Induction of complete and mosaic sex-linked recessive lethal mutations by cigarette smoke filtrate in Drosophila melanogaster.

The genotoxic effects of cigarette smoke filtrate (SF) on the germ-line stages were examined in Drosophila melanogaster using the sex-linked recessive lethal test, which detects a broad spectrum of genetic alterations and proved to show correlations between mutagenicity and carcinogenicity of the tested chemicals. SF was extracted from fiberglass filter cartridges; each used in smoking 15 cigarettes. The proper SF concentrations (0.2 µL) in 0.45% NaCl saline were injected intraperitoneally in 2- to 3-day-old wild-type males, alongside with controls injected with 0.2 µL of saline. The genotoxicity effects of SF were examined in all spermatogenesis stages of treated males. Results showed that SF was toxic with an median lethal dose value of approximately 0.2% and induced significant sterility effects. The mutagenicity of SF (0.2%) was significantly stage specific and induced complete sex-linked recessive lethal mutations in the broods representing the spermatocytes and late and early spermatogonia, and induced mosaic mutations in the untreated progeny in the brood representing late spermatogonia. These results indicated, for the first time, that SF induces mosaic mutations, which could result from DNA instabilities and labile permutations that can be replicated and passed to future generations before being fixed into mutations in the untreated progeny of treated males, or originating from mutations that result in increasing hyperplasia of the gonad that subsequently produce the actual mutations in later cell cycles. Such delayed mutagenic effects of SF indicated that SF and, consequently, cigarette smoking have much greater genotoxicity than what was previously predicted.

Genetic properties of N-alpha-acetoxymethyl-N-methylnitrosamine in relation to the metabolic activation of N,N-dimethylnitrosamine.

A genetic study was undertaken in Drosophila with N-alpha-acetoxymethyl-N-methylnitrosamine, a precursor of the alpha-hydroxymethyl derivative of N,N-dimethylnitrosamine, to assess the role of alpha-carbon oxidation in toxicological activation. Genetic activity was measured for the whole testicular tissue with respect to the general response of the X chromosome (recessive lethals and visibles), as well as certain specific genic sites, including representatives of the RNA genes. The biological activity of the acetoxy compound proved to be considerably higher than that of the parent amine with respect to both cytotoxicity and mutagenicity. At low and equitoxic molarities of the 2 agents (0.1 to 1.0 and 1.0 to 10.0 mM, respectively), dose-dependence for all the investigated genetic functions followed identical patterns, which were best described by quadratic dose curves. However, the regression coefficients for the acetoxy derivative were at least 1 order of magnitude higher than the corresponding values for the amine, indicating a consistent level of mutagenic activation as a result of the alpha-acetoxymethyl substitution. At mutagenically equivalent doses, the 2 compounds gave statistically comparable frequencies of mosaicism among corresponding mutational classes and equal ribosomal DNA selectivity indices, indicating identical molecular mechanisms of mutagenesis. The higher mutagenicity of N-alpha-acetoxymethyl-N-methylnitrosamine compared to N,N-dimethylnitrosamine was paralleled by its higher carcinogenicity which would suggest that the same effective metabolites might be involved in both processes.

Assaying potential carcinogens with Drosophila.

Drosophila offers many advantages for the detection of mutagenic activity of carcinogenic agents. It provides the quickest assay system for detecting mutations in animals today. Its generation time is short, and Drosophila is cheap and easy to breed in large numbers. The simple genetic testing methods give 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 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. The recent findings that Drosophila is capable of carrying out the same metabolic activation reactions as the mammalian liver makes the organism eminently suitable for verifying results obtained in prescreening with fast microbial assay systems. An additional advantage in this respect is the capacity of Drosophila for detecting short-lived activation products, because intracellular metabolic activation appears to occur within the spermatids and spermatocytes.

Mutagenicity testing of diethylene glycol monobutyl ether.

The mutagenic potential of diethylene glycol monobutyl ether (diEGBE) was examined with a Tier I battery of in vitro assays followed by a Tier II in vivo Drosophila sex-linked recessive lethal assay. The in vitro battery consisted of: the Salmonella mutagenicity test, the L5178Y mouse lymphoma test, a cytogenetics assay using Chinese hamster ovary cells and the unscheduled DNA synthesis (UDS) assay in rat hepatocytes. Results of the Salmonella mutagenicity test, the cytogenetics test, and the rat hepatocyte assay were negative at concentrations up to 20 microL/plate, 7.92 microL/mL, and 4.4 microL/mL, respectively. Toxicity was clearly demonstrated at all high doses. A weak, but dose-related increase in the mutation frequency (4-fold increase over the solvent control at 5.6 microL/mL with 12% survival) was obtained in the L5178Y lymphoma test in the absence of metabolic activation. Results of the mouse lymphoma assay were negative in the presence of the S-9 activation system. The significance of the mouse lymphoma assay were negative in the presence of the S-9 activation system. The significance of the mouse lymphoma assay results were assessed by performing the Tier II sex-linked recessive lethal assay in Drosophila in which the target tissue is maturing germinal cells. Both feeding (11,000 ppm for 3 days) and injection (0.3 microL of approximately 14,000 ppm solution) routes of administration were employed in the Drosophila assay. Approximately 11,000 individual crosses with an equal number of negative controls were performed for each route of administration. diEGBE produced no increase in recessive lethals under these conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Genotoxicity modulation by cadmium treatment: studies in the Drosophila wing spot test.

The genotoxic activity of cadmium chloride (CC) has been evaluated in the somatic mutation and recombination test (SMART) in Drosophila melanogaster. In addition, its possible modulating effect on the genotoxicity of two known mutagenic agents, potassium dichromate (PDC) and ethyl methanesulfonate (EMS), was investigated. Three different types of combined treatments of CC with the two genotoxins were performed: pretreatment, cotreatment, and posttreatment. The SMART assay is based on the principle that loss of heterozygosity for the recessive markers, multiple wing hairs (mwh) and flare-3 (flr(3)), leads to the formation of mutant clones in the imaginal disks of larvae, which are expressed as mutant spots on the wings of adult flies. Thus, after adult emergence, the wings of the adult flies were scored for the presence of single and/or twin spots. Our results show that CC alone was not effective in increasing the frequency of any of the three categories of spots (small, large, and twin). In the cotreatment experiments, CC increased the genotoxicity of PDC but it decreased the genotoxicity of EMS. No effects of CC were observed in the pretreatment or posttreatment experiments; however, only low concentrations of CC, PDC, and EMS were tested in the pretreatment assays due to the high toxicity of the treatment. Although our results with PDC are consistent with the hypothesis that cadmium can interfere with repair mechanisms, the EMS data suggest that other modulating mechanisms are also involved in the genotoxicity of this metal.

Genotoxicity of two arsenic compounds in germ cells and somatic cells of Drosophila melanogaster.

Two arsenic compounds, sodium arsenite (NaAsO2) and sodium arsenate (Na2HAsO4), were tested for their possible genotoxicity in germinal and somatic cells of Drosophila melanogaster. For germinal cells, the sex-linked recessive lethal test (SLRLT) and the sex chromosome loss test (SCLT) were used. In both tests, a brood scheme of 2-3-3 days was employed. Two routes of administration were used for the SLRLT: adult male injection (0.38, 0.77 mM for sodium arsenite; and 0.54, 1.08 mM for sodium arsenate) and larval feeding (0.008, 0.01, 0.02 mM for sodium arsenite; and 0.01, 0.02 mM for sodium arsenate). For the SCLT the compounds were injected into males. Controls were treated with a solution of 5% sucrose which was employed as solvent. The somatic mutation and recombination test (SMART) was run in the w+/w eye assay as well as in the mwh +/+ flr3 wing test, employing the standard and insecticide-resistant strains. In both tests, third instar larvae were treated for 6 hr with sodium arsenite (0.38, 0.77, 1.15 mM), and sodium arsenate (0.54, 1.34, 2.69 mM). In the SLRLT, both compounds were positive, but they were negative in the SCLT. The genotoxicity of both compounds was localized mainly in somatic cells, in agreement with reports on the carcinogenic potential of arsenical compounds. Sodium arsenite was an order of magnitude more toxic and mutagenic than sodium arsenate. This study confirms the reliability of the Drosophila in vivo system to test the genotoxicity of environmental compounds.

An investigation of some genetic toxicological effects of the fungicide benomyl.

The widely used fungicide benomyl (methyl-1-(butylcarbamoyl)-2-benzimidazole carbamate) and its breakdown product methyl-2-benzimidazole carbamate (MBC) have been reported to have mutagenic activity in some organisms. In experiments with Drosophila melanogaster we found (i) there was no significant increase in recessive lethal frequency after feeding adult flies with Benlate did not increase chromosome breakage or loss significantly; (ii) there was a relatively high incidence of sterility when males of one strain (Oregon R) were fed Benlate or MBC. In experiments in which cultures of human lymphocytes were exposed in vitro to 0.5 mg/ml MBC we observed extreme contraction of the chromosomes but found no evidence of an increase in the number of cells with chromosome aberrations. We conclude that benomyl and MBC are unlikely to be strong mutagens, but more research is needed to exclude the possibility that they are capable of inducing genetic damage in the germ cells of higher organisms.

Comparison of expression of aldehyde dehydrogenase 3 and CYP1A1 in dominant and recessive aryl hydrocarbon hydroxylase-deficient mutant mouse hepatoma cells.

The mouse hepatoma cell line Hepa-1 is inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for both CYP1A1 (aryl hydrocarbon hydroxylase, AHH) and class 3 aldehyde dehydrogenase (ALDH3) enzymes. To test the hypothesis of a common regulatory mechanism, several AHH deficient mutants of Hepa-1 were studied for their ALDH3 activities and specific mRNA levels before and after TCDD treatment. The recessive (with respect to the wild-type Hepa-1) mutants have defects in Cypla-1 structural gene (mutant c1) or in the Ah (aryl hydrocarbon) receptor (mutants c2 and c6 with decreased levels of Ah receptor; mutant c4 defective in the DNA binding of the Ah receptor). The results with these mutants suggested that Ah receptor nuclear translocator protein, ARNT, is needed for ALDH3 expression. Two dominant mutants, one of which is characterized by preventing the binding of the Ah receptor complex to DNA, were also studied. Surprisingly, these mutants possessed elevated levels of ALDH3 mRNA and enzyme activities which were also inducible by TCDD. The binding of Ah receptor-ligand complex to DNA was thus not needed for the expression of ALDH3. A dominant repressor for Cypla-1 gene transcription did not prevent the derepression or induction of ALDH3. The results thus suggest that Aldh-3 gene is regulated by a mechanism independent of the Ah receptor.

Mutation frequencies of Drosophila melanogaster reared in glass and plastic vials.

The mutation frequency of the sex-linked recessive lethal assay resulting from the examination of 9032 tests of Drosophila melanogaster reared in plastic vials (0.198%) was approximately twice that of Drosophila melanogaster reared in glass vials (0.085%) after examination of 9365 tests. This difference was found to be significant (P less than 0.05). This significant increase in the mutation frequency may be due to styrene which is one of the components of the plastic vials. Also, the frequency of failure was greater when using plastic vials (1.19%) compared to glass vials (0.71%). In the following study we examined D. melanogaster SLRL mutation frequencies and failure rates when using glass and plastic vials. Our results showed a doubling in the SLRL mutation rate.

Evaluation of mutagenic effect of the fungicide fenaminosulf in Drosophila melanogaster.

Fenaminosulf (p-dimethylaminobenzenediazo sodium sulfonate, CAS registry No. 140-56-7) which is an active ingredient in several commercial fungicides was reported to be mutagenic in Salmonella typhimurium (McCann et al., 1975), Bacillus subtilis (Kada et al., 1974) and shown to cause chromosome aberrations in plants (Zutshi and Kaul, 1975). Since fenaminosulf has structural similarity to the potent carcinogen, butter yellow (p-dimethylaminoazobenzene, CAS registry No. 60-11-7), the present studies were undertaken to evaluate the mutagenic potential of this fungicide in Drosophila melanogaster. Fenaminosulf administered at 10 mg/100 ml food medium failed to induce sex-linked recessive mutations in Drosophila. Since Drosophila has drug-metabolizing enzymes similar to those of mammals (Vogel, 1975), it is suggested that the lack of mutagenic activity of fenaminosulf could be due to the conversion of fenaminosulf to non-mutagenic derivatives in Drosophila.

Mutagenicity of nickel sulphate in Drosophila melanogaster.

Nickel sulphate was injected into Drosophila melanogaster males at different concentrations in order to test the chemical for the induction of SLRL and SCL in germ cells. Nickel sulphate induced SLRL at concentrations tested, with the peak of activity at premeiotic and postmeiotic stages. It failed to produce SCL except at the highest concentration tested, where induction of XO males was significant for the pooled data.

Mutagenicity of 1,2-dibromo-3-chloropropane (DBCP) in the mouse spot test.

The spot test with 1,2-dibromo-3-chloropropane (DBCP) was carried out using male PW and female C57BL/6 mice. DBCP induced recessive colour spots in offspring with a significantly high frequency of 2.9%, showing that this chemical is mutagenic for somatic cells of mice in vivo.

Mutations induced by a hormonal imbalance in Drosophila melanogaster.

Pterin treatment of chrysalids in diapause modified the juvenile hormone- ecdysone ratio. The treatment of Drosophila donor mutant with a mixture of reduced folic acid, pterins and extract of Pierides chrysalids in diapause induced the formation of short sequences, by the intermediary of variations in hormonal balance. The effect of this variation was seen in the germinal lines especially at the gonial stage, where recessive, visible or lethal mutations were induced in the form of clusters. Genetically active fractions were found in the 4S-8S and 18S-28S sedimentation zones after saccharose gradient ultracentrifugation. The short DNA sequences coded for tRNA and rRNA. Consequently, it is there DNA sequences from the Drosophila donor mutant that altered the genetic information of the host. 2 types of recessive visible mutation appeared: those affecting the differentiation of the imaginal discs and those affecting the pigment biosynthetic chain. Recessive lethals were induced by treatment. 3 hypothesis are proposed: the first suggests the formation of a short DNA sequence complexing at a specific locus in the acceptor. The second involves transposable factors belonging to the acceptor itself, behaving as a particular transposable factor. The third supposes the induction of alterations at loci of rRNA and tRNA synthesis at the origin of perturbations in protein synthesis. The present data do not allow us to choose between the 3 hypotheses. In conclusion, it seems that a "hormonal imbalance" can have grave consequences not only for the individual itself but also for its descendants.

The sex-linked recessive lethal test for mutagenesis in Drosophila melanogaster. A report of the U.S. Environmental Protection Agency Gene-Tox Program.

The test for sex-linked recessive lethals (SLRL) in Drosophila melanogaster has been used to detect induced mutations since 1927. The advantage of the test for both screening and hazard evaluation is its objectivity in testing for transmissible mutations in the germ cells of a eukaryote. Statistical criteria for both positive and negative mutagenicity at the highest concentration tested under a particular exposure condition were developed by the Work Group, and a recommended protocol for future testing was agreed upon. For 421 compounds there were sufficient data available in the literature for analysis; 198 compounds were found to be positive and 46 negative at the highest concentration tested. Most experiments had been done for objectives of pure research rather than for deliberately screening for mutagenicity, although many of the 421 chemicals were selected for testing because of suspected mutagenicity. Therefore, the statement of 198 positive and 46 negative should not be taken as an example of the proportion of mutagens in the environment. In three sets of experiments with D. melanogaster that were done specifically for screening, one involving 40 compounds for the Environmental Protection Agency (EPA), the others involving 13 for the Food and Drug Administration (FDA), only 6 mutagens were discovered. After completion of the classification of compounds according to their response in the SLRL test, the compounds were classified as to their carcinogenic response according to the list of Griesemer and Cueto (1980). There were 62 compounds that could be classified as positive or negative for both carcinogenesis and mutagenesis. Of the 62 compounds, there was agreement between the carcinogenesis and mutagenesis classification in 56 (50 positive and 6 negative), or 90% would have been correctly classified as to carcinogenesis from only the SLRL test. Because of inadequate sample size, 177 compounds could not be classified as positive or negative according to the statistical criteria established by the Work Group. This large number of inadequately tested compounds reflects the fact that many of the experiments were not done for screening. Further work is needed on the compounds with inadequate sample size.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of methylation and ring size on mutagenicity of cyclic nitrosamines in Drosophila melanogaster.

The mutagenic activity of 7 nitrosopiperazines, 2 nitropyrrolidines, and 3 nitrosomorpholines was examined in the X-linked recessive-lethal assay of Drosophila melanogaster. Mutagenicity is also reported for a series of cyclic nitrosamines that differ in structure only in the number of carbon atoms in the ring. Of the 18 compounds tested, 6 (nitrosopiperazine; 2,3,5,6-tetramethyldinitrosopiperazine; nitrosoproline; 2,5-dimethylnitrosopyrrolidine; nitrosothiomorpholine; and nitrosooctamethyleneimine) were nonmutagenic. As we reported earlier in investigations with the nitrosopiperidines, substitutions with methyl groups at all of the alpha-carbon atoms reduce or eliminate the mutagenic activity of dinitrosopiperazine and nitrosopyrrolidine.

Metabolic inactivation of mutagens in Drosophila melanogaster.

The route of administration of a drug is a pharmacological factor to be reckoned with. In Drosophila, a whole-animal object for mutagenicity studies, the way in which a mutagen is applied can also be of crucial importance. In this study the mutagenicity of a number of directly acting agents was determined after feeding or injection of the mutagen. Methyl-p-toluenesulphonate (Me-Tos), ethyl-p-toluenesulphonate (Et-Tos) and nor-nitrogen mustard (NNM) were not mutagenic in a sex-linked recessive lethal test when fed to the adult flies. Injection, however, did produce significant mutagenicity. The absence of mutagenicity after oral application is not caused by chemical instability but is the result of metabolic de-activation, presumably in the gut and the fat body. Feeding of these compounds in combination with the inhibition of cytochrome P-450 by 1-phenylimidazole (PhI) allowed sufficient quantities of the mutagen to reach the gonads and to produce significant genetic damage. This resembles what is known in pharmacology as a 'first-pass effect'. Formaldehyde (FA) mutagenicity, which also is only observed after injection and not in feeding experiments, was not affected by either iproniazid (Ipr) or PhI pretreatment. Aspecific enhancement of mutagenicity is excluded as this effect was not observed with mutagens that are structurally related to the tosylates, such as methyl methanesulphonate (MMS), ethyl methanesulphonate (EMS) or hycanthone methanesulphonate (HyMS). A number of other inhibitors of metabolism did not influence metabolic de-activation in Drosophila.

Dominant cataract and recessive specific-locus mutations detected in offspring of procarbazine-treated male mice.

The induction of dominant cataract mutations by procarbazine was studied concomitantly with the induction of specific-locus mutations in treated male mice. The most effective dose in the specific-locus test, 600 mg/kg of procarbazine, and a fractionated dose of 5 X 200 mg/kg were used. The frequencies of dominant cataract mutations were higher, but not significantly different from the historical control. The ratio between the number of recovered specific-locus and dominant cataract mutations was in accordance with that found in our experiments with gamma-rays (Ehling et al., 1982; Kratochvilova, 1981) or in experiments with ethylnitrosourea (Favor, 1986). A total of 3 dominant cataract mutations were recovered in the offspring of procarbazine-treated spermatogonial stem cells. Two mutations had complete penetrance while the third exhibited a reduced penetrance of approximately 70%. The viability and fertility of the heterozygotes of all 3 mutations were not affected. Only 1 mutation was shown to be viable as a homozygote.

Effect of X-ray and ethylnitrosourea exposures separated by 24 h on specific-locus mutation frequency in mouse stem-cell spermatogonia.

Specific-locus mutation frequencies in mouse stem-cell spermatogonia were determined in 3 experiments in which mature male mice were exposed to 100,m 300, or 500 R of X-rays followed, 24 h later, by intraperitoneal injection of 100 mg/kg of ethylnitrosourea (ENU). The purpose was to find out if the mutation frequencies would be augmented over those expected on the basis of additivity of the effects of the separate treatments. Such augmentation had been observed in earlier work in which exposure to 100 or 500 R of X-rays was followed 24 h later by a second exposure of 500 R. No augmentation was observed for X-rays followed by ENU. The mutation frequencies in all 3 experiments actually fell below those expected on the basis of additivity, although the reductions were not statistically significant.

Dosage-response relationships for methyl methanesulfonate in Drosophila melanogaster spermatozoa: DNA methylation per nucleotide vs. sex-linked recessive lethal frequency.

Two different mechanisms for mutagenesis following treatment with methyl methanesulfonate (MMS) are suggested from the dose-response curve that is best fit by the linear quadratic model where m = 0.130D + 0.038D2 (D = dose measured as alkylations per nucleotide X 10(3), APdN; m = percent sex-linked recessive lethals, SLRL). A predominant component of the dose-response curve at moderate to high dose is the quadratic component which is interpreted as the result of two single-strand breaks. The distribution of methyl adducts in vivo is consistent with the previously determined in vitro distribution of methyl adducts on DNA following treatment with MMS. With the use of HPLC, 82% of the 3H-labeled adducts are found on the N-7 of guanine. It has previously been shown by both in vitro studies and in vivo correlation with mutagenesis that the N-7 alkyl guanine is not itself a predominately genotoxic lesion; however, N-7 alkyl guanine destabilizes guanine resulting in an increased rate of hydrolysis producing apurinic sites. In data presented in this paper, the loss of labeled adducts is shown to be at a rate consistent with hydrolysis of the destabilized alkyl guanine. The apurinic site thus produced should be converted to single-strand breaks by AP endonucleases once sperm has fertilized the egg. Single-strand breaks are repaired by excision repair which is not error-prone; however, multiple breaks producing a proximity effect should lead to double-strand breaks that are repaired by an error-prone process. Mutations that are induced by a proximity effect would account for the quadratic term. It is hypothesized that a proximity effect is produced when two breaks are sufficiently close together to prevent using the complementary strand as a template. The linear component of the dose-response curve is probably due to alkylation of oxygens in the purine or pyrimidine ring leading to mispairing. However, due to the low frequency of ring-oxygen alkylation following treatment with MMS, this important genotoxic site is not the predominant one observed at experimental levels normally used in the laboratory. From the dose-response curve, it is calculated that at mutation frequencies of 10 times the spontaneous frequency or higher, the predominant mechanism is the multi-hit component; however, at mutation induced frequencies of 0.1 of the spontaneous frequency, which are levels more likely to be encountered in man's exposure to environmental mutagens, the dominant mechanism is the linear component.(ABSTRACT TRUNCATED AT 400 WORDS)

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.