Mutagenic and antimutagenic effects of aqueous extract of rosemary (Rosmarinus officinalis L.) on meristematic cells of Allium cepa.

Polyphenolic compounds present in rosemary were found to have antioxidant properties, anticarcinogenic activity, and to increase the detoxification of pro-carcinogens. The aim of the study was to determine the effect the aqueous extract of rosemary (AER) on mutagenicity induced by methylmethane sulfonate in meristematic cells of Allium cepa, as well as to describe its mode of action. Anti-mutagenicity experiments were carried out with 3 different concentrations of AER, which alone showed no mutagenic effects. In antimutagenicity experiments, AER showed chemopreventive activity in cultured meristematic cells of A. cepa against exposure to methylmethane sulfonate. Additionally, post-treatment and simultaneous treatment using pre-incubation protocols were the most effective. Evaluation of different protocols and the percent reduction in DNA indicated bioantimutagenic as well desmutagenic modes of action for AER. AER may be chemopreventive and antimutagenic.

Antigenotoxic and antimutagenic effects of glutamine supplementation on mice treated with cisplatin.

We evaluated the effects of glutamine on clastogenic and genotoxic damage prevention caused by the administration of cisplatin. Forty Swiss mice were divided into 8 experimental groups: G1 and G2, which were control groups; G3, G4, and G5, which were administered [2 doses of glutamine (orally)] separated by a 24-h period (150, 300, and 600 mg/kg, respectively), and a dose of phosphate-buffered saline by intraperitoneal injection; G6, G7, and G8, which were treated in the same manner as the previous groups, but received cisplatin rather than phosphate-buffered saline. The antimutagenicity groups showed damage reduction percentages of 79.05, 80.00, and 94.27% at the time point T1, 53.18, 67.05, and 64.74 at time point T2 for the 150, 300, and 600 mg/kg doses of glutamine, respectively. Antigenotoxic activity was evident for all 3 doses with damage reduction percentages of 115.05, 119.06, and 114.38 for the doses of glutamine of 150, 300, and 600 mg/ kg, respectively. These results suggest that further studies are needed to confirm the clastogenic activity of glutamine. However, our results may lead to rational strategies for supplementation of this antioxidant as an adjuvant in cancer treatment or for preventing genomic lesions.

Cytotoxicity and mutagenicity of fluoxetine hydrochloride (Prozac), with or without vitamins A and C, in plant and animal model systems.

Fluoxetine, commonly known as Prozac, is the first representative of the so-called new generation of antidepressants that promise efficacy, with few side effects, against deep depression, nervous bulimia, and anxiety. As there is a growing number of people suffering from anxiety and depression; consequently, the use of fluoxetine is also increasing. Verifying absence of drug effects such as cytotoxicity or mutagenicity is of great importance. Certain vitamins, such as vitamin A (retinol, retinoids) and vitamin C (ascorbic acid) protect and are extremely active against mutagens. We evaluated the cytotoxic and mutagenic activity of fluoxetine, with and without concomitant administration of vitamin A or C, in Allium cepa meristem cells and Wistar rat bone marrow cells. The A. cepa meristem cells showed fluoxetine cytotoxicity; concomitant treatment with vitamin A or C proved non-protective. Treatment of Wistar rats with fluoxetine intraperitoneally or via gavage did not affect cell division or cause clastogenic effects. Vitamin A and C did not affect the cytotoxicity or mutagenicity of fluoxetine in the rat cells.

Evaluation of the antimutagenic activity and mode of action of the fructooligosaccharide inulin in the meristematic cells of Allium cepa culture.

This study evaluated the mutagenicity and antimutagenicity of inulin in a chromosomal aberration assay in cultures of the meristematic cells of Allium cepa. The treatments evaluated were as follows: negative control--seed germination in distilled water; positive control--aqueous solution of methyl methanesulfonate (10 µg/mL MMS); mutagenicity--aqueous solutions of inulin (0.015, 0.15, and 1.50 µg/mL); and antimutagenicity--associations between MMS and the different inulin concentrations. The antimutagenicity protocols established were pre-treatment, simultaneous simple, simultaneous with pre-incubation, and post-treatment. The damage reduction percentage (DR%) was 43.56, 27.77, and 55.92% for the pre-treatment; -31.11, 18.51, and 7.03% for the simultaneous simple; 30.43, 19.12, and 21.11% for the simultaneous with pre-incubation; and 64.07, 42.96, and 53.70% for the post-treatment. The results indicated that the most effective treatment for inhibiting damages caused by MMS was the post-treatment, which was followed by the pre-treatment, suggesting activity by bioantimutagenesis and desmutagenesis. The Allium cepa assay was demonstrated to be a good screening test for this type of activity because it is easy to perform, has a low cost, and shows DR% that is comparable to that reported studies that evaluated the prevention of DNA damage in mammals by inulin.

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.

Evaluation of the mutagenicity and antimutagenicity of soy phytoestrogens using micronucleus and comet assays of the peripheral blood of mice.

Studies show that soy imparts many favorable properties in the human body, including the prevention of chronic diseases such as osteoporosis, heart disease, cancer, and diabetes. Soy is rich in isoflavones, and it is a candidate for the chemoprevention of diseases owing to its low toxicity. In this study, a soy phytoestrogen (with high levels of the isoflavones genistin and daidzein) was tested in mice to investigate its mutagenicity and genotoxicity using micronucleus and comet assays of mouse peripheral blood. Phytoestrogen (0.083, 0.83 and 8.3 mg/kg body weight) was evaluated with and without the chemotherapeutic agent cyclophosphamide. For the micronucleus assay, blood was collected before treatment and after 24 and 48 h. For the comet assay, blood was collected only after 24 h. Phytoestrogen was not mutagenic and reduced cyclophosphamide-induced DNA damage. The results from the comet assay revealed a reduction of DNA damage; however, phytoestrogen did induce genotoxic damage during the 24-h treatment. This genotoxic damage could have been repaired and was therefore not identified in the micronucleus assay, which detects mutations. The results suggested that the reduction of DNA damage observed in associated treatments could also reduce the side effects of chemotherapy. Moreover, they suggested that phytoestrogen might be a candidate of interest for the chemoprevention of cancer because it protects against DNA damage.

Cyclosporin A treatment and decreased fungal load/antigenemia in experimental murine paracoccidioidomycosis.

Paracoccidioidomycosis (PCM) is a systemic mycosis caused by the fungus Paracoccidioides brasiliensis (Pb). The cyclosporin A (CsA) is an immunosuppressant drug that inhibits calcineurin and has been described as a potential antifungal drug. The present study investigated the effect of CsA on the immune response, fungal load/antigenemia in experimental murine PCM. It was used four groups of BALB/c mice: (a) infected with 1 x 105 Pb18 yeast cells (Pb), (b) infected and treated with CsA every other day 10 mg/kg of CsA (s.c.) during 30 days (Pb/CsA), (c) treated with CsA (CsA) and (d) no infected/treated (PBS). The immune response was evaluated by lymphocyte proliferation, DTH assays to exoAgs, ELISA for IgG anti-gp43 (specific immune responses) and cytokine serum levels (IFN-γ, TNF-α, IL-4 and IL-10). Fungal load was determined by lung colony-forming units (CFU) counts, lung and liver histopathology analysis and antigenemia determined by inhibition-ELISA. As expected, CsA was able to inhibit the specific cellular and humoral immune response (P < 0.05), with decrease in serum IFN-γ, TNF-α and IL-4 levels (P < 0.05). Cyclosporin A treatment also resulted in significantly decreased lung Pb CFU (P < 0.05) as well as a lower number of yeasts in the lung and liver (P < 0.05) by histopathology. In concordance, the decreased antigenemia was observed in Pb/CsA group (P < 0.05). In conclusion, even with immunosuppressive action, treatment with CsA results in decreased lung fungal load/antigenemia in experimental PCM in BALB/c mice. Further study is required to determine whether this represents less severe disease or protection by CsA.

Brazilian natural dietary components (annatto, propolis and mushrooms) protecting against mutation and cancer.

Considering the high number of new cancer cases in Brazil (approximately 470000 cases in 2005) and the remarkable differences in the incidence of this disease around the world, the development of chemopreventive strategies using foods widely consumed would have a huge impact, both medically and economically. This review summarizes some of our studies conducted to verify the anti-mutagenic and anti-carcinogenic potential of some Brazilian natural dietary constituents (annatto, mushrooms, and propolis). Overall data have shown a clear role for these compounds in preventing mutation and specific preneoplastic lesions. Taken together, these agents indicate a favorable side-effect profile and may prove to be a promising alternative for cancer prevention strategies, although more investigation is needed to fully explore this issue.

Cytotoxic and genotoxic butanolides and lignans from Aiouea trinervis.

The ethanolic extracts from the roots, the underground trunk and the leaves of Aiouea trinervis were active in the brine shrimp (Artemia salina) lethality assay (LD (50): 1.93, 0.92 and 262.1 microg/mL, respectively). Fractionation of the extracts led to the isolation of four butanolides, namely (-)-epilitsenolides C (2) and C (1) ( 1 and 2), isoobtusilactone A ( 3) and obtusilactone A ( 4), two of which ( 1 and 2) are reported for the first time as genuine natural products. The lignans (+)-sesamin ( 5) and (+)-methylpiperitol ( 6) and polyprenol-12 ( 7) were isolated as well. Their structures were determined with spectral methods (1D-, 2D-NMR and MS). Compounds 1, 2, 3, 5 and 6 were tested for their cytotoxic activities in Hep (2) human cancer cells. The butanolides 2 and 3 were the most active (IC (50): 5.96 microg/mL and 4.95 microg/mL, respectively) whereas the other compounds showed moderate IC (50) values ranging from 12.20 microg/mL to 25.64 microg/mL. The genotoxic properties of the crude ethanolic extracts and of compounds 3 and 5 were also evaluated in this study on CHO K1 and HTC mammalian cells with single-cell gel electrophoresis (comet assay). The crude extracts as well as the compounds tested induced DNA migration in this assay, which was indicative of DNA damage (genotoxic effect).

Possible modulating actions of plant extracts on the chromosome breaking activity of MMC and Ara-C in human lymphocytes in vitro.

Plants popularly used as medicine have been seen as promising natural agents by the pharmaceutical industry. In the present study the action of Psidium guajava L. (Pg) and Achillea millefolium L. (Am) infusions on chromosomal aberration formation in human lymphocyte system in vitro was assessed, associating them with the alkylating agent mitomycin C (MMC) and the DNA repair inhibitor cytosine-beta-arabin-furanoside (Ara-C). The cells were cultivated for 72 h and treated continuously with Pg and the Am infusions at dosages of 2.62 x 10(-4) g and 3.5 x 10(-4) g/ml culture medium, respectively. Treatments with MMC (0.30 microg/ml) or Ara-C (5 x 10(-7) microg/ml) were administered after 48 h of cell culture. Each samples (five individual) were exposed to nine treatments (control with PBS; Pg; Am; MMC; MMC+Pg; MMC+Am; Ara-C; Ara-C+Pg; and Ara-C+Am) and 100 cells were analyzed per cell culture. The used doses of each infusion did not cause clastogenic effects significantly different to the negative control (control=1%; Pg=2.2%; Am=1.8%). Nevertheless, the aberrant cell frequency after MMC treatment was significantly increased by the Am infusion (MMC=32.4%; MMC+Pg=36.2%; MMC+Am=44%), especially when the chromatid break types number was scored (MMC=151; MMC+Pg=173; MMC+Am=249). Regarding DNA repair inhibition by Ara-C, the Pg infusion caused a significant reduction in aberrant cell frequency (Ara-C=15.8%; Ara-C+Pg=11%; Ara-C+Am=14.4%), only when the chromatid break types number was scored (Ara-C=63; Ara-C+Pg=40; Ara-C+Am=58). These results indicate that the plant infusions per se do not have clastogenic activity, but can influence the clastogenic action of MMC and Ara-C on DNA break induction, in vitro.

Evaluation of the genotoxic potential of the Casearia sylvestris extract on HTC and V79 cells by the comet assay.

Casearia sylvestris is common in tropical America growing wild in Brazil in the states of Amazonas and São Paulo. Its leaves are used in Brazilian folk medicine for several diseases. The present investigation was carried out to examine the genotoxic effects of a C. sylvestris crude ethanolic extract on Hepatoma Tissue Culture (HTC cells) of Rattus norvegicus and Chinese hamster V79 cells in culture, using the comet assay. For the genotoxic evaluation the cells were treated with three different concentrations (0.5, 1 and 2 mg/ml) of extract prepared from a 25 mg/ml aqueous solution. The positive control was cyclophosphamide for HTC cells and methyl methanesulfonate for V79 cells. The duration of the treatment was 2 h. The results showed that the extract of C. sylvestris presented no genotoxic effects and not modified effect inducing DNA damage by alkylating agents cyclophosphamide and methyl methanesulfonate in HTC and V 79 cells respectively.

Mechanism of anticlastogenicity of Agaricus blazei Murill mushroom organic extracts in wild type CHO (K(1)) and repair deficient (xrs5) cells by chromosome aberration and sister chromatid exchange assays.

Agaricus blazei Murill is a medicinal mushroom native to Brazil. The present work assessed the clastogenic and anticlastogenic potential of organic extracts (ethanol and chloroform/methanol) from the lineage AB97/11 in chinese hamster CHO-K(1) (wild type) and CHO-xrs5 (repair deficient) cells using the chromosome aberration (CA) and sister chromatid exchange (SCE) assays. In these experimental conditions were observed: (a) anticlastogenic effect at concentrations of 0.06 and 0.09% of the EtOH extract and at the 0.03 and 0.06% concentrations of the C/MetOH extract in CHO-K(1); (b) absence of protector effect on CHO-xrs5 cells; and (c) absence of protector effect in the SCE assay. These results indicate that organic extracts of A. blazei lineage AB97/11 present bio-antimutagenic type protective activity.

Antimutagenic effects of the mushroom Agaricus blazei Murrill extracts on V79 cells.

Agaricus blazei Murrill, a native mushroom in Brazil, has been widely consumed in different parts of the world due to its medicinal power. Its anticarcinogenic activity has been shown in experimental animals, and antimutagenic activity has been demonstrated only in Salmonella. In this work, the mutagenic and antimutagenic activities of mushroom teas of strains AB96/07, AB96/09 and AB97/11 were evaluated in Chinese hamster V79 cells, using the comet assay and the micronucleus test. The cells were treated with three different concentrations (0.05, 0.1 and 0.15) of teas prepared from a 2.5% aqueous solution, under three different temperatures: (1) room (20-25 degrees C); (2) ice-cold (2-8 degrees C); and (3) warm (60 degrees C). The teas were applied in co-, pre- and post-treatments in combination with the mutagen methyl methanesulfonate (MMS; 1.6x10(-4) and 4x10(-4)M). The duration of the treatment was 1h in the comet assay and 2h in the micronucleus test. The results showed that the mushroom was not mutagenic itself. Nevertheless, the mushroom is an efficient antimutagen against the induction of micronuclei by MMS in all concentrations and preparations tested. The observed reductions in the frequencies of micronuclei ranged from 61.5 (room temperature 0.1% tea in post-treatment) to 110.3% (co-treatment with warm and ice-cold 0.15% tea). In the comet assay, the antimutagenic activity was detected only when the cells were pre-treated with the following teas: warm 0.1 and 0.15%, room temperature 0.05% and ice-cold 0.1%. The results indicate that the mushroom A. blazei extracts are antimutagenic when tested in V79 cells.