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.     

Genotoxicity of Casiopeina III-Ea in mouse bone marrow cells

Casiopeina III-Ea® (Cas III-Ea®) is a chelated copper complex with antineoplastic activity that is capable of reducing tumor size and inducing antiproliferative and apoptotic effects. However, little is known about its in vivo genotoxic effects. Therefore, this study evaluated two cytogenetic and two proliferative parameters 24?h after the administration of Casiopeina III-Ea® to male CD-1 mice. Three doses of Cas III-Ea® were administered by intraperitoneal injections of 1.69, 3.39 and 6.76?mg/kg (corresponding to 1/8, 1/4 and 1/2 of LD₅₀, respectively). A reduction in the mitotic index (MI) and an increased numbers of cells with structural chromosomal aberrations (SCA) were detected. Additionally, a low but significant increase in the frequency of sister chromatid exchange (SCE) was observed at the highest dose. Changes in the DNA replication index (RI) were not observed. These results indicate that Casiopeina III-Ea® shows cytotoxic and clastogenic activity in bone marrow cells from treated mice.     

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.     

Effect of [Cu(4,7-dimethyl-1,10-phenanthroline)(acetylacetonato)]NO3, Casiopeína III-Ea,on the activity of cytochrome P450

Casiopeína III-Ea (Cas III-Ea¹) is a copper complex with antiproliferative and antitumor activities, designed to act via alternative mechanisms of action different from Cisplatin. This compound has also been well characterized in preclinical test and pharmacokinetic analysis, being a good candidate for clinical phases. Since very little is known about the processes of biotransformation of therapeutic metal based drugs, this paper report the first approach to the study of the interaction between metal complex Cas III-Ea and cytochromes P450 with the aim to find out possible biotransformation pathways for this complexes and feasible drug–drug interactions. Results showed that Cas III-Ea is a strong irreversible competitive inhibitor of CYP1A1 (IC₅₀ = 7.5 ± 1.0 μM; Ki = 240 nM). The magnitude of values indicate that it is necessary to be taken into account such effect when analyzing possible drug interactions with these new drugs in order to prevent adverse reactions derived from this inhibition.     

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.     

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.     

Genotoxic assessment of oxcarbazepine and carbamazepine in drosophila wing spot test

In this study, different concentrations of two antiepileptic drugs, carbamazepine (CBZ) and oxcarbazepine (OXC), have been evaluated for genotoxicity in the wing spot test of Drosophila melanogaster. The wing spot test detects different kinds of somatic mutations and allows detection of mitotic recombinations. Third-instar larvae trans-heterozygous for two genetic markers mwh and flr, were treated at different concentrations of the drugs. Oxcarbazepine exposure concentrations were 1.88, 3.75, 7.50 and 15mug/ml. Carbamazepine exposure concentrations were 5, 10, 20 and 40mug/ml. In addition, the observed mutations were classified according to size and type of mutation per wing. CBZ was genotoxic in terms of total mutations per wing in the highest two doses; the same was true for OXC in the highest three doses. Survival rates of flies used in the experiments were significantly lower than that of the control group showing both drugs to have toxic effects to Drosophila melanogaster larvae. Clone formation frequency for 10(5) cells was lower in OXC than CBZ. However this was lower than the critical genotoxicity frequency of 2.0.     

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.     

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.     

Genotoxicity of cobalt nanoparticles and ions in Drosophila

Abstract

Nanogenotoxicology is an emergent area of research, relevant for estimating the potential carcinogenic risk of nanomaterials. Since most of the approaches use in vitro studies, and neglecting the whole organism limits the accuracy of the obtained results, we have used Drosophila melanogaster to study the possible genotoxic potential of cobalt nanoparticles (Co NPs). The wing somatic mutation and recombination test has been the test of choice. This test is based on the principle that the 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 clone cells in growing up larvae, which are expressed as mutant spots on the wings of adult flies. Co NPs, as well as the ionic form cobalt chloride, were given to third instar larvae through the food, at concentrations ranging from 0.1 to 10 mM. The results obtained indicate that both cobalt forms are able to induce significant increases in the frequency of mutant clones. Although at low concentrations only Co NPs were genotoxic, the level of genetic damage obtained at the highest dose tested of cobalt chloride (10 mM) showed a significant higher increase in the frequency of total spots than those observed after the treatment with cobalt nanoparticles. As conclusion, our results indicate that Co NPs were able to induce genotoxic activity in the wing-spot assay of D. melanogaster, mainly via the induction of somatic recombination. The differences observed in the behaviour of the two selected cobalt forms may result from differences in the uptake.     

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 some organophosphate insecticides in the Drosophila wing spot test.

In this study, different concentrations of some organophosphate insecticides (methyl parathion, azamethiphos, dichlorvos and diazinon) 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 (1 ppm, 3 ppm, 5 ppm, 7 ppm, 10 ppm) of the test compounds. A positive correlation was observed between total mutations and the number of wings having mutations. In addition, the observed mutations were classified according to size and type of mutation per wing. Chemicals used were ranked in decreasing order according to their genotoxic effects as diazinon, dichlorvos, methyl parathion, azamethiphos.     

Assessment of the in vitro and in vivo genotoxicity of extracts and indole monoterpene alkaloid from the roots of Galianthe thalictroides (Rubiaceae)

Roots of Galianthe thalictroides K. Schum. (Rubiaceae) are used in folk medicine in the State of Mato Grosso do Sul, Brazil, for treating and preventing cancer. To gain information about the genotoxicity of extracts (aqueous and EtOH), the CHCl₃ phase resulting from partition of the EtOH extract and the indole monoterpene alkaloid 1 obtained from this plant. The genotoxicity of 1 and extracts was evaluated in vivo through the Drosophila melanogaster wing Somatic Mutation and Recombination Test – SMART, while in vitro cytotoxic (MTT) and Comet assays were performed only with alkaloid 1. The results obtained with the SMART test indicated that the aqueous extract had no genotoxic activity. The EtOH extract was not genotoxic to ST descendants but genotoxic to HB ones. The CHCl₃ phase was genotoxic and cytotoxic. Alkaloid 1 showed significant mutational events with SMART, in the cytotoxicity assay (MTT), it showed a high cytotoxicity for human hepatoma cells (HepG2), whereas for the Comet assay, not showing genotoxic activity. The ethanol extract was shown to be genotoxic to HB descendants in the SMART assay, while the results obtained in this test for the monoterpene indole alkaloid 1 isolated from this extract.     

Antimutagenic activity of two medicinal phytoextracts in somatic cells of Drosophila melanogaster

Context and Objective: We used the wing somatic assay in Drosophila melanogaster to test the hypothesis that two phytoextracts from Cecropia obtusifolia Bertol (Cecropiaceae) and Equisetum myriochaetum Schlecht. et Cham (Equisetaceae), which are used in folk medicine to treat type 2 diabetes mellitus, could detoxify the mutagen hydrogen peroxide.

Materials and methods: Third instar larvae from standard (ST) and high-bioactivation (HB) crosses were chronically exposed to different concentrations of the phytoextracts. Hydrogen peroxide was used to induce oxidative stress and was chronically tested in both crosses. Catalase activity was measured in larvae of both strains 48?h after treatment with hydrogen peroxide. A pretreatment protocol was devised to test the antimutagenic potency of the medicinal extracts.

Results: The present study showed that neither of the phytoextracts were genotoxic in Drosophila. Interestingly, the antioxidant enzyme activity levels were different between the larvae. Hydrogen peroxide resulted in a significant genotoxic effect in the ST cross, whereas a detoxification of hydrogen peroxide was found in the HB cross. Thus, catalase was stimulated in the HB cross, which was indicative of a cellular defense mechanism mounted against a xenobiotic hazard. We found that the percentage of inhibition of spots produced by E. myriochaetum was much higher than that induced by Cecropia obtusifolia.

Discussion and conclusions: These results are in agreement with the uses of these phytoextracts in traditional medicine. Indeed, the lack of genotoxicity and the antimutagenic activity observed for both phytoextracts validates their use as a therapeutic modality to treat diabetic patients. Moreover, these extracts are suitable for consumption as teas and/or phytomedicines.     

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.     

Genotoxic evaluation of sodium fluoride in the Somatic Mutation and Recombination Test (SMART)

In this study, genotoxic effect of sodium fluoride (NaF) was investigated in Drosophila melanogaster Somatic Mutation and Recombination Test. Third-instar larvae trans-heterozygous for two genetic markers mwh and flr, were treated at different concentrations (2.5 μg/ml, 5 μg/ml and 10 μg/ml) of the test compounds. After the treatment the observed mutations were classified according to size and type of mutation per wing. 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. NaF has genotoxic and toxic effects for concentrations of 5 and 10 μg/ml. The present study shows that NaF may have genotoxic and toxic effects.     

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.     

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 studies in the ST cross of the Drosophila wing spot test of sunflower and soybean oils before and after frying and boiling procedures

Sunflower and soybean oils were tested for genotoxicity in the Drosophila wing somatic mutation and recombination assay. Results indicate that both oils produce genotoxic effects when tested without any previous frying or boiling processes. Boiling sunflower oil during fifteen, thirty and sixty minutes significantly increased its genotoxic response; nevertheless, after frying potatoes this oil showed a significant decrease in the genotoxic activity. On the other hand, boiling and frying soybean oil in the same conditions results in a decrease of its genotoxic potential. We have also detected that the amount of total polar materials increases significantly in oils submitted to frying or boiling process. Nevertheless, in oils obtained after frying potatoes, the amount of TPM was higher than after boiling. It is suggested that this effect is probably due to the amount of non-volatile TPM, the fatty acid composition of the oils, the types of frying oil, the high frying temperature and time, and the number of boiling and frying. This is the first study reporting genotoxicity data in Drosophila for the boiling and frying of both sunflower and soybean oils.     

Lilium compounds kaempferol and jatropham can modulate cytotoxic and genotoxic effects of radiomimetic zeocin in plants and human lymphocytes In vitro

Organisms are constantly exposed to the detrimental effect of environmental DNA‐damaging agents. The harmful effects of environmental genotoxins could be decreased in a viable way by antimutagenesis. One of the modern approaches to reduce the mutagenic burden is based on exogenous natural and synthetic compounds that possess protective and antimutagenic potential against genotoxins. The natural compounds kaempferol and jatropham isolated from Lilium candidum were tested with respect to their potential to protect cells against the radiomimetic zeocin, as well as to their cytotoxic and genotoxic activities in two types of experimental eukaryotic test systems: Hordeum vulgare and human lymphocytes in vitro. Mitotic index (MI) was used as an endpoint for cytotoxicity; the frequency of chromosome aberrations (MwA) and the number of induced micronuclei (MN), as endpoints for genotoxicity/clastogenicity. Formation of aberration “hot spots” was also used as an indicator for genotoxicity in H. vulgare. Both kaempferol and jatropham were shown to possess a potential to modulate and decrease the cytotoxic and genotoxic/clastogenic effect of zeocin depending on the experimental design and the test system. Our data could be useful for health research programs, particularly in clarifying the pharmacological potential and activity of natural plant compounds. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 751–764, 2016.