Additional data in support of the quadruplicated white-ivory reversion system to test for somatic genotoxicity in Drosophila melanogaster

Cyclophosphamide, ethyl methanesulfonate, propyleneimine and tritiated water were tested in a new short-term somatic mutation bioassay, previously described by Green and coworkers (1986), to evaluate the suitability of the quadruplicated white-ivory system of Drosophila melanogaster for genotoxicity testing of chemicals. A 2.9-kb tandemly duplicated sequence of w⁺ within a w⁺ gene is responsible for the white-ivory phenotype. Reversion of wⁱ to w⁺ is in general, associated with the loss of the appended 2.9 kb or other alterations affecting this duplicated region. The appearance of light (white or nearly white) phenotypes could be due to the loss of some wⁱ copies. Thus, in the eyes of adult males resulting from treated larvae, we can detect 2 types of sectors (red and light) on the orange-yellow background. Our results indicate that the genetic system used in this somatic assay is sensitive to the genotoxic effects of the 4 compounds tested. All 4 compounds tested were positive. Tritiated water had weak effects, cyclophosphamide was characterized by small revertant clones and ethyl methanesulfonate and propyleneimine by large clone size. From the available data, we feel that the wⁱ system can be considered of potential value for genotoxicity testing.     

Genotoxic potency of monofunctional alkylating agents in E. coli: comparison with carcinogenic potency in rodents

A quantitative correlation between carcinogenicity and genotoxicity was investigated by a comparison between the carcinogenic potency in rodents and the mutagenic (M), recombinogenic (R) and SOS-inducing (I) potencies in a bacterial test (E. coli multitest) for 9 monofunctional alkylating agents: N-nitroso-N-methylurethane, N-nitroso-N-ethylurea, epichlorohydrin, N-nitroso-N-methylurea, N-nitroso-N-methyl-N'-nitroguanidine, methyl methanesulfonate, diethylsulfate, dimethylsulfate, ethyl methanesulfonate. A significant positive correlation between the carcinogenic potency and the product of the mutagenic and recombinogenic potencies was found for all tested compounds. Thus, the E. coli multitest may be used as a simple test to search for correlations between carcinogenicity and genotoxicity of DNA-damaging agents.     

Comparison of Somatic Reversions between the Ivory Allele and Transposon-Caused Mutant Alleles at the White Locus of DROSOPHILA MELANOGASTER after Larval Treatment with X Rays and Ethyl Methanesulfonate

Somatic reversion of strains with the ivory (wi) allele, a mutation associated with a tandem duplication of a DNA sequence at the white locus, increased with the age of larvae at the time of X-irradiation as expected from the increase in the number of target cells. In contrast, two independently isolated strains with unstable w+ loci associated with insertion of transposable elements showed higher reversion frequencies after treatment with X rays or ethyl methanesulfonate (EMS) at early larval stages than at late stages. Nevertheless, both the wi strain and the two unstable w+ strains reverted at nearly equal rates after treatment with X rays or EMS at early larval stages. Possible similarity in "hot spot" structure for the high reversibility of the two types of mutations is discussed in relation to production of presumed "mutator-type" cofactors specific to the transposon-caused mutations at early larval stages.     

A small tandem duplication is responsible for the unstable white-ivory mutation in Drosophila

The white-ivory (wi) mutation, an unstable allele of the white locus in Drosophila, reverts to wild-type at frequencies of 5 X 10(-5) in homozygous females, and 5 X 10(-6) in males and deletion heterozygous females. We show by molecular cloning and Southern blot analysis of DNA from wi flies that a 2.9 kilobase tandem duplication within the white locus is responsible for the mutation. Phenotypic reversion appears, in most cases, to be due to an exact excision of the extra copy of the sequence. Two derivative alleles of wi, one phenotypically wild-type, the other a partial revertant, carry insertions of moderately repetitive DNA from outside the locus, in addition to suffering deletions of some white locus DNA. Earlier genetic data preclude unequal crossing-over between homologs as an explanation for the precise reversions. Rather, an intrachromosomal meiotic event seems to be responsible. Our results suggest that intrachromosomal recombination may be responsible in other systems for a larger number of rearrangements than has been suspected, and that interallelic recombination frequencies in Drosophila do not always correlate in a simple way with DNA length or extent of homology.     

Effects of artichoke (Cynara scolymus) leaf and bloom head extracts on chemically induced DNA lesions in Drosophila melanogaster

The genotoxicity of bloom head (BHE) and leaf (LE) extracts from artichoke (Cynara scolymus L.), and their ability to modulate the mutagenicity and recombinogenicity of two alkylating agents (ethyl methanesulfonate – EMS and mitomycin C – MMC) and the intercalating agent bleomycin (BLM), were examined using the somatic mutation and recombination test (SMART) in Drosophila melanogaster. Neither the mutagenicity nor the recombinogenicity of BLM or MMC was modified by co- or post-treatment with BHE or LE. In contrast, co-treatment with BHE significantly enhanced the EMS-induced genotoxicity involving mutagenic and/or recombinant events. Co-treatment with LE did not alter the genotoxicity of EMS whereas post-treatment with the highest dose of LE significantly increased this genotoxicity. This enhancement included a synergistic increase restricted to somatic recombination. These results show that artichoke extracts promote homologous recombination in proliferative cells of D. melanogaster.     

The w/w+ SMART assay of Drosophila melanogaster detects the genotoxic effects of reactive oxygen species inducing compounds

The somatic mutation and recombination w/w+ eye assay has been used for genotoxic evaluation of a broad number of chemicals with different action mechanisms yielding high values of sensitivity, specificity and accuracy. The aim of this work was to determine the utility of this assay in the evaluation of reactive oxygen species inducers. For this, we have tested eight compounds: diquat, paraquat, menadione, juglone, plumbagin, streptonigrin, tert-butyl hydroperoxide and 4-nitroquinoline 1-oxide, using the Drosophila Oregon K strain which had previously shown advantageous conditions to test this type of compounds. Diquat was the only chemical for which the results were clearly negative, probably because its high toxicity, whereas indications of a marginal genotoxicity raised for menadione. The remaining compounds were evaluated as positives. The conclusion of these experiments is that the w/w+ assay is capable to detect genotoxic effects induced by compounds that generate reactive oxygen species through different action mechanisms.     

The white-ivory somatic mutation test in Drosophila. The effects of larval age, increasing the number of copies of wi and the response to some reference mutagens.

The white-ivory somatic mutation test is based on reversion of the X-linked, recessive eye-colour mutation white-ivory to wild-type. Somatic reversion of white-ivory leads to clones of red facets in the white-ivory eyes of eclosing adult flies. The alkylating agents MMS, EMS and ENU and the mutagen DEB all induce high levels of white-ivory reversion. The response of different larval instars has been investigated and dose-response data for MMS and EMS is reported. Strains with additional copies of the white-ivory mutation are more sensitive to reversion but the increase in reversion is not as high as might be expected given the number of white-ivory mutations present. Females, having twice the number of white-ivory copies, tend to show higher levels of reversion than males of the same strain.     

Somatic cell mutagenesis in Drosophila: recovery of genetic damage in relation to the types of DNA lesions induced in mutationally unstable and stable X-chromosomes

This paper reports the results of a study on the genotoxic activities of 12 mutagens and clastogens of widely differing mode of action in somatic cells in vivo, i.e., in the eye primordia of Drosophila larvae. After emergence, adult flies were monitored for aberrantly colored sectors in the compound eyes of the following genotypes: UZ males and females (zeste) carrying a genetically unstable transposable element, SZ males and females (zeste) carrying a partial duplication of the w+ locus plus a transposon insert, white-coral/white (wco/w) females, w+/w females and w+ males. The UZ and SZ marker sets make it possible to monitor shifts from zeste to red (scored as mosaic red spots, RS) and for loss of the white locus (light spots, LS). wco/w+ females were scored for mosaic twin spots (TS) and LS, w+ genotypes for just LS. The genotoxins analyzed were methyl methanesulfonate (MMS), dimethyl sulfate (DMS) and ethylnitrosourea (ENU) (alkylating), adriamycin (AM) and daunomycin (DM) (intercalating), Trenimon, Thio-TEPA and cisplatin (DDP) (cross-linking), bleomycin (strand-breaking), 7,12-dimethylbenz[a]anthracene (DMBA) and 9,10-dimethylanthracene (DA) (bulky monoadducts) and cytosine arabinofuranoside (inhibition of DNA synthesis). The relative mutabilities with frequencies of mosaic light spots (LS) in w+/w female as the standard (relative mutability = 1) vs. genotypes UZ (RS in male) vs. SZ (RS in male) vs. w+ (LS in male) were 1:0.6:0.2:0.3 for MMS, 1:0.09:0.05:0.7 for DDP, and 1:1.6:0.2:1.0 for ENU, ENU showed exceptional behavior in that it was the only compound for which mutational response, measured by the induction of red spots, was highest with the UZ marker set. Occurrence of large light spots (LS) in male but not in female genotypes was negatively correlated with efficiency of agents for chromosomal damage, suggesting that in the hemizygous condition, as in males, selection of damaged cells and mitotic delay may have played a significant role. In general, the results indicate that there is no association between the ability of an agent to act as a clastogen and the recovery of aberrant (red spots) sectors in either the UZ or the SZ strain, and of single light spots (LS) in w+, UZ and SZ males. The possibility is considered that the process causing the genetic instability in the UZ strain is under genetic control, and that strong point mutagens such as ENU through efficient gene mutation induction can interfere with it.     

Possible repair action of Vitamin C on DNA damage induced by methyl methanesulfonate, cyclophosphamide, FeSO4 and CuSO4 in mouse blood cells in vivo

Interaction between Vitamin C (VitC) and transition metals can induce the formation of reactive oxygen species (ROS). VitC may also act as an ROS scavenger and as a metal chelant. To examine these possibilities, we tested in vivo the effect of two doses of VitC (1 and 30 mg/kg of mouse body weight) on the genotoxicity of known mutagens and transition metals. We used the alkaline version of the comet assay to assess DNA damage in peripheral white blood cells of mice. Animals were orally given either water (control), cyclophosphamide (CP), methyl methanesulfonate (MMS), cupric sulfate or ferrous sulfate. A single treatment with each VitC dose was administered after treatment with the mutagens or the metal sulfates. Both doses of VitC enhanced DNA damage caused by the metal sulfates. DNA damage caused by MMS was significantly reduced by the lower dose, but not by the higher dose of VitC. For CP, neither post-treatment dose of VitC affected the DNA damage level. These results indicate a modulatory role of Vitamin C in the genotoxicity/repair effect of these compounds. Single treatment with either dose of VitC showed genotoxic effects after 24 h but not after 48 h, indicating repair. Double treatment with VitC (at 0 and 24 h) induced a cumulative genotoxic response at 48 h, more intense for the higher dose. The results suggest that VitC can be either genotoxic or a repair stimulant, since the alkaline version of the comet assay does not differentiate "effective" strand breaks from those generated as an intermediate step in excision repair (incomplete excision repair sites). Further data is needed to shed light upon the beneficial/noxious effects of VitC.     

Genotoxic profile of inhibitors of topoisomerases I (camptothecin) and II (etoposide) in a mitotic recombination and sex-chromosome loss somatic eye assay of Drosophila melanogaster

Genotoxic carcinogens which interact with DNA may produce double-strand breaks as normal intermediates of homologous mitotic recombination, and may give rise to structural chromosome aberrations and inter-chromosomal deletion-recombination. The genotoxic profile of two inhibitors of DNA topoisomerases were evaluated using an in vivo somatic w/w+ eye assay of Drosophila melanogaster for the detection of loss of heterozygosity (LOH) by homologous mitotic recombination, intra-chromosomal recombination and structural chromosomal aberrations. We studied camptothecin (CPT) as a topoisomerase-I-interactive agent and etoposide (ETOP) as a topoisomerase II inhibitor. These drugs act by stabilizing a ternary complex consisting of topoisomerases covalently linked to DNA at single-strand or at double-strand breaks, thereby preventing the relegation step of the breakage/rejoining reaction mediated by the enzyme. The genotoxic profiles were determined from the appearance of eye tissue in adult flies, in which LOH and expression of the reporter gene white produced light clones. The results demonstrated that both compounds were significantly genotoxic, with CPT being more effective than ETOP. Inter-chromosomal mitotic recombination was the major mechanism responsible for the induction of light spots by both compounds in XX females. Loss of the ring X chromosome (rX), was significantly enhanced by CPT, and this topoisomerase blocker also produced intra-chromosomal recombination (XY males).     

Characterization of the genotoxicity of anthraquinones in mammalian cells.

Naturally occurring 1,8-dihydroxyanthraquinones are under consideration as possible carcinogens. Here we wanted to elucidate a possible mechanism of their genotoxicity. All three tested anthraquinones, emodin, aloe-emodin, and danthron, showed capabilities to inhibit the non-covalent binding of bisbenzimide Hoechst 33342 to isolated DNA and in mouse lymphoma L5178Y cells comparable to the topoisomerase II inhibitor and intercalator m-amsacrine. In a cell-free decatenation assay, emodin exerted a stronger, danthron a similar and aloe-emodin a weaker inhibition of topoisomerase II activity than m-amsacrine. Analysis of the chromosomal extent of DNA damage induced by these anthraquinones was performed in mouse lymphoma L5178Y cells. Anthraquinone-induced mutant cell clones showed similar chromosomal lesions when compared to the topoisomerase II inhibitors etoposide and m-amsacrine, but were different from mutants induced by the DNA alkylator ethyl methanesulfonate. These data support the idea that inhibition of the catalytic activity of topoisomerase II contributes to anthraquinone-induced genotoxicity and mutagenicity.     

Genotoxicity evaluation of five tricyclic antidepressants in the wing somatic mutation and recombination test in Drosophila melanogaster

Five tricyclic antidepressants were tested for genotoxicity using the somatic mutation and recombination test (SMART) in wing cells of Drosophila melanogaster. Three-day-old larvae trans-heterozygous for 2 linked recessive wing hair mutants (multiple wing hairs and flare) were fed the test compounds in water mixed with a standard dry food for 48 h. Wings of the emerging adult flies were scored for the presence of spots of mutant cells which can be the consequence of either somatic mutation or mitotic recombination. Desipramine and imipramine were clearly genotoxic at concentrations above 1 mM whereas amitriptyline, nortriptyline and protriptyline were not genotoxic at concentrations up to 100 mM. This seems to implicate the nitrogen atom at position 5 in the 7-membered ring of the tricyclic molecule as being responsible for the genotoxic property of the compounds in Drosophila.     

Comparison of chemically induced chromosome loss in a diploid, triploid, and tetraploid strain of Saccharomyces cerevisiae.

Triploid and tetraploid strains of Saccharomyces cerevisiae were constructed and the spontaneous loss during mitosis of one, two or three copies of chromosome VII was determined. In one strain, a triploid (VM2) in which expression of the recessive alleles can be observed only after loss of two copies of chromosome VII (3N-2), the spontaneous frequency of chromosome loss was lower than in the diploid D61.M. In another strain, a tetraploid (VM4) that also requires the loss of two copies of chromosome VII for observation (4N-2) of the recessive alleles, the spontaneous frequency was slightly higher than in the diploid D61.M. The spontaneous frequency of other genetic events (that is, mutation, recombination or chromosome breakage) were lower by 2-3 orders of magnitude than in the diploid strain D61.M. Induction of chromosome loss and other genetic events by nocodazole, ethyl acetate, hydroxyurea and ethyl methanesulfonate was determined in D61.M, VM2, and VM4, and the results were compared. Nocodazole and ethyl acetate induced chromosome loss in both the triploid and the tetraploid strains at lower concentrations than required in the diploid. These compounds also induced elevated frequencies of other genetic events in both the triploid and the tetraploid strains but not in the diploid. Hydroxyurea induced elevated frequencies of chromosome loss in the diploid and the tetraploid. Frequencies of chromosome loss in the triploid treated with hydroxyurea, although elevated, are based on observation of very few colonies of the correct phenotype. Ethyl methanesulfonate failed to induce chromosome loss in any of the three strains. Hydroxyurea and ethyl methanesulfonate did, however, induce very high frequencies of other genetic events.     

Modifying action of gamma-radiation in mutagenesis of E. coli WU36-10 induced by ethylene oxide, ethyl methanesulfonate and methyl methanesulfonate

The influence of gamma-radiation pre-exposure on ethylene oxide, ethyl methanesulfonate and methyl methanesulfonate mutagenesis in Escherichia coli WU36-10 was studied. Pretreatment with gamma-radiation resulted, in the case of subsequent treatment with ethylene oxide and ethyl methanesulfonate, in a decrease of the frequency of leu+ revertants, and in the case of subsequent treatment with methyl methanesulfonate, in an increase of this mutation frequency.     

Distribution and evolutionary significance of mitochondrial plasmids in Neurospora spp.

The lethal and mutagenic effects of various mutagens on Neisseria gonorrhoeae were investigated. Lethality studies demonstrated that N. gonorrhoeae was relatively sensitive to ethyl methanesulfonate, UV light, and methyl methanesulfonate. Although N. gonorrhoeae was readily mutated by ethyl methanesulfonate and N-methyl-N'-nitro-N-nitrosoguanidine for the three genetic markers assayed, no increase in the mutation frequency was observed for any of the selective markers after UV irradiation or methyl methanesulfonate treatment. These results suggest that N. gonorrhoeae lacks an error-prone repair mechanism.     

The mutagenicity of natural products from marine algae.

5 polyhalogenated hydrocarbon natural products isolated from the marine red alga Plocamium spp. were tested for mutagenicity in the Ames reversion assay. All 5 of the compounds induced revertants in Salmonella typhimurium strains TA100 and TA1535, indicating the mutational events involved base substitutions. One of the compounds, designated cross-conjugated ketone, was shown to be almost 200 times more effective as a mutagen than was ethyl methanesulfonate.     

Induction of homologous recombination following in utero exposure to DNA-damaging agents

Much of our understanding of homologous recombination, as well as the development of the working models for these processes, has been derived from extensive work in model organisms, such as yeast and fruit flies, and mammalian systems by studying the repair of induced double strand breaks or repair following exposure to genotoxic agents in vitro. We therefore set out to expand this in vitro work to ask whether DNA-damaging agents with varying modes of action could induce somatic change in an in vivo mouse model of homologous recombination. We exposed pregnant dams to DNA-damaging agents, conferring a variety of lesions at a specific time in embryo development. To monitor homologous recombination frequency, we used the well-established retinal pigment epithelium pink-eyed unstable assay. Homologous recombination resulting in the deletion of a duplicated 70 kb fragment in the coding region of the Oca2 gene renders this gene functional and can be visualized as a pigmented eyespot in the retinal pigment epithelium. We observed an increased frequency of pigmented eyespots in resultant litters following exposure to cisplatin, methyl methanesulfonate, ethyl methanesulfonate, 3-aminobenzamide, bleomycin, and etoposide with a contrasting decrease in the frequency of detectable reversion events following camptothecin and hydroxyurea exposure. The somatic genomic rearrangements that result from such a wide variety of differently acting damaging agents implies long-term potential effects from even short-term in utero exposures.     

Effect of light quality on somatic embryogenesis of quince leaves

The effect of light quality on somatic embryogenesis in quince BA 29 was investigated. 2,4-D induced leaves were exposed for 25 days to the following light quality treatments: dark, far-red, far-red+blue, far-red+red, blue, white, red+blue, red. After a further 20 days of white light exposure, somatic embryo production was recorded. Somatic embryogenesis was highest in cultures subjected to red light treatment, and decreased progressively with the transition to red+blue and to white. Overall, embryogenic competence showed a correlation with photoequilibrium. Phytochrome appeared to be inductive although this effect was adversely influenced by the blue absorbing photoreceptor, in particular at low photoequilibrium. Independently of light treatments applied, somatic embryos frequently showed severe morphological abnormalities. Conversion of somatic embryos to plantlets was not observed. This revised version was published online in June 2006 with corrections to the Cover Date.     

DNA sequence of the white locus of Drosophila melanogaster

The DNA sequence of the white locus of Drosophila melanogaster is presented. This 14,100 base-pair sequence includes the region of the locus required for wild-type levels of expression and control of expression. We also report the sequence of a complementary DNA clone which established the position of the 3' end of the white RNA on this genomic sequence. The probable exon-intron structure of the gene has been predicted from the DNA sequence of the regions known to be represented in the RNA. The amino acid sequence of the protein which would be produced by translation of this RNA suggests that the white locus gene product may be a membrane protein. The DNA sequence rearrangements associated with seven insertion mutants (white-dominant-zeste-like (wDZL), white-spotted (wsp), white-honey (wh), white-zeste-mottled (wzm), white-apricot (wa), white-buff (wbf) and white-hd81b11 (whd81b11)), one deletion mutant (white-spotted 4 (wsp4)) and one internal duplication mutant (white-ivory (wi)) have been determined and positioned on the wild-type sequence. The positions of these insertions and those of previously characterized insertions associated with six other mutations suggest that some insertions within an intron may still allow the production of correctly spliced RNA, but affect the amount, and correspondingly the expression of the w locus.