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.     

Protective effects of proanthocyanidins of grape (Vitis vinifera L.) seeds on DNA damage induced by Doxorubicin in somatic cells of Drosophila melanogaster

Proanthocyanidins (PAs), also known as condensed tannins, are naturally occurring oligomers and polymers of flavan-3-ol monomer units widely found in the leaves, flowers, fruits, seeds, nuts and barks of many plants. Grape seed proanthocyanidins (GSPs) have been used as nutritional supplements, as antioxidants, in preventing atherosclerosis and cardiovascular diseases, and for dislipidemy treatment. The anthracycline antibiotic adriamycin (Doxorubicin, DXR) is a cancer chemotherapeutic agent that interferes with the topoisomerase II enzyme and generates free radicals. In the present study, GSPs (1.680, 3.375, or 6.750 mg/mL) alone were examined for genotoxicity, and combined with DXR (0.125 mg/mL) for antigenotoxicity, using the standard (ST) and high bioactivation (HB) versions of the wing somatic mutation and recombination test in Drosophila melanogaster. The results observed in both crosses were rather similar. GSPs themselves did not show genotoxicity at the doses used. GSPs suppressed the DNA damage induced by DXR in a dose-dependent manner. Comparison of the frequencies of wing spots in the marker-heterozygous (MH) flies and balancer-heterozygous (BH) flies from both crosses, indicated that induced recombination was the major response for the treatments with DXR alone. The co-treatments demonstrated that GSPs have some anti-mutagenic activity; however, anti-recombinagenic activity was the major response.     

Lack of mutagenic effect by multi-walled functionalized carbon nanotubes in the somatic cells of Drosophila melanogaster

Carbon nanotubes (CNTs) are formed by rolling up a single graphite sheet into a tube. Among the different types of CNTs, the multi-walled carbon nanotubes (MWCNTs) comprise a set of concentric nanotubes with perfect structures. Several uses for MWCNTs have been suggested to be included in biological applications such as manufacturing of biosensors, carriers of drugs. However, before these materials can be put on the market, it is necessary to know their genotoxic effects. Thus, this study aims to evaluate the mutagenicity of multi-walled carbon nanotubes (MWCNTs) functionalized in somatic cells of Drosophila melanogaster, using the somatic mutation and recombination test (SMART). This assay detects the loss of heterozygosity of marker genes expressed phenotypically on the wings of the fly. Larvae of three days were used, resulting from ST cross, with basal levels of the cytochrome P450 and larvae of high metabolic bioactivity capacity (HB cross). They were treated with different concentrations of MWCNTs functionalized. The MH descendants, analyzed in both ST and HB crosses, had no significant effects on the frequency of mutant. Based on the results and on the experimental conditions mentioned in this study, it was concluded that MWCNTs were not mutagenic in D. melanogaster.     

Assessment of the mutagenic,recombinagenic and carcinogenic potential of orlistat in somatic cells of Drosophila melanogaster

In this study the mutagenic, recombinagenic, carcinogenic and anticarcinogenic potential of orlistat was assessed using the somatic mutation and recombination test (SMART) and the epithelial tumor detection test (wts). The experiments were conducted on Drosophila melanogaster. In the assessment using SMART, larvae, descendants from the standard (ST) cross and the high bioactivation (HB) cross, were treated chronically with three orlistat concentrations. The results revealed a recombinagenic effect, associated with orlistat, in the descendants of the HB cross, at all three levels of concentration. Homologous recombination can function as a determinant at different stages of carcinogenesis. For verification, larvae from the wts test, descendants of the wts/TM3 virgin female and mwh/mwh male cross, were treated with the same three orlistat concentrations separately and in association with mitomicin C (0.1 mM). The results did not, however, provide evidence that orlistat has carcinogenic potential nor was it associated with the reduction of tumors induced by mitomicin C in D. melanogaster.     

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.     

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)     

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.     

Modulating effect of losartan potassium on the mutagenicity and recombinogenicity of doxorubicin in somatic cells of Drosophila melanogaster

Losartan potassium is an antihypertensive drug in the angiotensin II receptor antagonist (ARA) class. Some studies claim that, in addition to regulating blood pressure, this class of drug has anticancer properties. The objective of this study was to evaluate the genotoxic and antigenotoxic potential of losartan potassium using the SMART (Somatic Mutation and Recombination Test) assay on the somatic cells of Drosophila melanogaster, as well as the possible modulating effects of this drug, when associated with doxorubicin (DXR). Third instar larvae, descendents of standard and high bioactivation (ST and HB) crosses, were chronically treated with different concentrations of losartan potassium (0.25; 0.5; 1; 2; and 4 mM) alone or in association (co-treatment) with doxorubicin (DXR 0.125 mg/mL). The results showed an absence of a mutagenic effect of losartan potassium. In the co-treatment of losartan with DXR, the results showed that losartan is capable of reducing the number of mutant spots induced by DXR without altering the recombinogenic effect of the chemotherapeutic agent. Antiproliferative action appears to be the main mechanism involved in reducing the frequency of mutant spots and consequent modulation of alterations induced by DXR, although this parameter has not been directly assessed in this study.     

Modulating effect of simvastatin on the DNA damage induced by doxorubicin in somatic cells of Drosophila melanogaster

Simvastatin is an antilipemic drug that promotes inhibition of HMG-CoA reductase. Simvastatin can also inhibit the formation of other products, such as isoprenoids, conferring additional benefits to this drug, which include antiproliferative, anti-invasive and pro-apoptotic effects. This study was carried out with the aim of evaluating the mutagenic/recombinogenic effect of simvastatin as well as the possible modulatory effects of this statin on the DNA damage induced by doxorubicin (DXR). This analysis was performed using the somatic mutation and recombination test (SMART) in Drosophila melanogaster. To study these effects, larvae descendants of both crosses (ST and HB) were chronically treated with five concentrations of simvastatin, separately and in association with DXR. The results revealed no mutagenic/recombinogenic effect of simvastatin for any of the concentrations tested. A modulating effect of simvastatin was also observed on DNA damage induced by DXR. The reduction of total mutant frequency was observed for spots from descendants of both crosses, but the inhibition was more effective in descendants from the standard cross (ST). It is believed that this modulating effect is mainly associated with the antioxidant activity of this class of drugs, although this parameter has not been directly assessed in this study.     

Antimutagenic effect of sage tea in the wing spot test of Drosophila melanogaster

The present study assayed the antimutagenic potential of Salvia officinalis (sage) in the form of tea infusion, by the somatic mutation and recombination test (SMART) on Drosophila melanogaster. The use of herbal infusions is much common in the human diet, so the aim of the present study was to estimate the antimutagenic effects of the S. officinalis tea rather than essential oils. Methyl methanesulphonate (MMS) was used as the mutagen and positive control. Several types of treatment were performed: short acute treatment with sage infusion or MMS, longer (chronic) treatment with sage solution or MMS, and two combined treatments, i.e. short treatment with sage followed by a longer treatment with MMS and vice versa. Sage infusion used in our experiments showed a clear antimutagenic effect, reducing the frequency of mutations induced by MMS. The inhibition effect of sage tea is obtained and confirmed when pre- or post-treatments with mutagen were used. The results indicate that although sage in this regime decreases the number of mutation events, it is not efficient enough in case of the 2 h sage pre-treatment. Antioxidant activity, suppression of metabolic activation, could be mechanisms through which sage or some of its components act as desmutagen.     

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.     

The effect of the dibenzylbutyrolactolic lignan (−)-cubebin on doxorubicin mutagenicity and recombinogenicity in wing somatic cells of Drosophila melanogaster

The dibenzylbutyrolactolic lignan (−)-cubebin was isolated from dry seeds of Piper cubeba L. (Piperaceae). (−)-Cubebin possesses anti-inflammatory, analgesic and antimicrobial activities. Doxorubicin (DXR) is a topoisomerase-interactive agent that may induce single- and double-strand breaks, intercalate into the DNA and generate oxygen free radicals. Here, we examine the mutagenicity and recombinogenicity of different concentrations of (−)-cubebin alone or in combination with DXR using standard (ST) and high bioactivation (HB) crosses of the wing Somatic Mutation And Recombination Test in Drosophila melanogaster. The results from both crosses were rather similar. (−)-Cubebin alone did not induce mutation or recombination. At lower concentrations, (−)-cubebin statistically reduced the frequencies of DXR-induced mutant spots. At higher concentrations, however, (−)-cubebin was found to potentiate the effects of DXR, leading to either an increase in the production of mutant spots or a reduction, due to toxicity. These results suggest that depending on the concentration, (−)-cubebin may interact with the enzymatic system that catalyzes the metabolic detoxification of DXR, inhibiting the activity of mitochondrial complex I and thereby scavenging free radicals. Recombination was found to be the major effect of the treatments with DXR alone. The combined treatments reduced DXR mutagenicity but did not affect DXR recombinogenicity.     

Inhibitory effects of water extract of propolis on doxorubicin-induced somatic mutation and recombination in Drosophila melanogaster.

Propolis is a substance produced by honeybees (Apis mellifera L.). Its components are strong antioxidants and free radical scavengers. The aim of this study was to evaluate the protective effects of a water extract of Brazilian green propolis (WEP) combined with the antitumor agent doxorubicin (DXR) on Drosophila melanogaster wing cells through the somatic mutation and recombination test (SMART). Two different crosses were used: The standard (ST) cross and the high bioactivation (HB) cross. The HB cross is characterized by a constitutively enhanced level of cytochrome P450 which leads to an increased sensitivity to a number of promutagens and procarcinogens. Larvae obtained from these two crosses were chronically treated with different concentrations of WEP (12.5,25.0 and 50.0 mg/mL) alone or combined with DXR (0.125 mg/mL). The results obtained with the two different crosses were rather similar. Neither toxicity nor genotoxicity were observed in WEP treated series. Simultaneous treatment with different concentrations of WEP and DXR led to a reduction in the frequency of recombination compared to the treatment with DXR alone. This anti-recombinogenic effect was proportional to the concentrations applied, indicating a dose-response correlation and can be attributed to the powerful scavenger ability of WEP.     

Ameliorative effects of gallic acid,quercetin and limonene on urethane-induced genotoxicity and oxidative stress in Drosophila melanogaster

The main objective of our present work was to ascertain the efficacy of Drosophila melanogaster model for assessing antigenotoxic and antioxidant effects of dietary phytochemicals gallic acid (GA), quercetin (QC) and limonene (Lim) against urethane (URE), a genotoxic environmental carcinogen. Oregon-K (ORK) adult male flies were fed GA, QC and Lim in combination with URE (20?mM) in 10% sucrose for 72?h. Third instar larvae were fed instant medium containing the above phytochemicals and URE for 24?h. Sex-linked recessive lethal (SLRL) test and assays for estimating glutathione content (GSH), glutathione S-transferase (GST), catalase (CAT), superoxide dismutase (SOD) and lipid peroxidation (MDA content) were performed. Adult feeding experiments demonstrated that co-treatment of flies with URE and the test phytochemicals has significantly decreased the frequencies of SLRL mutations in all the germ cell stages when compared to that with URE alone. Larval feeding experiments also showed a similar pattern. The above results correlate well with antioxidative potentials of the test agents where we observed the elevated enzymatic levels with a significant reduction in MDA level in Drosophila larvae. The results further suggest that the dietary phytochemicals have an antioxidant and antimutagenic property which can be assessed using D. melanogaster.     

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.     

Genotoxicity of metacid established through the somatic and germ line mosaic assays and the sex-linked recessive lethal test in drosophila

The mutagenic potential of metacid (methyl parathion), an anticholinesterase organophosphate pesticide, has been studied in the Drosophila eye, wing and female germ line assays and the sex-linked recessive lethal tests. Larvae 24 h, 48 h and 72 h old, heterozygous for various recessive genetic markers on the first and third chromosomes, were exposed to the LD₅₀ and half of this dose for different periods of time. The eyes and wings were checked for the presence of mosaic spots and the eggs laid by the females for germ line mosaicism. The M-5 technique was used to detect the induction of sex-linked recessive lethals. It is concluded that metacid is mutagenic in somatic and germ line cells of Drosophila and induces sex-linked recessive lethals in immature male germ cells.     

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.     

临床表葡菌gyrA、gyrB、grlA和grlB基因突变与其对环丙沙星耐药性的关系

用平皿二倍稀释法测定18株表葡菌对环丙沙星的MIC（0．125—128μg／ml）。分别针对表葡菌中gyrA、gyrB、grlA、grlB等四种基因进行引物设计与合成，提取基因组DNA，PCR扩增，对PCR产物进行克隆、鉴定并测序，用DNASIS软件分析测序结果。在表葡菌的GdA中，只有Ser80→Phe、Ser80→Tyr或Asp84→Asn、Asp84→Ala、Asp84→Tyr的改变。在GyrA中，存在ser84→TYr、ser84→Phe、Glu88→Lys的改变；在任一株表葡菌中未见有gyrB或grlB的突变。在被检测的18株临床分离表葡菌中有3个分离株对环丙沙星的MICs≤0．5μg／ml，在GyrA或GrlA的决定区没有变化。对环丙沙星的MICs≥2μg／ml的15个分离株中，GyrA中有Ser84和Glu88及GdA中的Ser80和Asp84的氨基酸改变。1株MIC为2μg／ml的GdA中发生一个氨基酸改变，而GyrA中无变化。说明拓朴异构酶Ⅵ可能是环丙沙星作用于表葡菌的首要靶位酶。在高水平耐药株中，在GdA和GyrA中均存在多位点的突变，提示耐药水平的高低与GdA、GyrA位点的突变直接相关。