The capacity of Drosophila for detecting relevant genetic damage

For the detection and study of mutagenic agents, Drosophila offers many advantages. It is a higher organism with a short generation time that is cheap and easy to breed in large numbers. The simple genetic testing methods provide unequivocal answers about the whole spectrum of relevant genetic damage. A comparison of the detection capacity of assays sampling different kinds of genetic damage revealed that various substances are highly effective in inducing mutations, but do not produce chromosome breakage effects at all, or only at much higher concentrations than those required for mutation induction. Of the different assay systems available, the classical sex-linked recessive lethal test thus deserves priority, in view of its superior capacity to detect mutagens. Of practical importance is also its high sensitivity, because a large number of loci in one-fifth of the genome is tested for newly induced forward mutations, including small deletions.Drosophila is capable of carrying out the same metabolic activation reactions as the mammalian liver. An additional advantage, in this respect, is the capacity of Drosphila for detecting short-lived activation products, because intracellular activation occurs within the spermatids and spermatocytes. These properties make the test for recessive sex-linked lethals a useful tool for verifying results obtained in the pre-screening of potential mutagens with fast microbial assay systems.In studies on non-disjunction, detailed genetic analysis of the induced changes is possible, and these may shed light on the mechanisms involved.A new adaptation of the bithorax transvection method by Mendelson permits the recovery of high yields of chromosome aberrations in a fast one-generation test.     

Effects of N-nitrosopiperidine substitutions on mutagenicity in Drosophila melanogaster.

N-Nitrosopiperidine (NP) and various derivatives were fed to Drosophila melanogaster males over a wide concentration range in order to assess their mutagenic potency in the induction of X-linked recessive lethals and chromosome loss. NP was effective in inducing lethals, as were its halogen and methyl-substituted derivatives, with the exception of 2,6-dimethyl NP. (Methyl substitutions at the alpha carbon atoms reduce or eliminate mutagenic activity.) Substitution of halogen groups on the piperidine ring enhanced the mutagenic activity, with the 3-chloro compound being the most mutagenic. In contrast, substitutions with a hydroxyl, carboxyl, or keto group resulted in a loss of mutagenicity. None of the compounds tested increased the frequency of chromosome loss or breakage in mature sperm.     

Evidence for the absence of a mutagenic effect of methadone in germ cells of Drosophila melanogaster.

The compound, methadone, used as a modality in the treatment of heroin addiction, was tested for mutagenic activity in germ cells of Drosophila. Results were negative in tests for sex-linked recessive lethals using feeding and injection procedures. Similarly, results from tests for chromosome breakage and nondisjunction failed to provide evidence of a mutagenic effect.     

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.     

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.     

The main effect of chromosomal rearrangement is changing the action of regulatory genes

Effect of chromosomal rearrangements on the expression of mutations was studied in Drosophila melanogaster regulatory genes. These were facultative dominant lethals and recessive lethals on the X chromosome obtained by the classical Muller-5 method. Chromosomal rearrangements drastically changed the expression of regulatory gene mutations. Rearrangements either caused the lethal effect of mutations or suppressed the already present lethality. The action of rearrangements exhibited the maternal or paternal effect. Irrespective of the presence in the genome of mutations at regulatory genes, a rearrangement acted as a factor decreasing fertility of the organism. The rearrangement effect is identical to the expression of regulatory genes per se. It is concluded that the chromosomal rearrangement affects the examined regulatory genes indirectly through a change in the operation of regulatory genes located within the rearrangement. Thus, rearrangements gain great importance for the definition of the pattern of genome functional activity. Widespread distribution of rearrangements in individual genotypes and their effectivity in the process of speciation are thus explained.     

Genetic analysis of recessive lethal mutations induced by the influenza virus in the X chromosome of Drosophila melanogaster

Mutagenic potential of the influenza virus was evaluated. Based on its capacity of inducing recessive lethal mutations in the X chromosome of Drosophila melanogaster, the influenza virus can be classified as a moderate-activity mutagen. Its mutagenicity does not depend on ability to reproduce in the cell system. This virus was shown to disrupt formation of the wing, particularly wing vein M1 + 2. Cytogenetic examination of polytene X chromosomes bearing recessive lethal mutations in Drosophila salivary glands did not reveal chromosome rearrangements. These lethals are assumed to be small deletions or point mutations. The determination of the lethal activity stage of these mutations showed that they disrupt the expression of genes functioning at various developmental stage of Drosophila. Two of them were conditionally lethal (temperature-sensitive). Two of 15 mutations analyzed were mapped to region 2B9-10-3C10-11.     

The Main Effect of Chromosomal Rearrangement Is Changing the Action of Regulatory Genes

Effect of chromosomal rearrangements on the expression of mutations was studied in Drosophila melanogaster regulatory genes. These were facultative dominant lethals and recessive lethals on the X chromosome obtained by the classical Muller-5 method. Chromosomal rearrangements drastically changed the expression of regulatory gene mutations. Rearrangements either caused the lethal effect of mutations or suppressed the already present lethality. The action of rearrangements exhibited the maternal or paternal effect. Irrespective of the presence in the genome of mutations of regulatory genes, a rearrangement acted as a factor decreasing fertility of the organism. The rearrangement effect is identical to the expression of regulatory genes per se. It is concluded that the chromosomal rearrangement affects the examined regulatory genes indirectly through a change in the operation of regulatory genes located within the rearrangement. Thus, rearrangements gain great importance for the definition of the pattern of genome functional activity. Widespread distribution of rearrangements in individual genotypes and their effectivity in the process of speciation are thus explained.     

Autosomal recessive lethal mutations in two mutator stocks of Drosophila ananassae.

The frequency of recessive lethals in the 2nd chromosome was examined in two mutator stocks of Drosophila ananassae, ca and ca; px. They are characterized respectively by possessing an extrachromosomal clastogenic mutator in males, and by the retrotransposon "tom", which induces Om mutability only in females. The frequencies of recessive lethal mutations in the 2nd chromosome among progenies from males and females of the ca; px stock are 0.35 and 0.34 percent, respectively. Similarity of these frequencies indicates that tom does not induce recessive lethals in females. In contrast to the ca; px stock, the frequency of recessive lethals in males of the ca mutator stock was estimated to be 1.54 percent for the 2nd chromosome. No visible mutants except Minutes were recovered. Some recessive lethals derived from ca stock males were associated with chromosomal rearrangements. Being consistent with its high rate of Minute mutation it was demonstrated that the ca clastogenic mutator also induced recessive lethals.     

Mutagenicity of hycanthone in drosophila: Additional results and a comparison with some analogs

The data reported in this paper extend earlier results on the effects of hycanthone in Drosophila. The main findings are the following. (1) A refined brood-pattern analysis of hycanthone-induced sex-linked recessive lethals confirmed the specific sensitivity of mid- and late spermatids. Injection of young males (0–20 h old) did not cause a shift in the brood pattern, but tended to produce higher rates of recessive lethals than injection of 4-day-old males, although the difference was not significant. (2) An autosomal recessive lethal test (chromosome 2) similarly showed a low sensitivity of premeiotic stages. (3) Feeding of hycanthone was much less effective than injection. This difference was not observed for the methyl analog lucanthone. From the observation that hycanthone- and lucanthone-induced mutations exhibited different germ-cell-stage sensitivity patterns, it was concluded that lucanthone does not (at least not exclusively) act via metabolic activation to hycanthone. (4) After injection, the hycanthone analogs IA-4-N-oxide and IA-4-N-oxide were marginally mutagenic. (5) It was shown previously that hycanthone was ineffective in producing breakage events, in Drosophila. In this report, hycanthone is shown to be weakly active in inducing ring-X chromosome loss. This emphasizes the relat ive sensitivity of the ring-X-loss test, in comaprison with the tests that etect translocations or dominant lethals.     

Vinyl chloride mutagenesis in Drosophila melanogaster.

In inhalation experiments, Drosophila males were exposed to vinyl chloride at concentrations of 200, 850, 10,000 30,000 or 50,000 ppm for 2 days, and to 30 or 850 ppm for 17 days. VCM was mutagenic in the recessive-lethal test both after short-term and long-term exposures. The lowest effective concentration (LEC) was 850 ppm after 2 day exposure, and this value could be lowered to 30 ppm by prolonging the exposure time to 17 days. With the concentration levels tested, the mutation frequency increased with concentrations and reached a plateau at 10,000 ppm. This indicates a substrate saturation effect. In contrast with the recessive lethal assay, negative results were obtained when tests on dominant lethals, translocations, entire and partial sex-chromosome loss were carried out with VCM at 30,000 ppm for 2 days. This finding of a false negative seems a logical consequence of the observed saturation effect, and strengthens the concept that there exist two effective concentrations for point mutations vs the induction of chromosome breakage events. Vinyl chloride monomer provides another example to support our view that chromosome breakage is not a reliable measure of mutagenic activity.     

Induction of sex-linked recessive lethals and autosomal translocations by beta-propiolactone in Drosophila: influence of the route of administration on mutagenic activity.

Beta-propiolactone (BPL) was tested for the induction of sex-linked recessive lethals and autosomal translocations in Drosophila melanogaster. The compound was administered to adult males either by oral application or by abdominal injection. When injected, BPL was a potent inducer of sex-linked recessive lethals. When BPL was given by feeding, its mutagenic activity was detectable only when the flies were starved and when the BPL-containing solutions were renewed several times. Nevertheless, the recessive-lethal frequency was one order of magnitude higher with injection. This difference in effects is attributed to (1) rapid decomposition of the compound in aqueous feeding solutions, and to (2) rapid degradation in vivo which restricts the activity of BPL mainly to the site of application. These data are compared with other studies in which both routes of application were applied. BPL induced translocations in stored spermatozoa when injected, but not when fed. This finding seems a logical consequence of (1) the difference in effectiveness of the two routes of application for BPL, and (2) the existence of different LECs for mutation induction (recessive lethals) and for chromosome breakage (translocations). In Drosophila, the breakage capacity of BPL was one order of magnitude lower than that of MMS, when a comparison was made on the basis of equal recessive-lethal frequencies.     

Genetic Analysis of Recessive Lethal Mutations Induced by the Influenza Virus in the X Chromosome of Drosophila melanogaster

Mutagenic potential of the influenza virus was evaluated. Based on its capacity of inducing recessive lethal mutations in the X chromosome of Drosophila melanogaster, the influenza virus can be classified as a moderate-activity mutagen. Its mutagenicity does not depend on ability to reproduce in the cell system. This virus was shown to disrupt formation of the wing, particularly wing vein M_{1 + 2}. Cytogenetic examination of polytene X chromosomes bearing recessive lethal mutations in Drosophilasalivary glands did not reveal chromosome rearrangements. These lethals are assumed to be small deletions or point mutations. The determination of the lethal activity stage of these mutations showed that they disrupt the expression of genes functioning at various developmental stages of Drosophila.Two of them were conditionally lethal (temperature-sensitive). Two of 15 mutations analyzed were mapped to region 2B9-10–3C10-11.     

O-alkylation in DNA does not correlate with the formation of chromosome breakage events in D. melanogaster

Postmeiotic cell stages of repair-proficient ring-X (RX) males were treated with methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS), diethylnitrosamine (DEN) or ethylnitrosourea (ENU) and then mated to either repair-defective (mei-9L1) or to repair-competent females (mei-9+). Absence of the mei-9+ function resulted in a hypermutability effect to all alkylating agents (AAs) when they were assayed for their ability to induce chromosomal aberrations (chromosome loss; CL), irrespective of marked differences in distribution of DNA adducts brought about by these AAs. This picture is different from that described previously for the induction of point mutations (Vogel et al., 1985a). There, evidence was presented indicating that reduction in DNA excision repair does not affect point mutation induction (recessive lethals) by those AAs most efficient in ring-oxygen alkylation such as ENU, DEN, N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG), and isopropyl methanesulfonate (iPMS): the order of hypermutability of AAs with mei-9L relative to mei-9+ was MMS greater than MNU greater than DMN = EMS greater than iPMS = ENU = DEN = ENNG. When the percentage of lethal mutations induced in mei-9L1 females were plotted against those determined for mei-9+ females, straight lines of following slopes were obtained: MMS = 7.6, MNU = 5.4, DMN = 2.4, EMS = 2.4, and iPMS = ENU = DEN = ENNG = 1. Those findings, together with the recent observation that AAs do not split into two groups when assayed for their ability to cause CL, point to the involvement of different DNA alkylation products in ENU- and DEN-induced chromosome loss vs. that of point mutations. It is concluded that with ENU and DEN chromosomal loss results from N-alkylation products whereas point mutations (SLRL) are the consequence of interactions with oxygen-sites in DNA. Thus, as a consequence of a very dominating role of O-ethylguanine (and possibly O4-alkylation of thymine), N-alkylation in DNA does not contribute measurably to mutation induction in the case of ENU-type mutagens while O-alkylation, very clearly, does not show a positive correlation with the formation of chromosome breakage events in Drosophila. Conversely, it appeared that with MMS-type mutagens (MMS; dimethyl sulfate, DMS; trimethyl phosphate, TMP), alkylation products such as 7-methylguanine and 3-methyladenine, if unrepaired or misrepaired, are potentially mutagenic lesions causing both mutations and chromosomal aberrations.     

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.     

Mutagenicity of BCNU and related chloroethylnitrosoureas in Drosophila.

BCNU and 10 related chloroethylnitrosoureas were tested for their ability to induce sex-linked recessive lethals in Drosophila spermatozoa. All chloroethylnitrosoureas tested were potent mutagens. Among the substances with one chloroethylnitrosourea group, chlorozotocin, BCNU and methanesulfonyloxyethyl chloroethylnitrosourea exhibited the strongest mutagenic effects. Two hydroxyalkyl chloroethylnitrosoureas behaved as potent mutagens too, although the mutation frequencies obtained were one order of magnitude lower relative to the other substances. Among the compounds with two chloroethylnitrosourea groups, bisCNU-ethane and bisCNU-diphenylmethane were most active. When the interconnecting polymethylene chain was elongated from 2 methylene groups (bisCNU-ethane) to 6 methylene groups (bisCNU-hexane), the mutagenic activity decreased by a factor of 2. The mutagenic activity of polymethylene bischloroethylnitrosoureas with connecting chains of intermediate length was not different from bisCNU-hexane. Differences in mutagenic activity were supposed to reflect different concentrations reaching the target cells, possibly in part as a result of differences in transportability of the substances.     

Detection of antigenically active mutant HGPRT after mutagenesis with Simian virus 40.

BCNU and 10 related chloroethylnitrosoureas were tested for their ability to induce sex-linked recessive lethals in Drosophila spermatozoa. All chloroethyl-nitrosoureas tested were potent mutagens.Among the substances with one chloroethylnitrosourea group, chlorozotocin, BCNU and methanesulfonyloxyethyl chloroethylnitrosourea exhibited the strongest mutagenic effects. Two hydroxyalkyl chloroethylnitrosoureas behaved as potent mutagens too, although the mutation frequencies obtained were one order of magnitude lower relative to the other substances.Among the compounds with two chloroethylnitrosourea groups, bisCNU-ethane and bisCNU-diphenylmethane were most active. When the interconnecting polymethylene chain was elongated from 2 methylene groups (bisCNU-ethane) to 6 methylene groups (bisCNU-hexane), the mutagenic activity decreased by a factor of 2. The mutagenic activity of polymethylene bischloroethylnitrosoureas with connecting chains of intermediate length was not different from bisCNU-hexane.Differences in mutagenic activity were supposed to reflect different concentrations reaching the target cells, possibly in part as a result of differences in transportability of the substances.     

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.     

The Intercellular Distribution of Mutations Induced in Oocytes of DROSOPHILA MELANOGASTER by Chemical and Physical Mutagens

When females of Drosophila melanogaster are treated with chemical or physical mutagens, not only in one but also in both of the two homologous X chromosomes of a given oocyte, a recessive sex-linked lethal mutation may be induced. A method is described that discriminates between such "single" and "double mutations". A theory is developed to show how a comparison between the expected and the observed frequency of double mutations yields an indication of the intercellular distribution (random or nonrandom) of recessive lethal mutations induced by mutagenic agents in oocytes and, consequently, of the distribution (homogeneous or nonhomogeneous) of those agents.--Three agents were tested: FUdR (12.5, 50.5 and 81.0 micrograms/ml), mitomycin C (130.0 micrograms/ml) and X rays (2000 R, 150 kV). After FUdR feeding, no increase in the mutation frequency usually observed in D. melanogaster without mutagenic treatment was obtained (u = 0.13%, namely three single mutations among 2332 chromosomes tested). After mitomycin C feeding, 104 single and three double mutations were obtained. All of the 50 mutations observed after X irradiation were single mutations. The results obtained in the mitomycin C and radiation experiments favor the assumption of a random intercellular distribution of recessive lethal mutations induced by these two agents in oocytes of D. melanogaster. Reasons are discussed why for other types of mutagenic agents nonrandom distributions may be observed with our technique.     

Genetic Damage at Specific Gene Loci in Drosophila with Betatron X-Rays

The mutation rate was determined for mature sperm at eight specific gene loci on the third chromosome of Drosophila melanogaster using the low ion density radiations of 22 Mev betatron X-rays. A dose of 3000 rads of betatron X-rays produced a mutation rate of 4.36 x 10^-8 per rad/locus. Among the mutations observed, 66% were recessive lethals and 34% viable when homozygous. Only one of the 24 viable mutations was associated with a chromosome aberration. Among the 47 recessive lethals, no two-break aberrations were detected in 48.9% of the lethals, deletions were associated with 42.2%, inversions with 6.7% and translocations with 2.2%.—When these genetic results are compared to those for 250 KV X-rays, the mutation rate for betatron treatments was slightly lower (.76), the recessive lethal rate among induced mutations was higher, and the chromosome aberrations among lethal mutations were slightly lower than with 250 KV X-rays. Although the two types of irradiations differ by an ion density of approximately ten, the amount and types of inheritable genetic damage induced by the two radiations in mature sperm were not significantly different.