Genotoxic analysis of silver nanoparticles in Drosophila

Abstract

Health risk assessment of nanomaterials is an emergent field, genotoxicity being an important endpoint to be tested. Since in vivo studies offer many advantages, such as the study of the bioavailability of nanomaterials to sensitive target cells, we propose Drosophila as a useful model for the study of the toxic and genotoxic risks associated with nanoparticle exposure. In this work we have carried out a genotoxic evaluation of silver nanoparticles in Drosophila by using the wing somatic mutation and recombination test. This test is based on the principle that loss of heterozygosis and the corresponding expression of the suitable recessive markers, multiple wing hairs and flare-3, can lead to the formation of mutant clones in larval cells, which are expressed as mutant spots on the wings of adult flies. Silver nanoparticles were supplied to third instar larvae at concentrations ranging from 0.1–10 mM. The results showed that small but significant increases in the frequency of total spots were observed, thus indicating that silver nanoparticles were able to induce genotoxic activity in the wing spot assay of D. melanogaster, mainly via the induction of somatic recombination. These positive results obtained with silver nanoparticles contrast with the negative findings obtained when silver nitrate was tested.     

Genotoxic analysis of silver nanoparticles in Drosophila

Health risk assessment of nanomaterials is an emergent field, genotoxicity being an important endpoint to be tested. Since in vivo studies offer many advantages, such as the study of the bioavailability of nanomaterials to sensitive target cells, we propose Drosophila as a useful model for the study of the toxic and genotoxic risks associated with nanoparticle exposure. In this work we have carried out a genotoxic evaluation of silver nanoparticles in Drosophila by using the wing somatic mutation and recombination test. This test is based on the principle that loss of heterozygosis and the corresponding expression of the suitable recessive markers, multiple wing hairs and flare-3, can lead to the formation of mutant clones in larval cells, which are expressed as mutant spots on the wings of adult flies. Silver nanoparticles were supplied to third instar larvae at concentrations ranging from 0.1-10 mM. The results showed that small but significant increases in the frequency of total spots were observed, thus indicating that silver nanoparticles were able to induce genotoxic activity in the wing spot assay of D. melanogaster, mainly via the induction of somatic recombination. These positive results obtained with silver nanoparticles contrast with the negative findings obtained when silver nitrate was tested.     

Genotoxicity testing of four food preservatives and their combinations in the Drosophila wing spot test

In this study, four food preservatives (sodium nitrate, sodium nitrite, potassium nitrate and potassium nitrite) and there five combinations at a concentration of 25 mM have been evaluated for genotoxicity in the somatic mutation and recombination test (SMART) of Drosophila melanogaster. Three-day-old larvae trans-heterozygous including two linked recessive wing hair mutations (multiple wing hairs and flare) were fed at different concentrations of the test compounds (25, 50, 75 and 100 mM) in standard Drosophila Instant Medium. Wings of the emerging adult flies were scored for the presence of spots of mutant cells, which can result from either somatic mutation or mitotic recombination. Also lethal doses of food preservatives used were determined in the experiments. A positive correlation was observed between total mutations and the number of wings having mutation. In addition, the observed mutations in each wing were classified according to the size and type of the mutation. For the evaluation of genotoxic effects, the frequencies of spots per wing in the treated series were compared to the control group, which is distilled water. Chemicals used were ranked as sodium nitrite, potassium nitrite, sodium nitrate and potassium nitrate according to their genotoxic and toxic effects. Moreover, the genotoxic and toxic effects produced by the combined treatments were considerably increased, especially when the four chemicals were mixed. The present study shows that correct administration of food preservatives/additives may have a significant effect on human health.     

Assessment of the genotoxic potential of two zinc oxide sources (amorphous and nanoparticles) using the in vitro micronucleus test and the in vivo wing somatic mutation and recombination test

In this study, we evaluated the toxic and genotoxic potential of zinc oxide nanoparticles (ZnO NPs) of 20 nm and the mutagenic potential of these ZnO NPs as well as that of an amorphous ZnO. Toxicity was assessed by XTT colorimetric assay. ZnO NPs were toxic at concentrations equal to or higher than 240.0 μM. Genotoxicity was assessed by in vitro Cytokinesis Block Micronucleus Assay (CBMN) in V79 cells. ZnO NPs were genotoxic at 120.0 μM. The mutagenic potential of amorphous ZnO and the ZnO NPs was assayed using the wing Somatic Mutation and Recombination Test (SMART) of Drosophila melanogaster. In the Standard cross, the amorphous ZnO and ZnO NPs were not mutagenic. Nevertheless, Marker trans-heterozygous individuals from the High bioactivation cross treated with amorphous ZnO (6.25 mM) and ZnO NPs (12.50 mM) displayed a significant increased number of mutant spots when compared with the negative control. In conclusion, the results were not dose related and indicate that only higher concentrations of ZnO NPs were toxic and able to induce genotoxicity in V79 cells. The increase in mutant spots observed in D. melanogaster was generated due to mitotic recombination, rather than mutational events.     

Genotoxicity testing of four food preservatives and their combinations in the Drosophila wing spot test

In this study, four food preservatives (sodium nitrate, sodium nitrite, potassium nitrate and potassium nitrite) and there five combinations at a concentration of 25 mM have been evaluated for genotoxicity in the somatic mutation and recombination test (SMART) of Drosophila melanogaster. Three-day-old larvae trans-heterozygous including two linked recessive wing hair mutations (multiple wing hairs and flare) were fed at different concentrations of the test compounds (25, 50, 75 and 100 mM) in standard Drosophila Instant Medium. Wings of the emerging adult flies were scored for the presence of spots of mutant cells, which can result from either somatic mutation or mitotic recombination. Also lethal doses of food preservatives used were determined in the experiments. A positive correlation was observed between total mutations and the number of wings having mutation. In addition, the observed mutations in each wing were classified according to the size and type of the mutation. For the evaluation of genotoxic effects, the frequencies of spots per wing in the treated series were compared to the control group, which is distilled water. Chemicals used were ranked as sodium nitrite, potassium nitrite, sodium nitrate and potassium nitrate according to their genotoxic and toxic effects. Moreover, the genotoxic and toxic effects produced by the combined treatments were considerably increased, especially when the four chemicals were mixed. The present study shows that correct administration of food preservatives/additives may have a significant effect on human health.     

Genotoxicity testing of four benzyl derivatives in the Drosophila wing spot test.

Food flavourings are an essential element in foods. Benzyl derivatives are the food additives which are used for increasing the taste of foods and beverages. In this study, different concentrations of four benzyl derivatives (benzaldehyde, benzyl acetate, benzyl alcohol and benzoic acid) used as flavour ingredients have been evaluated for genotoxicity in the wing somatic mutation and recombination test (SMART) of Drosophila melanogaster. Third-instar larvae trans-heterozygous for two genetic markers mwh and flr, were treated at different concentrations (0.1, 0.5, 1, 10, 25 and 50mM) of the test compounds. Wings of the emerging adult flies were scored for the presence of spots of mutant cells, which can result from either somatic mutation or mitotic recombination. Also lethal doses of benzyl derivatives used as flavour ingredients were determined in the experiments. For the evaluation of genotoxic effects, the frequencies of spots per wing in the treated series were compared to the control group, which is distilled water. Chemicals used were ranked as benzaldehyde, benzyl acetate, benzyl alcohol and benzoic acid according to their genotoxic effects. The present study shows that intensive administration of benzyl derivatives used as flavouring agents may have a significant genotoxic effects.     

Evaluation of genotoxic and antigenotoxic effects of boron by the somatic mutation and recombination test (SMART) on Drosophila

Objective: In this study, different concentrations of boron have been evaluated for genotoxic and antigenotoxic properties by using the somatic mutation and recombination test (SMART) on Drosophila melanogaster. Study Design: The treatment concentrations were chosen to a pretest. Third-instar larvae trans-heterozygous for two genetic markers, multiple wing hair (mwh) and flare (flr3), were treated at different concentrations (0.1, 5, 10, 20, and 40?mg/mL) of boron. In addition to investigating antigenotoxic effects, the same boron concentrations were co-administered with 0.1?mM Ethyl Methane Sulfonate (EMS). Distilled water was used as a negative control; 0.1?mM of EMS was used as a positive control. For the chronic feeding study, small plastic vials were prepared with 1.5?g of dry Drosophila Instant Medium and 5?mL of the respective test solution. Hundreds of trans-heterozygous larvae were embedded into this medium. Feeding ended with pupation of the surviving larvae. After metamorphosis, all surviving flies were collected and stored in a 70% ethanol solution. Preparation and microscopic analyses of wing were made after the treatment. Then the observed mutations were classified according to size and type of mutation per wing. Results: Results indicated that there is no significant genotoxic effect with all of the boron concentrations. In addition, the antigenotoxic activities of boron against EMS were tested. Results indicated that all boron concentrations (0.1, 5, 10, 20 and 40?mg/mL) were able to abolish the genotoxic effects induced by the EMS. Conclusion: It is suggested that the observed effects can be linked to the antioxidant properties of boron. Moreover, these in vivo results will contribute to the antigenotoxicity database of boron.     

Cytotoxic and genotoxic actions of Casiopeina III-Ea (Cas III-Ea) in somatic and germ cells of Drosophila melanogaster

Casiopeinas® are a group of newly synthesized drugs designed to treat cancer. These copper (Cu) complexes exhibit cytostatic, cytotoxic, genotoxic, and antineoplastic activities through different mechanisms of action. To evaluate the influence of these compounds, some in vivo studies were performed using predominantly somatic cells. The aim of the present study was to examine the cytotoxic and genotoxic actions of Casiopeina III-Ea (Cas III-Ea) in somatic as well as germ cells of Drosophila melanogaster. For cytotoxicity, the productivity and some morphometric parameters were measured and genotoxicity was assessed by means of the somatic mutation and recombination test assay in the wing. For this purpose, second-instar larvae of the Canton-S strain were treated with different concentrations of Cas III-Ea. The emerged adults were weighed, the area of the wings determined, and the number of trichomes of the region C? counted. The productivity of treated males was measured by a brood method to monitor the influence of Cas III-Ea on spermatozoa, meiotic stage cells, and spermatogonia. For genotoxicity, mwh + /+ flr³ larvae 48 hr age were chronically treated within the same concentration range. Results indicated that Cas III-Ea at all concentrations tested significantly increased the productivity per couple in Brood III (spermatids) while at 1 mM a marked elevation was noted in the three broods tested. In contrast, the weight and size of individuals as well as the size and number of cells in the wing were decreased significantly. Data suggest that Cas III-Ea is a weak genotoxic but selective mutagen. Failure to obtain a dose-related genotoxic response suggests that one of the preferred mechanisms of action of Cas III-Ea is to induce apoptosis.     

Aristolochic acid is mutagenic and recombinogenic in Drosophila genotoxicity tests

Aristolochic acid (AA) has been tested for genotoxic activity in three different assays with Drosophila melanogaster (i–iii). AA induced sex-linked recessive lethals (i) and chromosome losses (ii) in male germ cells. In a newly developed fast assay with somatic cells of larvae (iii), AA induced mutant single spots as well as twin spots. The data indicate that in addition to the mutagenic activity, AA also possesses recombinogenic activity leading to somatic recombination in mitotically active cells. The experimental labor involved to detect the genotoxic activity of AA was lowest with the somatic cell assay. Part of this work was presented as a poster at the 15th Annual Meeting of the Union of the Swiss Societies for Experimental Biology, Freiburg, Switzerland, March 17–18, 1983 (Frei et al. 1983)     

Investigation of coffee in Drosophila genotoxicity tests

Two preparations of coffee (instant coffee and freeze-dried home-brew coffee) were tested in different mutagenicity assays in germ cells as well as in somatic cells of Drosophila melanogaster. The three end-points assayed in germ cells were sex-linked recessive lethals (mainly gene mutations and small chromosome aberrations), dominant lethals (cytotoxic effects as well as genotoxic effects) and sex-chromosome losses (chromosome breakage and non-disjunction). The aqueous coffee solutions were fed either to adult male flies for 3 days or to growing larvae during the whole larval development. Treated males were crossed with appropriately marked females, and the different genetic end-points were analysed in the F1 or F2 generation. The test concentrations (instant coffee 4% (w/v), home-brew coffee 3%) were acutely toxic in adult males (killing approximately 75 and 90% of the exposed flies, respectively). No increase in deaths was caused in larvae by the same concentrations. Only cytotoxic effects were observed in the test for dominant lethals. No conclusive genotoxic effects could be detected in any of the three germ cell assays. The coffee preparations were also tested for induction of mutation and mitotic recombination in somatic cells of the wing imaginal disc. Larvae trans-heterozygous for two recessive wing hair markers were fed high concentrations of the coffees for varying periods of time. Wings of surviving adult flies were analysed for mosaic spots. Twin spots exhibiting both mutant phenotypes are produced by mitotic recombination; single spots showing one or the other phenotype are the result of somatic mutation, such as gene mutation or deletion, or of mitotic recombination. Both coffees had weak effects on normal (repair-proficient) cells as well as on excision repair-defective cells in this assay. Additional experiments with pure caffeine and decaffeinated coffee show that these weak effects in somatic cells were most probably caused by the caffeine present in the two coffees.     

Mutagenicity of natural anthraquinones from Rubia tinctorum in the Drosophila wing spot test

Mutagenicity of anthraquinone aglycones from Rubia tinctorum L. (Rubiaceae) was examined using the somatic mutation and recombination test in Drosophila melanogaster. Larvae heterozygous for recessive wing trichome mutations, multiple wing hairs (mwh), and flare (flr3) were exposed to test compounds and wings of emerged mwh/flr3 females were inspected for the presence of phenotypically mutant mosaic spots. No significant increase in the frequency of mutant spots was observed after the treatment of Drosophila larvae with pure alizarin, xanthopurpurin, and lucidin, or with the crude mixture of anthraquinone aglycones. In contrast, the naphthohydroquinone mollugin induced mainly single spots that can originate either from somatic mutation or from mitotic recombination. Twin spots, consisting of both the mwh and flr3 subclones and originating exclusively from mitotic recombination, were also enhanced, but the increase was only marginally significant. We suggest that mollugin exhibits both the mutagenic and recombinagenic activities.     

Genotoxic evaluation of antiepileptic drugs by Drosophila somatic mutation and recombination test

The study examines the potential genotoxicity of three antiepileptic drugs (phenytoin sodium, pregabalin, gabapentin) using the wing somatic mutation and recombination test (SMART) in Drosophila melanogaster. Trans-heterozygous (two genetic markers mwh and flr) third-instar larvae of D. melanogaster were treated with different concentrations of the test compounds. A positive correlation was observed between total mutations and the number of wings with morphologically detectable mutations. The observed mutations were classified according to size and type of mutation per wing. Phenytoin clearly increased the frequency of total spots at all concentrations above 1.25 μg/ml. Gabapentin also increased the frequency of total spots at concentrations of 40 and 80 μg/ml. This study shows that phenytoin and gabapentin have genotoxic effects according to the SMART test; however, pregabalin displays lower genotoxicity in the SMAR assay when compared with the other two antiepileptics. The results also show that all AED concentrations lower the survival rate of the flies.     

Comparative genotoxicity evaluation of imidazolinone herbicides in somatic cells of Drosophila melanogaster.

In the present study, five analogous herbicides, namely Imazapyr (IMZR), Imazapic (IMZC), Imazethapyr (IMZT), Imazamox (IMZX) and Imazaquin (IMZQ), were evaluated for genotoxicity (mutagenic and recombinagenic activity) in the wing somatic mutation and recombination test (SMART) of Drosophila melanogaster. They are classified as imidazolinone (IMI) herbicides and their mode of action is to inhibit acetohydroxyacid synthase (AHAS), an enzyme involved in the biosynthesis of the amino acids leucine, isoleucine and valine. Two crosses were used: the standard (ST) cross and the high bioactivation (HB) cross. The latter is characterized by high levels of cytochrome P450 conferring increased sensitivity to promutagens and procarcinogens. Three-day-old larvae were exposed by chronic feeding (48 h) to four different concentrations of these herbicides (2.5, 5.0, 10.0 or 20.0 mM). For the evaluation of genotoxic effects, the frequencies of spots per individual in the treated series were compared to the concurrent negative control series (ultrapure water). Imazapyr, Imazapic and Imazethapyr gave negative results with both crosses of the wing spot test. In the ST cross, Imazamox showed positive results only for large single spots (20.0 mM IMZX) and weak positive results for total spots (10.0 and 20.0 mM IMZX), while Imazaquin showed positive results only for large single spots (5.0 and 20.0mM IMZQ) and a weak positive result for total spots (20.0 mM IMZQ). These positive results are mainly due to induced recombination and to a minor extent to mutations. In the HB cross, only Imazamox (5.0 mM IMZX) showed a weak positive result for small single spots. The positive control urethane, a promutagen, caused an increase in the number of all types of spots in both crosses. In conclusion, the results of chronic treatments performed at high doses (toxicity was observed at higher doses) shows the existence of a genotoxic risk for IMZX and IMZQ exposure under these experimental conditions, and indicate the need for further research to delineate the exact mechanisms involved.     

Assessment of the mutagenic,recombinogenic, and carcinogenic potential of amphotericin B in somatic cells of Drosophila melanogaster

Amphotericin B (AmB) is an antifungal antibiotic extracted from Streptomyces nodosus. Its fungicidal activity depends primarily on its binding to the sterol group that is present in fungal membranes. In view of the toxicity of this drug, the purpose of this study was to evaluate its mutagenic, carcinogenic, and recombinogenic activity, based on the wing somatic mutation and recombination test (SMART) and the epithelial tumor detection test (wts) applied to Drosophila melanogaster. Larvae were chronically treated with different concentrations of AmB (0.01, 0.02, and 0.04?mg/mL). The results revealed that AmB is a promutagen exhibiting increase in the number of spots on individuals from high bioactivation (HB) cross with a high level of cytochrome P450. The results also indicate that the main genotoxic event induced by AmB is recombinogenicity. Homologous recombination can act as a determinant at different stages of carcinogenesis. For verification of carcinogenic potential of this compound, larvae from the wts/mwh and wts/ORR, flr³ were treated with the same three AmB concentrations used in the SMART assay. The results did not provide evidence that AmB has carcinogenic potential in wts/mwh individuals. However, individuals from wts/ORR, flr³ developed tumors at the highest concentration tested.     

The effect of royal sun agaricus, Agaricus brasiliensis S. Wasser et al., extract on methyl methanesulfonate caused genotoxicity in Drosophila melanogaster

The effect of culinary-medicinal Royal Sun Agaricus (Agaricus brasiliensis) hot water extract on methyl methane sulfonate (MMS) induced mutagenicity/genotoxity in Drosophila melanogaster was studied using a quick and broadly applicable in vivo assay, i.e., the wing somatic mutation and recombination test. We used 2nd instar larvae, trans-heterozygous for the third chromosome recessive markers, i.e., multiple wing hairs (mvh) and flare-3 [flr (3)], and fed them for 24 h with the aqueous extract of A. brasiliensis. For antigenotoxicity studies a 24-h pretreatment with the extract was done, followed by a 48-h treatment of the then 3rd instar larvae with MMS. The frequency of mutations of the wing blade changes (i.e., of the number of wing spots of different sizes) induced in somatic cells was determined as a parameter of genetic changes of the wing imaginal discs. The results showed that A. brasiliensis extract did not cause any genotoxic or mutagenic effects. No antigenotoxic and/or protective effect against the induction of mutations by MMS was observed. Instead, a possible enhanced mitotic recombination frequency by MMS was seen after pretreatment of the larvae with A. brasiliensis extract. Possible mechanisms of action are discussed.     

Assessment of genotoxicity of Lidocaine,Prilonest and Septanest in the drosophila wing-spot test

The scope of this study was to characterize the likely interaction Lidocaine^®, Prilonest^® and Septanest^® have with DNA, with a view to quantitatively and qualitatively establishing mutagenic, clastogenic, and/or recombinagenic activity of those compounds. The wing somatic mutation and recombination test in Drosophila melanogaster, which detects simultaneously point and chromosomal mutations as well as recombination induced by the activity of genotoxins of direct and indirect action, was used. Each of the anesthetics was tested at different concentrations, administered orally for 48 h to 3rd-stage larvae, in two independent experiments, with concurrent negative controls. The results obtained revealed that only Prilonest^® exhibits genotoxic activity in somatic cells, being able to induce exclusively homologous recombination. Additionally, it was possible to conclude that the genotoxic effect attributed to Prilonest^® is not related to metabolites produced via the P450-type enzymes. However, both Lidocaine^® and Septanest^® are unable to induce either events related to gene and chromosomal mutation, or reciprocal recombination.     

Genotoxicity of two mouthwash products in the Drosophila Wing-Spot Test

In this study, genotoxicity of two mouthwash products (chlorexidin, benzidamine–HCl) were investigated in the Drosophila Wing-Spot Test which makes use of the wing cell markers multiple wing hairs (mwh) and flare (flr) and detects both mitotic recombination and various types of mutational events. Induced mutations are detected as single mosaic spots on the wing blade of surviving adults that show either the multiple wing hairs or flare phenotype. Induced recombination leads to mwh and flr twin spots and also, to some extent, to mwh single spots. Recording of the frequency and the size of different spots is allowed for a quantitative determination of the mutagenic and recombinogenic effects. Trans-heterozygous third-instar larvae were treated at different concentrations of the mouthwash products. Chlorexidin exposure concentrations were 0.5, 1 and 2 mg/ml. Benzidamine–HCl exposure concentrations were 0.38, 0.75 and 1.5 mg/ml. In addition, the observed mutations were classified according to size and type of mutation per wing. Both chlorexidin and benzidamine–HCl were genotoxic in terms of total mutations per wing at the highest doses. Survival rates of flies used in the experiments were significantly lower than those of the control group, with both mouthwash products showing toxic effects on Drosophila melanogaster larvae.     

Genotoxicity and cytotoxicity of ZnO and Al2O3 nanoparticles

Abstract

Objectives: Metal oxide nanoparticles (ZnO-NPs and Al₂O₃-NPs) are used in many fields, including consumer products and biomedical applications. As a result, exposure to these NPs is highly frequent, however, no conclusive information on their potential cytotoxicity and genotoxicity mechanisms are available. For this reason, we studied cytotoxic and genotoxic effects of ZnO-NPs and Al₂O₃-NPs on human peripheral blood lymphocytes.

Materials and methods: We obtained our goals by using MTT assay, Annexin V-FITC flow cytometry, and alkaline, neural and pH 12.1 versions of comet assay.

Results: Exposure of lymphocytes to both NPs for 24?h slightly decreased viability of lymphocytes at ≥0.5?mM. For the first time, we revealed using the comet assays that both ZnO-NPs and Al₂O₃-NPs caused a concentration-dependent increase of DNA single-strand breaks, but not alkali-labile sites. Treatment with DNA glycosylases showed that the NPs induced oxidative DNA damage. DNA damage caused by both nanoparticles at 0.05?mM was removed within 120?min, however lymphocytes did not repair DNA damage induced by 0.5?mM NPs. Studied nanoparticles did not induce apoptosis in lymphocytes.

Conclusion: Our results suggest that ZnO-NPs and Al₂O₃-NPs at concentration up to 0.5?mM did not exhibit cytotoxic effect but may exert genotoxic effect on lymphocytes, at least partially by the generation of oxidative DNA damage and strand breaks.     

Genetic Toxicology of Dental Composite Resin Extracts in Somatic Cells In Vivo

Abstract: The aim of this study was to assess the potential genetic toxicity associated to nine aqueous extracts from dental composite resins (Charisma, Fill Magic, Fill Magic Flow, Durafill, TPH Spectrum, Concept, Natural Look, Filtek Z250 and Filtek P60) and one random extract. Homologous mitotic recombination, point and chromosomal mutation effects were determined in somatic proliferative cells of Drosophila melanogaster exposed to aqueous extracts of the clinically used composites. Reproducible increases in clone mutant spot frequencies induced by diluted extract of Fill Magic Flow were observed. These increments were exclusively associated to the induction of homologous recombination – a genetic phenomenon involved in the loss of heterozygosis. The other eight composite resins and the random extract had no statistically significant effect on total spot frequencies – suggesting that they are non‐genotoxic in the somatic mutation and recombination test assay, which agrees with the applications they have in dentistry. These findings – supported by numerous studies showing a positive correlation between carcinogenicity in man and genotoxicity in the Drosophila wing spot test – point to the potential risks some composite resins pose to the health of patients and dentistry personnel.     

Evaluation of titanium dioxide nanocrystal-induced genotoxicity by the cytokinesis-block micronucleus assay and the Drosophila wing spot test

Titanium dioxide nanocrystals (TiO₂ NCs) crystalline structures include anatase, rutile and brookite. This study evaluated the genotoxic effects of 3.4 and 6.2 nm anatase TiO₂ NCs and 78.0 nm predominantly rutile TiO₂ NCs through an in vitro micronucleus (MN) assay using V79 cells and an in vivo somatic mutation and recombination test in Drosophila wings. The MN assay was performed with nontoxic concentrations of TiO₂ NCs. Only anatase (3.4 nm) at the highest concentration (120 μM) induced genotoxicity in V79 cells. In the in vivo test, Drosophila melanogaster larvae obtained from standard (ST) or high bioactivation (HB) crosses were treated with TiO₂ NCs. In the ST cross, no mutagenic effects were observed. However, in the HB cross, TiO₂ NCs (3.4 nm) were mutagenic at 1.5625 and 3.125 mM, while 78.0 nm NCs increased mutant spots at all concentrations tested except 3.125 mM. Only the smallest anatase TiO₂ NCs induced mutagenic effects in vitro and in vivo. For rutile TiO₂ NCs, no clastogenic/aneugenic effects were observed in the MN assay. However, they were mutagenic in Drosophila. Therefore, both anatase and rutile TiO₂ NCs induced mutagenicity. Further research is necessary to clarify the TiO₂ NCs genotoxic/mutagenic action mechanisms.