Tannic acid is not mutagenic in germ cells but weakly genotoxic in somatic cells of Drosophila melanogaster

Tannic acid (TA) was tested for genotoxic activity in threedifferent assays (1–3) in Drosophila melanogaster by feedingof larvae or adult flies. TA did not induce sex-linked recessivelethals (1) nor sex-chromosome loss, mosaicism or non-disjunction(2) in male germ cells. In the wing somatic mutation and recombinationtest (SMART) (3) TA was found to be toxic for larvae of thehigh bioactivation cross and produced a weak positive response.These results suggest that this compound, when administeredorally to larvae or adults of D.melanogaster, is not mutagenicand clastogenic in male germ cells, but weakly genotoxic insomatic cells of the wing imaginal disk. ³To whom correspondence should be addressed     

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. © 1995 Wiley-Liss, Inc.     

Absence of genotoxicity of a phytotherapeutic extract from Stryphnodendron adstringens (Mart.) Coville in somatic and germ cells of Drosophila melanogaster

Stryphnodendron adstringens (Mart.) Coville, a medicinal plant that grows in the “cerrados” (a savanna ecosystem) of Brazil, popularly known as “Barbatimão,” is an important source of tannins (polyphenols). In Brazil, it is used in industry (mainly as vegetable tanning) and also in traditional medicine for the treatment of various diseases. In the present study, a phytotherapeutic extract from S. adstringens stem bark was evaluated for mutagenic and recombinagenic effects using the wing spot test of Drosophila melanogaster (somatic mutation and recombination test, SMART), and for chromosome damage in germ cells using the Drosophila sex‐chromosome loss test (ring‐X loss). For SMART, the standard as well as the high bioactivation fly crosses were used; the latter cross is characterized by a high sensitivity to promutagens and procarcinogens. Third‐instar larvae from these two crosses were treated for 48 hr with different concentrations (66%, 75%, and 100%) of the phytotherapeutic extract. The wings of the emerging adults were analyzed for the occurrence of different types of mutant spots. No statistically significant differences in spot frequencies between controls and treated series were observed. For the ring‐X loss test, adult males were fed with the same concentrations of the extract as in the wing spot test. No statistically significant increases in ring‐X losses were observed. The results of our experiments suggest that the phytotherapeutic extract from S. adstringens stem bark is not genotoxic in somatic and germ cells of D. melanogaster. Environ. Mol. Mutagen. 41:293–299, 2003. © 2003 Wiley‐Liss, Inc.     

Genotoxicity testing of Plantago major extracts in somatic cells of Drosophila melanogaster

Plantago major is used in many parts of the world for the treatment of diseases and to promote the healing of wounds. In the present study, the somatic mutation and recombination test (SMART) in Drosophila melanogaster was used to evaluate the genotoxic activity of an aqueous extract of P. major. The following Drosophila crosses were made: standard (ST) cross, in which virgin flare females (flr3/TM3, Bds) were mated with mwh/mwh males, and high-bioactivation (HB) cross, in which virgin ORR females (ORR/ORR; flr3/TM3, Bds) were mated with mwh/mwh males. Each cross produced two types of descendents, marker-transheterozygous (MH) (mwh +/+ flr3) and balancer-heterozygous (BH) (mwh +/+ TM3, Bds) flies. Three-day-old larvae of both types of descendents were treated with undiluted and diluted (1:1 and 1:2 in water) aqueous extracts of P. major. The extracts were genotoxic in both crosses, producing similar induced frequencies in ST and HB flies. Comparison of the frequencies of wing spots in the BH and MH descendents indicated that recombination was a major response. The results indicate that, under these experimental conditions, aqueous extracts of P. major are genotoxic (recombinagenic).     

Delta signaling from the germ line controls the proliferation and differentiation of the somatic follicle cells during Drosophila oogenesis

The body axes of Drosophila are established during oogenesis through reciprocal interactions between the germ line cells and the somatic follicle cells that surround them. The Notch pathway is required at two stages in this process: first, for the migration of the follicle cells around the germ line cyst and, later, for the polarization of the anterior-posterior (A-P) axis of the oocyte. Its function in these events, however, has remained controversial. Using clonal analysis, we show that Notch signaling controls cell proliferation and differentiation in the whole follicular epithelium. Notch mutant follicle cells remain in a precursor state and fail to switch from the mitotic cell cycle to the endocycle. Furthermore, removal of Delta from the germ line produces an identical phenotype, showing that Delta signals from the germ cells to control the timing of follicle cell differentiation. This explains the axis formation defects in Notch mutants, which arise because undifferentiated posterior follicle cells cannot signal to polarize the oocyte. Delta also signals from the germ line to Notch in the soma earlier in oogenesis to control the differentiation of the polar and stalk follicle cells. The germ line therefore regulates the development of the follicle cells through two complementary signaling pathways: Gurken signals twice to control spatial patterning, whereas Delta signals twice to exert temporal control.     

The ovo locus is required for sex-specific germ line maintenance in Drosophila

Mutations at the ovo locus result in a defective female germ line. The male germ line is not affected. Adult females homozygous for loss-of-function alleles have no germ line stem cells. The sex-specific phenotype is evident at late blastoderm and early gastrula stages when the pole cells of embryos homozygous for a loss-of-function allele begin to die. This is the only zygotically acting gene known that is required specifically for embryonic germ line survival. Females heterozygous for dominant alleles or homozygous for alleles reducing gene activity exhibit a range of defects in oogenesis. We have mapped the ovo locus to position 4E1-2 of the salivary gland X chromosome by using a set of cytologically visible deletions.     

Taxanes: the genetic toxicity of paclitaxel and docetaxel in somatic cells of Drosophila melanogaster

In this study, the taxanes, paclitaxel and docetaxel were investigated for genotoxicity in the wing spot test of Drosophila melanogaster. These relatively new drugs are used in cancer therapy and show great promise in the treatment of a variety of cancers. Their major cellular target is the alpha,beta-tubulin dimer but, unlike other spindle poisons, they stabilize microtubules by a shift towards assembly, producing nonfunctional microtubule bundles. The Drosophila wing Somatic Mutation and Recombination Test (SMART) provides a rapid means to evaluate agents able to induce gene mutations and chromosome aberrations, as well as rearrangements related to mitotic recombination. We applied the standard version of SMART (with normal bioactivation) and a variant version with increased cytochrome P450-dependent biotransformation capacity. In the standard assay, docetaxel was found to be aneuploidogenic; this was effectively abolished by a high cytochrome P450-dependent detoxification capacity. This suggests, as previously reported, the involvement of this family of enzymes in the detoxification of docetaxel rather than in its activation. In contrast, paclitaxel was clearly non-genotoxic at the same (millimolar) concentrations as used for docetaxel in both crosses. The weak responsiveness of SMART assays to aneugenic compounds, the weaker ligand and assembly action of paclitaxel and the more rapid reversibility of the microtubules formed with this compound, may have caused the negative response observed in the present study.     

DNA-damaging potency and genotoxicity of aflation M1 in somatic cells in vivo of Drosophila melanogaster

Aflatoxin M₁ (AFM₁), a metabolic hydroxylation product of aflatoxinB₁ (AFB₁), and the parent compound were comparatively assayedfor DNA-damaging potency and genotoxicity in vivo in Drosophilamelanogaster using, respectively, the mei-9^a mei-41^D5 DNA repairtest and the mwh/flr³ wing spot test. In the repair test, larvalstock, consisting of meiotic recombination-deficient doublemutant mei-9^a mei-41^D5 males and repair-proficient females,was exposed to the test agents, and the preferential killingof the mutant larvae was taken as evidence of the DNA-damagingeffect. In this test, AFM₁ was registered as a DNA-damagingagent with an activity 3-fold lower than that of AFB₁. In thewing spot test, where larval flies, trans-heterozygous for thesomatic cell markers mwh and flr³, were treated and the wingswere inspected at adulthood for spots manifesting the phenotypesof the markers, AFM₁ exerted a genotoxic effect compatible tothat of AFB₁. Based on these results and other data, we predictthat AFM₁ may be genotoxic in mammalian in-vivo systems as well. ⁵To whom correspondence should be addressed     

Effect of nucleotide excision repair on ENU-induced mutation in female germ cells of Drosophila melanogaster

The role of nucleotide excision repair (NER) in the repair of alkylation damage in the germ cells of higher eukaryotes has been studied mainly by treating postmeiotic male germ cells. Little is known about repair in actively repairing female germ cells. In this study, we treated NER-deficient (ner(-)) mus201(D1) Drosophila females with N-ethyl-N-nitrosourea (ENU) and determined both the mutant frequencies in the multiple locus recessive lethal (RL) test and in the single locus vermilion gene and determined the ENU mutation spectrum in the vermilion gene. The results show that ENU is mutagenic in all cell stages and that the induced frequencies increase with cell maturation, from oogonia to mature oocytes. In addition, the induced spectrum consists mainly of A:T-->T:A transversions (43.8%), A:T-->G:C transitions (21.9%), and A:T-->C:G transversions (15.6%). G:C-->A:T (3.1%) transitions, other transversions (9.4%), frameshifts (3.1%), and deletions (3.1%) were also found. Comparison of these results with those previously obtained for repair-proficient (ner(+)) female germ cells reveal: 1) Differences in the RL and vermilion mutation frequencies for ner(+) and ner(-) germ cells, indicating that NER is involved in the repair of ENU-induced damage to these cells. 2) At least 15.6% of mutations in ner(-) cells may be the consequence of N-ethylation damage and mutations of this type were not detected in ner(+) cells. 3) Although differences were found in transition frequencies between ENU-treated ner(+) and ner(-) germ cells (52.2% vs. 25%), suggesting that a functional NER is involved in processing O-ethylated damage, the role of NER in repairing O-ethylated adducts is uncertain.     

Genotoxic effects of cisplatin in somatic tissue of Drosophila melanogaster

Third instar larvae of Drosophila melanogaster transdihybrid for mwh and flr were exposed to varying concentrations of cisplatin by feeding on dry media wetted with aqueous solutions of the test compound. Larval feeding continued until pupation, and surviving transdihybrid adults were collected seven days following commencement of feeding. Wings of adults were removed and scored under 400X magnification for the presence of twin spots and single spots comprised of clones of cells possessing malformed wing hairs. Cisplatin was found to induce both twin spots and single spots, and significant (p less than 0.05) linear concentration-response relationships were obtained with respect to the induction of all endpoints. Induction of twin spots demonstrates that cisplatin induces mitotic recombination in the somatic tissue of Drosophila larvae. This capacity to induce mitotic exchange in the somatic tissue of Drosophila compares well with the compound's reported ability to induce chromosome breaks in Drosophila germ cells [Brodberg et al. 1983]. However, not all compounds possess similar genotoxic profiles in the somatic and germ tissue of Drosophila.     

Effect of vanillin on toxicant-induced mutation and mitotic recombination in proliferating somatic cells of Drosophila melanogaster

Vanillin (VA; C8H8O3) is a flavoring agent that in previous studies has both increased and decreased the genotoxicity of chemical agents, depending on the nature of both the agent and the genetic event measured. The ability of VA to modulate the mutagenicity and recombinogenicity of three different monoalkylating agents, N-ethyl-N-nitrosourea (ENU), N-methyl-N-nitrosourea (MNU), and ethyl methanesulfonate (EMS), and the intercalating agent bleomycin (BLEO) was examined using the somatic mutation and recombination test (SMART) in Drosophila melanogaster. While neither the mutagenicity nor the recombinagenicity of ENU or MNU was modified by posttreatment with VA, EMS-induced genetic toxicity was enhanced by as much as 30%. This overall enhancement included a synergistic increase in mitotic recombination and a lesser decrease in mutation. Posttreatment with VA also produced an increase in the genotoxicity of BLEO, which was characterized by increases of 120% and 180% for 0.5% and 1% VA, respectively. This enhancement was restricted to an increase in recombinational events, since no alteration in BLEO-induced mutation was observed. The data suggest that the major VA-modulatory action on genotoxicity in D. melanogaster is related to its synergistic effects on somatic recombination, which has a greater consequence on overall genotoxicity than its antimutagenic effects. Since the SMART assay is specifically sensitive to mitotic crossing-over, our data suggest that VA promotes toxicant-induced homologous recombination, at least in the proliferative cells of Drosophila.     

Recombinagenic activity of integerrimine,a pyrrolizidine alkaloid from Senecio brasiliensis,in somatic cells of Drosophila melanogaster

Integerrimine (ITR), a pyrrolizidine alkaloid from Senecio brasiliensis, was tested for genotoxicity using the wing somatic mutation and recombination test (SMART) in Drosophila melanogaster. The compound was administered by chronic feeding (48 hours) of 3-day-old larvae. Two different crosses involving the markers flare (flr) and multiple wing hairs (mwh) were used, that is, the standard (ST) cross and the high bioactivation (HB) cross, which has a high cytochrome P450-dependent bioactivation capacity. In both crosses, the wings of two types of progeny were analyzed, that is, inversion-free marker heterozygotes and balancer heterozygotes carrying multiple inversions. ITR was found to be equally potent in inducing spots in a dose-related manner in the marker heterozygotes of both crosses. This indicates that the bioactivation capacity present in larvae of the ST cross is sufficient to reveal the genotoxic activity of ITR. In the balancer heterozygotes of both crosses, where all recombinational events are eliminated due to the inversions, the frequencies of induced spots were considerably reduced which documents the recombinagenic activity of ITR. Linear regression analysis of the dose response relationships for both genotypes shows that 85% to 90% of the wing spots are due to mitotic recombination. Environ. Mol. Mutagen 29:91–97, 1997 © 1997 Wiley-Liss, Inc.     

Genotoxicity of vesicular fluid and saline extract of Taenia solium metacestodes in somatic cells of Drosophila melanogaster

Neurocysticercosis, the most common parasitic disease of the central nervous system, is caused by cysticerci of the helminth Taenia solium, which is prevalent in developing countries and is reemerging in affluent societies. This helminth is associated with brain tumors and hematological malignancies in humans. In the present study, we analyzed the genotoxicity of vesicular fluid (VF) and a saline extract (SE) of T. solium metacestodes in the Drosophila melanogaster wing somatic mutation and recombination test (SMART). Third-instar larvae derived from standard and high bioactivation crosses were treated for approximately 48 hr with 12.5, 25.0, and 50.0 microg/ml of VF and SE of T. solium metacestodes. Negative (phosphate buffered saline) and positive (10 mM urethane) controls were also included. The results showed that the two test compounds were genotoxic in both crosses of Drosophila. Nevertheless, further research is needed to determine the genotoxic potential of specific compounds present in VF and SE and their role in the development of cancer.     

The formation and dissolution of the bridge-partitioning complex in intercellular bridges of dividing germ cells in the testis of the golden hamster

Intercellular bridges (IBs) connecting mammalian germ cells get transiently closed during cell divisions by a structure termed the bridge-partitioning complex (BPC). It is composed of a compact stack of transverse endomembrane cisterns that are laterally anchored to the thickened bridge wall (bridge density). Studying male gonads of the golden hamster, the formation of the BPC during prophase/prometaphase transition and its dissolution in late telophase of germ cell divisions was followed. 1) There are strong indications for the involvement of a microtubule-based transport mechanism in the movement of membrane-bound vesicles toward and away from the site of the BPC during its formation or dissolution, respectively. 2) The BPC is built up exactly within the central segment of the IB carrying the bridge density. This feature suggests a specific interaction between components of the (forming) BPC and the material constituting the bridge density. The bridge density might be involved in locally directing the process of BPC formation within germ cell IBs.     

Protective effects of a mixture of antioxidant vitamins and minerals on the genotoxicity of doxorubicin in somatic cells of Drosophila melanogaster

Antioxidant vitamins are able to deactivate highly bioactive molecules, such as free radicals, that are generated during cellular biochemical processes. Doxorubicin (DXR) is a cancer chemotherapeutic agent that generates free radicals as a byproduct. In the present study, the Drosophila melanogaster somatic wing spot test was used to evaluate the effects of a mixture of vitamins (Vitamins C, E, and beta-carotene) and minerals (copper, selenium, and zinc), commercially known as Vitergan Zinc Plus, on the genotoxicity of DXR in standard and high-bioactivation crosses of flies. 12.5, 25, and 50 mg/ml of the vitamin/mineral mixture by itself was nongenotoxic in the trans-heterozygous descendants of both crosses, while the mixture produced a significant reduction in the genotoxicity produced by 0.125 mg/ml DXR in the trans-heterozygous descendants of both crosses. The protective effect was observed when the larvae received either pre- or cotreatments of the multivitamin/mineral (MV) mixture. The results indicate that, under these experimental conditions, the MV mixture is not genotoxic; however, it protects against the genotoxic effects of the chemotherapeutic free-radical generator DXR.     

Effects of tannic acid on spontaneous and induced somatic mutations in Drosophila melanogaster

The effects of tannic acid (TA) alone and in combination with direct acting chemical genotoxins and gamma-radiation were investigated in Somatic Mutation and Recombination Tests (SMARTs) using Drosophila melanogaster. Treatment with TA alone (2.5-15 mmol/l) resulted in a moderate but dose dependent induction of mosaic spots in males and females, indicating that the compound possesses mutagenic and recombinogenic activity. When TA (10 mmol/l) was given simultaneously with MMS, 4-NQO and cis-DDP, a potentiating effect on their mutagenicity was observed in males whereas in females no such increase was measured. The frequency of 4-NQO induced mosaic spots in males was raised more than threefold in presence of TA, for MMS and cis-DDP the enhancement was approximately 2-fold; with gamma-radiation no synergistic effect occurred. The different response in the two sexes indicates that TA preferentially induces gene mutations and deletions but has no enhancing effect on the number of mosaic spots which are formed as a consequence of recombinogenic events.     

On the use of excision repair defective cells in the wing somatic mutation and recombination test in Drosophila melanogaster

Ten chemical mutagens were tested in the wing somatic mutation and recombination test in Drosophila melanogaster. This assay makes use of genetic markers expressed on the wing of adult flies. Larvae which are trans-heterozygous for mwh (multiple wing hairs) and flr (flare) were fed with the compounds either acutely (2, 4, or 6 hr) or chronically (48 or 72 hr), or were treated by inhalation (1 hr). Genetic changes induced in the somatic cells of the wing imaginal discs lead to the formation of mutant clones on the wing (mwh and/or flr). Single spots are produced by point mutation, chromosome breakage, and mitotic recombination; twin spots are produced exclusively by mitotic recombination. All 10 mutagens belonging to different chemical classes were clearly positive in this assay. However, the choice of the optimal treatment conditions (concentration of compound, duration of treatment, age of larvae at treatment) is essential. Eight of the compounds were also tested in excision repair defective cells by introducing the mei-9L1 mutation into the test system. This seems not to improve the detection capacity of the assay, mainly because only small spots are found in excision repair defective wings. In addition, the frequencies of spots in these wings are enhanced four to five times, which makes the scoring more tedious. For these and other practical reasons the use of this specific cross is not recommended in the wing spot test for routine screening purposes.     

O-ethylthymidine adducts are the most relevant damages for mutation induced by N-ethyl-N-nitrosourea in female germ cells of Drosophila melanogaster

Responses to genotoxic agents vary not only among organisms, test systems, and cellular stages, but also between sexes; little, however, is known about the mutagenic consequences of chemical exposures to female germ cells. In this study, the mutagenicity of N-ethyl-N-nitrosourea (ENU) was analyzed in female germ cells of Drosophila melanogaster using the recessive-lethal test and the vermilion system, which simultaneously generates information on induced mutation frequency and mutation spectrum. ENU was mutagenic in all stages of oogenesis, although there were differences among the stages. In mature and immature oocytes, ENU-induced mutations in the vermilion locus were 43.5% A:T-->G:C transitions, 39.1% A:T-->T:A transversions, 8.7% G:C-->A:T transitions, and 8.7% A:T-->C:G transversions, indicating that the most important premutagenic lesions induced by this chemical are O(4)-ethylthymine and O(2)-ethylthymine. The low frequency of mutation involving O(6)-ethylguanine (i.e., G:C-->A:T transitions) could be a consequence of the repair of these lesions by O(6)-methylguanine DNA methyltransferase. Comparison of these results with those previously obtained in male germ cells stresses the importance of the repair activity of the analyzed cells, because the mutation spectrum in female germ cells was similar to the spectrum obtained with repair-proficient spermatogonial cells and different from repair-deficient postmeiotic cells. The results also indicate that studies with female germ cells could be an alternative to the use of premeiotic male germ cells, especially when the analysis of these cells is difficult or almost impossible and when studies of in vivo DNA repair in premeiotic germ cells are performed.