Interaction between mobile DNA-element-induced lethal mutations and chemical mutagens in the hybrid dysgenic system of Drosophila melanogaster

Using the sex-linked recessive lethal mutation screen, a synergistic interaction is observed for mutations induced by chemical mutagens (ethyl methanesulfonate and dimethylnitrosamine) and the transposable DNA-element system of hybrid dysgenesis in spermatogonial cells of Drosophila melanogaster. Although the mechanism of this interaction is unknown, these results suggest that some chemical mutagens may induce transpositions, hybrid dysgenic cells may be more sensitive to chemically induced genetic damage, or hybrid dysgenesis may inhibit the efficiency of repair of chemically induced lesions.     

A comparative study of the antimutagenic effects of antioxidants on chemical mutagenesis in Drosophila melanogaster

The 1,4-dihydropyridine derivative 2,6-dimethyl-3,5-diethoxycarbonyl-4-(Na carboxylate)-1,4-dihydropyridine (1,4-DHP) was studied for antimutagenic effects in the dominant lethal test and in the sex-linked recessive lethal test of Drosophila melanogaster. The observed effects were compared with those of the radioprotectors cysteine and cysteamine and with those of the phenolic antioxidant butylated hydroxytoluene (BHT). In a wide range of concentrations, including low ones, 1,4-DHP reduces the frequency of EMS-induced genetic damage (point mutations and chromosome breakage). A reduction of the mutation rate induced by EMS in adults could be observed independently of the developmental stages (larvae or imago) pretreated with 1,4-DHP. The protective effect of this new antimutagen against the alkylating agent depended on both the 1,4-DHP dose and the level of the EMS-induced mutation rate. The effect of 1,4-DHP was more pronounced than that of the studied radioprotectors. It is concluded that dihydropyridine-type compounds are able to protect eukaryote germs cells from genetic damage produced by direct-acting mutagens such as EMS.     

Testing the mutagenicity of malondialdehyde and formaldehyde by the Drosophila mosaic and the sex-linked recessive lethal tests

The mutagenicities of malondialdehyde and formaldehyde were tested by screening each for genetic mosaics of Drosophila melanogaster and by the Muller-5 test for sex-linked recessive lethal mutations. For comparison, the effects of X-rays were also assayed by the above technique. Malondialdehyde, a degradation product of polyunsaturated fatty acids, was found to be a weak mutagen by the above criteria; it induced point mutations and chromosome exchanges at low frequency, as proved by the mosaic test, but failed to induce detectable sex-linked lethality. Formaldehyde was more mutagenic than malondialdehyde; beside induction of mosaic spots it induced sex-linked recessive lethal mutations, but only in the larval testes of Drosophila. Formaldehyde also induced disintegration of the clones. Formaldehyde treatment (feeding larvae with formaldehyde-containing food for about 4 days) was 5 times more mutagenic than malondialdehyde treatment and 5 times less effective than irradiation by 1000 R of X-rays. Wing mosaicism offers a more sensitive way to detect mutagenesis as compared with eye mosaicism. It is suggested that aldehyde-induced mosaic spots derive from mitotic recombination and point mutations.     

Sensitivity of individual Drosophila to the mutagenic action of ethyl methanesulfonate

The genetic effect of some factors is generally evaluated as an average response of all individuals, without taking into account their potential differences. The presence of individual sensitivity in separate Drosophila organisms to the mutagenic influence of ethyl methanesulfonate was shown when analysing recessive sex-linked lethal mutations in germ cells of males. Different sensitivity of separate individuals to mutagens reflects the existence of cryptic genetic variability in Drosophila strains on a large scale. It is advisable to take into account individual sensitivity of organisms to mutagenic factors, when conducting mutation research and studying genetic consequences of biosphere pollution.     

Genotoxicity testing of extracts of a Swedish moist oral snuff

The present study was designed to investigate the potential genotoxicity of aqueous and methylene chloride extracts of Swedish moist oral snuff. The test systems were selected to provide optimal data for the prediction of carcinogenicity in rodents and included assays for the induction of mutation in bacteria, sister-chromatid exchanges (SCE) in human lymphocytes, of chromosome aberrations and gene mutations in V79 Chinese hamster cells and of micronuclei in mouse bone marrow cells. In addition, the methylene chloride extract was tested for the induction of sex-linked recessive lethal mutations in Drosophila melanogaster. The aqueous extract of 'Snus' induced SCE in human lymphocytes and chromosome aberrations in V79 cells, the latter effect being observed both with and without metabolic activation. No induction of point mutations was detected with the Ames test or in V79 cells and the micronucleus test in mice was negative. It was demonstrated that the induction of chromosome aberrations without metabolic activation may be due to a high salt concentration, indicating that the clastogenic agent(s) in this extract required metabolic activation. The methylene chloride extract showed genotoxicity in the Ames test, the SCE test and the chromosome aberration test, whereas no induction of gene mutations in V79 cells was observed. Once again, the results suggested that metabolism is required for genotoxicity. The methylene chloride extract did not cause induction of micronuclei in mice or of sex-linked recessive lethal mutations in Drosophila melanogaster. These combined data on genotoxicity were analyzed using various models for the prediction of carcinogenicity. In a sequential testing model, the probabilities that the aqueous and methylene chloride extracts of 'Snus' are carcinogenic due to a genotoxic mechanism were both predicted to be low. Using carcinogenicity prediction by battery selection (CPBS), the probabilities of the methylene chloride and aqueous extracts being correctly identified as non-carcinogens are 71 and 77%, respectively. Up to date, the CPBS approach has been validated primarily for individual compounds, so some caution should at present be exercised in interpreting the results using this method. Based on these results, the carcinogenic potential of Swedish 'Snus' should be considered to be low, a conclusion in agreement with the low incidence of oral cancer in Sweden compared to other countries.     

Some aspects of the detection of potential mutagenic agents in Drosophila.

The Drosophila system is a valuable test for detecting and characterizing mutagenic agents. Tester strains are available or can be synthesized for determining almost all types of genetical change ranging from gene mutations to chromosome rearrangements in a great variety of cell types of both sexes. Metabolic activation of all groups of indirect mutagens tested so far (aryldialkyltriazenes, cyclophosphamides, nitrosamines, azo-, hydrazo- and azoxyalkanes, aflatoxins, and polycyclic hydrocarbons; about 35 representatives in all), gives strong although indirect support for the considerable metabolizing ability of Drosophila. This capability would be expected from comprehensive biochemical data on bioactivation of foreign compounds in other insects. From a comparison of which types of genetical change are induced at high, low and threshold concentrations, it appears that lethal tests remain the most reliable method for any screening program. Mutagenic agents such as diethylnitrosamine, hycanthone and certain triazenes, which are highly efficient in the induction of recessive lethals (gene mutations and/or deficiencies), would not have been detected in Drosophila if chromosome breakage were the only indicator for mutagenic activity. Moreover, for several mono- and polyfunctional agents, the lowest dose which is still genetically active was definitely lowest for recessive lethals when compared with dominant lethals, chromosome rearrangements or loss. If a new mutagen is discovered by a screening procedure using Drosophila, an accurate picture of its ability to cause either or both gene mutations and chromosome aberrations can be drawn. Such work will be valuable in helping to clarify similar problems in mammalian systems. For instance, it was important to learn that mutagens of the nitrosamine type apparently fail to produce breakage events in Drosophila. Similarly, three cyclophosphamides appeared not to have chromosome breaking ability. However, from a more detailed study, in which a series of concentrations was used, it became obvious that a penetration effect or, more likely, a rate-limiting factor in bioactivation, was the cause of the negative results obtained with these agents.     

Mutagenicity of hycanthone in Drosophila melanogaster

The schistosomicidal agent hycanthone was tested for mutagenicity in Drosophila melanogaster. The compound was administered either by injection into adult males or by larval feeding. The following types of genetic damage were measured:(1) complete and mosaic sex-linked recessive lethal mutations; (2) II–III translocations; and (3) dominant lethals.In postmeiotic germ cells, especially in late spermatids, a pronounced increase was found in the frequency of sex-linked recessive lethals, both completes and mosaics. By contrast, translocations and dominant lethals were not induced.     

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.     

Farm-grade chlorpyrifos (Durmet) is genotoxic in somatic and germ-line cells of Drosophila.

The mutagenic potential of Durmet, a farm-grade formulation of chlorpyrifos, was studied in the Drosophila wing mosaic and sex-linked recessive lethal tests. Larvae of the 2nd or 3rd instar carrying suitable recessive genetic markers on chromosome 3 were exposed to different concentrations of the insecticide and the frequency of induction of mutant mosaic spots on the wings was noted. The Basc technique was followed to study the induction of sex-linked recessive lethals. On the basis of the frequency of induction of mosaic wing spots and sex-linked recessive lethals, it is concluded that Durmet is genotoxic in somatic cells as well as germ cells of Drosophila.     

Metronidazole (Flagyl): lack of induction of sister-chromatid exchanges in CHO-K1 cells

328 X-linked recessive lethal mutations induced in late spermatids by hycanthone methanesulfonate were tested for coverage by duplications that comprised, in total, about 24% of the euchromatic X chromosome; 78 lethals appeared to be covered. Crossover localization tests of a random sample of 38 non-covered lethals revealed 4 chromosomes carrying a lethal within a duplicated segment. Lethals localized to a particular region were crossed to reference deficiencies and single-locus mutations, and inter se, to ascertain their genetic extent. The proportion of multi-locus deletions among these 78 covered and 4 non-covered lethals was 3/48, 1/10 and 13/24 for the distal, medial and proximal regions, respectively. A storage period of 9 days did not noticeably influence these proportions. In the sample of 38 non-covered lethals, and among 17 of the covered single-site lethals, 4 cases of strong crossover suppression were detected. Comparison of these results with data obtained with other mutagens suggests that induction of multi-locus deletions, and possibly of other types of chromosome rearrangement, could in part depend on other mechanisms than those acting in the formation of translocations and chromosome loss. For the purpose of mutagen testing, these findings imply that, in Drosophila, results in the regular genetic tests for chromosome breakage events do not always accurately predict the capacity of a mutagen to induce multi-locus deletions. This is of importance since transmissible multi-locus deletions have been considered a significant source of genetic damage in man.     

Inhalation exposure in Drosophila mutagenesis assays: experiments with aliphatic halogenated hydrocarbons, with emphasis on the genetic activity profile of 1,2-dichloroethane

A series of mutation experiments was carried out with Drosophila melanogaster using inhalation exposure. 1,2-Dichloroethane (DCE) and 1,2-dibromoethane (DBE) were active in the sex-linked recessive lethal assay (SLRLT), whereas dichloromethane, dibromomethane, 1,2-dichloropropane and 1,3-dichloropropane were not. Compared to DBE, DCE is a less potent mutagen in the SLRL system. For both compounds, there is no evidence of a clear-cut dose-rate effect. DCE and dichloromethane were also investigated in the somatic mutation and recombination test (SMART), with results similar to those from the SLRLT. For DCE the genetic activity profile was further analyzed by carrying out a sex-chromosome loss assay and a complementation analysis of a series of induced recessive lethal mutations. A review of the use of inhalation in mutagenicity assays with Drosophila shows that this route of exposure is an effective one. Especially with chronic exposure times, rather low exposure concentrations can be detected. With compounds of intermediate volatility inhalation is not superior to other modes of administration; nor is it likely to be sensitive enough for in situ monitoring.     

Fluoride: interaction with chemical mutagens in Drosophila

Simultaneous feeding of sodium fluoride and some chemical mutagens to Drosophila has been reported to reduce the yield of induced mutations compared with feeding the mutagens alone. This observation has been interpreted as a genuine case of antimutagenesis in which fluoride specifically inhibits the induction of chromosome breaks. An alternative hypothesis is that the presence of fluoride inhibits the uptake of the mutagen solutions, producing the same effect as an antimutagen, but for a trivial reason. We have tested this hypothesis using radioactive labelled sucrose to measure the uptake of test solutions. The results show that differential uptake can account for the "antimutagenic" effects reported in Drosophila. Comparison of recessive lethal frequencies induced by Trenimon and PDT do not support the hypothesis that fluoride has any genuine antimutagenic action. Antimutagenic effects of fluoride have been reported in other systems. We cannot exclude the possibility of some genuine effects but we consider that these reports should be critically re-examined in the light of our present findings.     

Mutagenic activity of dioxydine]

A previous evaluation of mutagenic activity of some drugs and perspective substances is carried out using indicator microorganisms. The mutagenicity of dioxydine, a drag with discovered antibacterial activity, is investigated. Dioxydine is shown to induce reversions in mutant of Salmonella typhimurium TA-1950, the indicator strain which demonstrates mutagenic activity of agents, producing mutations of base pair substitution type. Dioxydine proved to affect logariphmiically growing bacterial culture with great activity. Mutageni effect of dioxydine is not modified itself in microsomal oxidation system in vitro. Some data concerning participation of excision reparation enzyme (uvr-B+ gene product) in repair of lethal damages induced by dioxydine, have been obtained. The dioxydine ability to cause bacterial gene mutations in host mediated assay as well as dominant and recessive sex-linked lethal mutations in Drosophila is demonstrated. Dioxydine is capable of inducing chromosome aberrations in bone marrow cells and dominant lethal mutations in mouse germ cells.     

Mutagenicity of dimecron studied in 3 mosaic assays and the sex-linked recessive lethal test in Drosophila melanogaster

The genotoxicity of dimecron, a systemic organophosphate pesticide, has been tested in the wing, eye and germ line mosaic assays and the sex-linked recessive lethal test in Drosophila melanogaster. Larvae heterozygous for recessive marker mutations were fed the compound for various periods of time. On emergence, the wings and eyes of the adults were screened for mosaic spots and the eggs laid by the females were checked for induction of female germ line mosaicism. Dimecron is mutagenic to the somatic and germ line cells of Drosophila and induces a high frequency of sex-linked recessive lethals.     

Radioprotective effect of six chemicals against X-ray-induced genetic damage in D. melanogaster

In Drosophila melanogater six chemicals were tested for radioprotectiveeffect against X-ray-induced genetic damage such as sex-linked recessive lethals and autosomal translocations using Oster's ring-X chromosome stock. A 2-day brood pattern was followed to score the damage induced at different spermatogenic stages separately. In all cases the chemicals were injected before X-irradiation. 10-mM solution of reduced glutathione (GSH) provided statistically significant protection against sex-linked recessive lethals in all broods. In translocation tests this chemical reduced the frequency in all broods but the result is not statistically significant. Cysteamine (MEA) did not show any protective effect but the frequency of lethals was slightly reduced in the first and fourth broods. 2-Aminoethyl isothiuronium Br·HBr (AET) showed a statistically significant protective effect when the data of the replicate experiments were pooled. Negative results were obtained for 5-hydroxytryptamine (5-HT) in sex-linked lethal tests. Aminoethyl phosphorothioate (AEPT) reduced the frequencies of both sex-linked lethals and autosomal translocations in all broods consistently but the results are not statistically significant. In tests for both lethals and translocations the reduction was largest in the stages with highest radiosensitivity. N(3-Aminopropyl)aminoethyl phosphorothioate (3AP-AEPT) gave no protection.     

The mutational spectrum of procarbazine in Drosophila melanogaster.

The antineoplastic agent Procarbazine was tested for the induction of genetic damage in Drosophila melanogaster. The compound was administered to adult males by oral application. The following types of genetic damage were measured: (1) sex-linked recessive lethals; (2) dominant lethals; (3) total and partial sex-chromosome loss; and (4) translocations. Procarbazine is highly mutagenic in causing recessive lethal mutations in all stages of spermatogenesis. In sperm a clear-cut concentration-effect relationship is not apparent, but in spermatids such a relationship is obtained for mutation induction at low levels of procarbazine exposure, while at high concentrations the induction of recessive lethals is not a function of concentration. A low induction of total sex-chromosome loss (X,Y) and dominant lethals was observed in metabolically active germ cells (spermatids), but procarbazine failed to produce well-defined breakage events, such as partial sex-chromosome loss (YL,YS) and II-III translocations. The results obtained in Drosophila melanogaster are discussed and compared with the mutational pattern reported in the mouse after procarbazine treatment.     

Comparative study of the clastogenic efficiency of ethyl methanesulfonate and diethyl sulfate in Drosophila melanogaster mature sperm

Chromosome loss and translocation tests were carried out in Drosophila melanogaster sperm, stored in untreated females for up to 24 days, to compare the clastogenicity of ethyl methanesulfonate (EMS) and diethyl sulfate (DES). The sex-linked recessive lethal test was used as a "biological dosimeter" and the following results were obtained: The yield of 2-3 translocations induced by both mutagens increased steadily with storage, being significantly higher after EMS than after DES treatment. The frequencies of partial losses induced by EMS and DES were similar and increased with storage. With up to 11 days' storage, the frequency of complete loss induced by DES was higher than that induced by EMS and remained unchanged when storage was extended to 24 days. Complete loss induced by EMS increased significantly with further storage (12-24 days). With DES, complete (but not partial) loss was detected with a dose at which EMS failed to modify the control values. These data suggest that the lower recovery of II-III translocations after treatment with DES does not result from a low breaking capacity but from a diminished or delayed rejoining of the induced breaks. This could be due to a physiological impairment of the treated cells by the high toxicity of DES or to an actual lower rejoinability of the broken ends. The differential recovery of complete and partial losses after DES treatment further suggests that the mechanisms leading to the fixation of both types of damage are somehow different, and that processes intervening in the recovery of partial losses are less affected, or not at all, by the proposed reduced rejoining of chromosome breaks.     

Mutagenicity studies in Drosophila melanogaster with Lannate 20

Lannate 20 a carbamate pesticide was evaluated for its mutagenicity in Drosophila melanogaster by the sex-linked recessive lethals and chromosome II-III translocation tests by continuous larval feeding. The 3 sublethal doses of 0.2, 0.4 and 0.6 microliter of Lannate per 100 ml of the food medium induced a significant (P less than 0.01) increase in the number of sex-linked recessive lethals over the controls. However, no translocations were observed either in the treated or the control series.     

Strong antimutagenic effects of fluoride on mutation induction by trenimon and 1-phenyl-3,3-dimethyltriazene in Drosophila melanogaster

After fluoride treatment of mature and immature oocytes of Drosophila females, a clear-cut dose-dependent decrease in fertility and fecundity was observed. The hatchability of mature oocytes was reduced by as much as 35%. When immature oocytes were treated, a pronounced dose-dependent reduction in fecundity occurred.

Exposure of mature sperm to NaF resulted in a slight decrease in fertility, comparable to the effect obtained with immature oocytes. Of the criteria used to measure possible mutagenic effects of NaF (sex-linked lethals, partial and total X- and Y-chromosome losses), only the rate of total losses was enhanced significantly.

The slight mutagenic effect of NaF on mature sperm was not related to the strong antimutagenic activity observed, when applied simultaneously with any of the several chemical mutagens. NaF treatment drastically reduced both the Trenimon-induced decrease in fertility and Trenimon-induced increases in recessive lethal mutation frequencies and rates of partial and total chromosome losses. The inhibitory effect of NaF was less pronounced with 1-phenyl-3,3-dimethyltriazene (PDT), a poor chromosome breaker in Drosophila, and absent for A 137, a weak mutagen which so far has failed to induce chromosomal aberrations in Drosophila. Therefore, the data are interpreted as being the result of a specific fluoride inhibition of chemically induced chromosomal breakage.

In mature and immature oocytes, the decreases in fertility and fecundity, and increase in recessive lethal frequency (mature oocytes) produced by Trenimon were also suppressed in the presence of fluoride. However, since Trenimon failed to produce a significant rise in X losses and NDJ in both stages, the effect of NaF on these mutational classes was, of course, not testable.     

Effect of glyoxal pretreatment on radiation-induced genetic damage in Drosophila melanogaster

The effects of glyoxal and of glyoxal pretreatments on radiation-induced genetic damage were investigated in Drosophila melanogaster mature sperm, by means of sex-linked recessive and dominant lethality, reciprocal translocation and chromosome loss tests. In addition, the possible mutagenic effect of glyoxal was assessed in postmeiotic cells up to 7 days after treatment. The results obtained show: (1) the frequencies of recessive lethals after glyoxal treatment were within control values, (2) no clastogenic effect of glyoxal was observed, (3) glyoxal pretreatment did not modify the frequency of recessive lethals induced by X-rays, (4) after pretreatment with glyoxal a consistent, though not significant, increase was seen in the frequency of reciprocal translocations in 3 replicate experiments, (5) the yield of dominant lethals and of complete and partial chromosome loss induced by radiation was significantly increased by pretreatments with glyoxal. It is suggested that the increase of the frequency of genetic endpoints resulting from chromosome breakage, when glyoxal was administered prior to irradiation, could be ascribed to: (a) a sensitizing action of glyoxal to the clastogenic effect of ionizing radiation; (b) the formation of reactive species by the interaction of glyoxal with radiation; and/or (c) interference of glyoxal with the normal handling of radiation-induced lesions in mature postmeiotic male cells.