert et al. (1992) that CHLN has no effect on the mutagenicity of direct acting MFFAs inferred from their work with MNU and ethylmethanesulfonate (EMS) in the V79 and Salmonella in vitro test systems. The results suggest the possibility that the action of CHLN need not include an inhibiting effect on metabolic activation.     

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.     

Modulatory effects of Duguetia furfuracea (A. St. Hil) Benth. and Hook. f. in Drosophila melanogaster somatic and germinative cells

Mutagenic and antimutagenic activities of the medicinal plant Duguetia furfuracea were assessed using SMART/wing and ring-X-loss tests. For the ring-X-loss test, 2- to 3-day-old Drosophila melanogaster ring-X-lineage males and virgin ywsn3 females received D. furfuracea infusion at doses of 0.085, 0.042, or 0.014 g/mL for 24 h. We found that D. furfuracea did not produce any mutagenic effects in D. melanogaster germinative cells. The somatic cells of D. melanogaster were analyzed using the SMART/wing test involving three lineages - mwh, flr3, and ORR - and the same doses of D. furfuracea infusion employed in the ring-X-loss test, as well as 20 mM urethane. The results of both standard (ST) and high bioactivation (HB) crosses showed absence of mutagenic activity of D. furfuracea. In contrast, in both ST and HB crosses, we observed a modulatory effect of D. furfuracea against the genotoxic activity of urethane.     

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.