Mauricio Lehmann

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.

Induced DNA Damage by Dental Resin Monomers in Somatic Cells

The present in vivo study investigated the genotoxicity of four dental resin monomers: triethyleneglycoldimethacrylate (TEGDMA), hydroxyethylmethacrylate (HEMA), urethanedimethacrylate (UDMA) and bisphenol A-glycidylmethacrylate (BisGMA). The Somatic Mutation and Recombination Test (SMART) in Drosophila melanogaster was applied to analyse their genotoxicity expressed as homologous mitotic recombination, point and chromosomal mutation. SMART detects the loss of heterozygosity of marker genes expressed phenotypically on the flys wings. This fruit fly has an extensive genetic homology to mammalians, which makes it a suitable model organism for genotoxic investigations. The present findings provide evidence that the mechanistic basis underlying the genotoxicity of UDMA and TEGDMA is related to homologous recombination and gene/chromosomal mutation. A genotoxic pattern can correspondingly be discerned for both UDMA and TEGDMA: their genotoxicity is attributed respectively to 49% and 44% of mitotic recombination, as well as 51% and 56% of mutational events, including point and chromosomal alterations. The monomer UDMA is 1.6 times more active than TEGDMA to induce mutant clones per treatment unit. BisGMA and HEMA had no statistically significant effect on total spot frequencies - suggesting no genotoxic action in the SMART assay. The clinical significance of these observations has to be interpreted for data obtained in other bioassays.

In Vivo Genotoxicity Evaluation of an Artichoke (Cynara scolymus L.) Aqueous Extract

UNLABELLED The Cynara scolymus (artichoke) is widely consumed as tea or food and shows important therapeutic properties. However, few studies have assessed the possible toxic effects of artichoke extracts. This study evaluates genotoxic and mutagenic activities of artichoke leaf aqueous extract in mice using the comet assay and the micronucleus test. Leaf extracts were given by gavage (500 mg/kg, 1000 mg/kg, and 2000 mg/kg) for 3 consecutive days. Extract composition was investigated using phytochemical screening and high-performance liquid chromatography (HPLC). In addition, antioxidant capacity was analyzed through the diphenyl-picrylhydrazyl (DPPH) and xanthine oxidase assay. Phytochemical screening detected the presence of phenolic compounds, flavonoids, and saponins. HPLC analyses indicated the presence of chlorogenic acid, caffeic acid, isoquercetrin, and rutin. Extracts showed a dose-dependent free radical scavenging effect of DPPH and an inhibitory effect of xanthine oxidase. The genotoxic results showed that leaf extracts did not increase micronuclei in peripheral blood cells. Compared to the control group, a significant increase in comet assay values was observed only in bone marrow of group treated with 2000 mg/kg, the highest dose tested, indicating that artichoke tea should be consumed with moderation. PRACTICAL APPLICATION This is the first report of in vivo mutagenic and genotoxic evaluation with C. scolymus. The present study revealed leaf aqueous extract from artichoke shows lack of mutagenicity in vivo, and low genotoxicity and antioxidant activity; indicating that artichoke tea should be consumed with moderation.

Evaluation of the genotoxicity of cisplatin, paclitaxel and 5-fluorouracil combined treatment in the Drosophila wing-spot test

The somatic mutation and recombination test in Drosophila melanogaster was applied to analyze the mutagenic and recombinagenic activity of the chemotherapeutic drugs cisplatin, paclitaxel, and 5-fluorouracil, comparing the effects observed in combinatory treatments with those observed in single administrations. The results obtained in two different genotypes allowed to quantitatively and qualitatively estimate the contribution of genotoxic effects. The results obtained with the individual drug treatments showed that cisplatin and 5-fluorouracil were genotoxic, being able to increase the frequency of total spots on both genotypes. While cisplatin preferentially induced DNA damage of recombinational origin, all the damages induced by 5-fluorouracil were caused by gene and/or chromosome mutations, and the aneuploidogenic compound paclitaxel was not genotoxic. The combination of these drugs does not exert a synergist genotoxic effect in both genotypes compared to the single-agent administration. Instead, it was observed a modification in the proportion of mutation and recombination to the final genotoxicity observed. The antiproliferative activity of PAC could be responsible for the non-synergic genotoxic effect observed. Based on our results it is possible to suggest that cisplatin/paclitaxel/5-fluorouracil treatment regimen cannot impose a higher risk of the development of genotoxicity-associated secondary tumors in comparison to their individual applications.

Artichoke induces genetic toxicity in the cytokinesis-block micronucleus (CBMN) cytome assay

Artichoke leaves are used in traditional medicine as an herbal medicament for the treatment of hepatic related diseases, as well as choleretic and diuretic. The aim of the present study was to evaluate the capacity of Cynara scolymus L. leaves extract (LE) to cause chromosomal instability and cytotoxicity in Chinese hamster ovary cells (CHO) employing the cytokinesis-block micronucleus (CBMN) cytome assay. Cells were treated with four concentrations of C. scolymus for two exposure times: 1h and 24h. Our findings showed that LE did not increase the frequencies of nucleoplasmic bridges (NPBs) and nuclear bud (NBUD). However, all concentrations of the extract produced increments in micronuclei frequencies (MNi) in both exposure times, when compared to the negative control. No significant differences were observed in the nuclear division cytotoxicity index (NDCI), reflecting the absence of cytotoxic effects associated to LE. The results demonstrated the ability of C. scolymus LE to promote chromosomal mutations which are, probably, a result of the pro-oxidant activity of LE constituents such as flavonoids and chlorogenic acids. The data obtained in this study suggests that high concentrations of artichoke can pose a risk associated to its consumption.

Hepatics alterations and genotoxic effects of Croton cajucara Benth (SACACA) in diabetic rats

CONTEXT Croton cajucara Benth is a plant found in Amazonia, Brazil and the bark and leaf infusions of this plant have been popularly used to treat diabetes and hepatic disorders. OBJECTIVES This study investigated effects hepatics alterations and genotoxic and antidiabetic effect of Croton cajucara Benth bark extracts treatment in streptozotocin-induced diabetic rats. METHODS Male Wistar rats were divided into six groups: control rats; control rats treated with Croton cajucara Benth extract during 5 and 20 days; diabetic rats, and diabetic rats treated with Croton cajucara Benth during 5 and 20 days. Diabetes was induced by a single intraperitoneal injection of streptozotocin (70 mg/kg). Eight weeks later we measured glucose, triglyceride, cholesterol and hepatic transaminases on blood. The bone marrow micronucleus assay was used to assess the genotoxic activity of Croton cajucara Benth. RESULTS Treatment with aqueous extrat of Croton cajucara was able to significantly reduce levels of triglycerides in diabetic animals, however, did not modify significantly the levels of glucose and cholesterol in these animals. There was no significant elevation in liver transaminases in the control group treated with Croton cajucara Benth, as there was no genotoxic effect of treatment in this model. Our results did not show a significant effect on glucose and cholesterol reduction, the treatment was able to significantly reduce triclycerides plasmatic level. There was no significant alterations on hepatic transferase in the animals from the control group treated with Croton cajucara Benth. It was observed no genotoxic effect of the treatment in the model studied. CONCLUSION In this study Croton cajucara bark extract showed absence of hepatotoxicity in this animal model and presented a hypolipidemic activity, and could be used to reverse dyslipidemia associated with diabetes and to prevent the cardiovascular complications that are very prevalent in diabetic patients.

Agents of earthy-musty taste and odor in water: Evaluation of cytotoxicity, genotoxicity and toxicogenomics

Considering the limited number of studies on the biological effects on human health of cyanobacterial compounds that cause taste and odor, the present study assessed the cytotoxic and genotoxic potentials of 2-methylisoborneol (2-MIB) and geosmin (GEO) using the MTT assay and the in vitro comet and cytokinesis-block micronucleus (CBMN-Cyt) assays in human HepG2 cells. The toxicogenomics of genes responsive to DNA damage and metabolization by the exposure of cells to 2-MIB and GEO were also investigated. The results showed that concentrations of 2-MIB and GEO above 100 and 75 μg/mL, respectively, were cytotoxic to HepG2 cells. Doses of 2-MIB (12.5, 25, 50, 75 and 100 μg/mL) and GEO (12.5, 25, 50, and 75 μg/mL) were unable to induce neither DNA damage nor events associated with chromosomal instability. Similarly, no concentration of each compound induced increments in the expression of CDKN1A, GADD45α, MDM2 and TP53 DNA damage responsive genes as well as in CYP1A1 and CYP1A2 metabolizing genes. Although cytotoxicity was observed, concentrations that caused it are much higher than those expected to occur in aquatic environments. Thus, environmentally relevant concentrations of both compounds are not expected to exhibit cytotoxicity or genotoxicity to humans.

Genotoxic and biochemical changes in Baccharis trimera induced by coal contamination.

The processing and combustion of coal in thermal power plants release anthropogenic chemicals into the environment. Baccharis trimera is a common plant used in folk medicine that grows readily in soils degraded by coal mining activities. This shrub bioaccumulates metals released into the environment, and thus its consumption may be harmful to health. The purpose of this study was to investigate the phytochemical profile, antioxidant capacity (DPPH), genotoxic (comet assay) and mutagenic potential (CBMN-cyt) in V79 cells of B. trimera aqueous extracts in the coal-mining region of Candiota (Bt-AEC), and in Bagé, a city that does not experience the effects of exposure to coal (Bt-AEB, a reference site). In the comet assay, only Bt-AEC was genotoxic at the highest doses (0.8mg/mL and 1.6mg/mL), compared to the control. For extracts from both areas, mutagenic effects were observed at higher concentrations compared to the control. The cell damage parameters were significantly high in both extracts; however, more striking values were observed for Bt-AEC, up to the dose of 0.8mg/mL. In chemical analysis, no variation was observed in the contents of flavonoids and phenolic compounds, neither the antioxidant activity, which may suggest that DNA damage observed in V79 cells was induced by the presence of coal contaminants absorbed by the plant.

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.

In vivo and in vitro genotoxicity assessment of 2-methylisoborneol, causal agent of earthy–musty taste and odor in water

The water eutrophication process by phosphorus and nitrogen allows cyanobacteria blooms which promote, among other effects, the generation and release of the metabolite 2-methylisoborneol (2-MIB) in the environment. This substance has been shown to be recalcitrant to conventional water treatment, degrading water quality. Considering the limited number of studies on the biological effects of 2-MIB in eukaryotic organisms, the present study assessed the genotoxicity of 2-MIB using the in vitro comet assay and cytokinesis block-micronucleus (CBMN-Cytome) assay on Chinese Hamster Ovary (CHO) cells and the in vivo Drosophila melanogaster Somatic Mutation and Recombination Test (SMART). The results showed that 2-MIB (125, 250 and 500 µg/mL) was unable to induce gene and chromosome mutations or events associated with mitotic recombination in the SMART. Similarly, four different concentrations (7.5, 15, 30 and 60 µg/mL) of 2-MIB did not induce increments in frequencies of micronuclei, nuclear buds, and nucleoplasmatic bridges in the CBMN-Cytome assay. In the comet assay, the positive results were restricted to the highest dose, 60 µg/mL of 2-MIB. The results obtained may help evaluate the genotoxic profile of extracellular algal products.