Mário Antônio Spanó

Detection of micronuclei in peripheral erythrocytes of Cyprinus carpio exposed to metallic mercury.

Cyprinus carpio fish (carp), exposed to elemental or metallic mercury (Hg0) at concentrations of 2.0, 20.0, and 200.0 mg per liter of water, were kept in concrete tanks for 159 days. Ten fish were used for each concentration level. Thirteen samples of peripheral blood were collected from each animal through gill puncture, 12 during the first 90 days of the experiment, and the last one at the end of the experiment. The micronucleus test (MNT) was designed to study dose and time yield effects of mercury after indirect exposure in vivo. The results indicated that for a concentration of 2.0 mg Hg0/l, there was no significant increase in frequency of micronuclei (MN), but at higher concentrations [20.0 and 200.0 mg Hg0/l] there was a significant increase in MN frequencies. This effect was higher after 31 days of exposure, followed by slight stabilization and gradual decrease. Environ. Mol. Mutagen. 30:293–297, 1997

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.

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.

Modulatory effects of the antioxidant ascorbic acid on the direct genotoxicity of doxorubicin in somatic cells of Drosophila melanogaster

In this study two different crosses involving the wing cell markers mwh and flr3 (standard (ST) cross and high bioactivation (HB) cross, the latter being characterized by a high constitutive level of cytochrome P450 which leads to an increased sensitivity to a number of promutagens and procarcinogens) were used to investigate the modulatory effects of ascorbic acid (AA) combined with the antitumor agent doxorubicin (DXR) in Drosophila melanogaster. We observed that the two different concentrations of AA (50 or 100 mM) had no effect on spots frequencies, while DXR treatments (0.2 or 0.4 mM) gave positive results for all types of spots, when compared to negative control. For marker-heterozygous (MH) flies, a protective effect was observed with the lower concentration of AA (50 mM) that was able to statistically decrease the frequency of spots induced by DXR (0.2 mM), while an enhanced frequency of spots induced by DXR was observed with the higher concentration of AA (100 mM), when compared to DXR treatment (p < 0.05). These results suggest that AA may interfere with free radicals generated by DXR and with other possible reactive metabolites. The efficiency of AA in protecting the somatic cells of D. melanogaster against mutation and recombination induced by DXR is dependent on the dose used and the protection is directly related to the activity of cytochrome P450 enzymes.

A comparative study of the modulatory effects of (−)-cubebin on the mutagenicity/recombinogenicity induced by different chemical agents

(-)-Cubebin (CUB) is a lignan isolated from dry seeds of Piper cubeba. We aimed to assess its genotoxic potential and influence on chromosomal damage (frequency of micronuclei - MN) induced by doxorubicin (DXR) in V79 cells and by urethane (URE) in somatic Drosophila melanogaster cells. Our findings indicate an absence of a CUB-mediated genotoxic effect at the concentrations tested. The results also revealed that CUB significantly reduced the frequency of MN induced by DXR, with a mean reduction of 63.88%. In a previous study, our research group demonstrated an absence of CUB-mediated mutagenic effects through the wing Somatic Mutation and Recombination Test (SMART) in Drosophila. In the present study, we used the standard and high bioactivation versions of the SMART to estimate the antigenotoxic effects of CUB associated with URE. At lower concentrations, the recombination level decreased, but at the highest concentration, the recombination level increased. Our data and previous studies suggest that CUB may act as a free radical scavenger at low concentrations, a pro-oxidant at higher concentrations when it interacts with the enzymatic system that catalyzes the metabolic detoxification of DXR or URE, and/or an inducer of recombinational DNA repair.

Modulatory effects of Tabebuia impetiginosa (Lamiales, Bignoniaceae) on doxorubicin-induced somatic mutation and recombination in Drosophila melanogaster

The wing Somatic Mutation and Recombination Test (SMART) in D. melanogaster was used to study genotoxicity of the medicinal plant Tabebuia impetiginosa. Lapachol (naphthoquinone) and β-lapachone (quinone) are the two main chemical constituents of T. impetiginosa. These compounds have several biological properties. They induce apoptosis by generating oxygen-reactive species, thereby inhibiting topoisomerases (I and II) or inducing other enzymes dependent on NAD(P)H:quinone oxidoreductase 1, thus affecting cell cycle checkpoints. The SMART was used in the standard (ST) version, which has normal levels of cytochrome P450 (CYP) enzymes, to check the direct action of this compound, and in the high bioactivation (HB) version, which has a high constitutive level of CYP enzymes, to check for indirect action in three different T. impetiginosa concentrations (10%, 20% or 40% w/w). It was observed that T. impetiginosa alone did not modify the spontaneous frequencies of mutant spots in either cross. The negative results observed prompted us to study this phytotherapeuticum in association with the reference mutagen doxorubicin (DXR). In co-treated series, T. impetiginosa was toxic in both crosses at higher concentration, whereas in the HB cross, it induced a considerable potentiating effect (from ~24.0 to ~95.0%) on DXR genotoxity. Therefore, further research is needed to determine the possible risks associated with the exposure of living organisms to this complex mixture.

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.

Mutagenic evaluation of combined paclitaxel and cisplatin treatment in somatic cells of Drosophila melanogaster

Recent studies have added paclitaxel (PAC) to traditional cisplatin (CIS) regimen to treat squamous cell carcinoma of the head and neck. The target of these antineoplastic agents is nuclear DNA for CIS and microtubules for PAC, although it is not restricted to malignant cells. In this study, the genotoxicity of the combined treatment of PAC and CIS was investigated using the standard version of the wing Somatic Mutation and Recombination Test (SMART) in Drosophila melanogaster. Quantitative and qualitative genotoxic effects of these compounds were estimated by comparing wing spot frequencies in marker-heterozygous to balancer-heterozygous flies. Two different concentrations of PAC (0.0025 and 0.005mM) and CIS (0.025 and 0.05mM) as well as combinations of them were employed. The results demonstrated that the spindle poison PAC alone was not genotoxic in this test system, while CIS was able to induce a high incidence of DNA damage in both genotypes, mainly related to somatic recombination. The data obtained for the combined treatments showed that its genotoxicity varied with the concentrations used. In small concentrations the number of total spots induced by combination was reduced in relation to CIS 0.025mM just for marker-heterozygous flies, showing that somatic recombination was the prevalent event involved. At higher concentrations the combined treatment showed significant reductions in the frequencies of large single spots, for both genotypes, and twin spots for marker-heterozygous flies, but did not significantly reduce the total spots frequency in either genotype. The data suggest that aneugenic activity of PAC could be responsible for the reduction in the genotoxicity of CIS.

Protection by Panax ginseng C.A. Meyer against the genotoxicity of doxorubicin in somatic cells of Drosophila melanogaster

Panax ginseng is one of the most widely prescribed herbal medicines for the treatment of cancer, diabetes, chronic inflammation, and neurodegenerative and cardiovascular diseases. Since the use of alternative medicines in combination with conventional therapy may increase the risk of unwanted interactions, we investigated the possible genotoxicity of a water-soluble form of the dry root of P. ginseng (2.5, 5.0 or 10.0 mg/mL) and its ability to protect against the genotoxicity of doxorubicin (DOX; 0.125 mg/mL) by using the Drosophila melanogaster wing somatic mutation and recombination test (SMART) with standard and high-bioactivation crosses of flies. Panax ginseng was not genotoxic at the concentrations tested, whereas DOX-induced genotoxicity in marker-heterozygous flies resulted mainly from mitotic recombination. At low concentrations, P. ginseng had antirecombinogenic activity that was independent of the concentration of extract used. Recombination events may promote cancer, but little is known about the ability of P. ginseng to inhibit such recombination or modulate DNA repair mechanisms.

Antigenotoxic effects of Mandevilla velutina (Gentianales, Apocynaceae) crude extract on cyclophosphamide-induced micronuclei in Swiss mice and urethane-induced somatic mutation and recombination in Drosophila melanogaster

A Mandevilla velutina crude extract was investigated using the mouse micronucleus test (MNT) and the Drosophila melanogaster somatic mutation and recombination test (SMART) using standard (ST) and high bioactivation (HB) crosses. The MNT used 10 mg, 20 mg or 40 mg per 100 g of body weight (bw) of extract with and without 0.2 mg per 100 g bw peritoneal cyclophosphamide. There was no genotoxicity in the negative control or extract only groups and, compared to the cyclophosphamide control, there was a significant reduction in micronucleated polychromatic erythrocytes in all the groups given extract plus cyclophosphamide. For SMART larvae were fed 5 or 10 mg mL-1 of extract for seven days with and without 0.89 mg mL-1 of urethane given on day seven. The ST and HB flies showed no significant differences in spots between the negative control and the extract only groups. The number of urethane-induced spots was reduced by the highest concentration of extract for the ST flies and by both concentrations of extract for the HB flies. The results suggest that M. velutina extract is not genotoxic but is antigenotoxic.