Gamma-linolenic Acid Alters Ku80, E2F1, and Bax Expression and Induces Micronucleus Formation in C6 Glioma Cells In Vitro

Gamma-linolenic acid (GLA) is an inhibitor of tumor cell proliferation in both in vitro and in vivo conditions. The aim of this study was to investigate the effects of 150 mu M GLA on the expression of E2F1, cyclin D1, bax, bcl2, Ku70, and Ku80 in C6 rat glioma cells. The Ku proteins were chosen as previous studies have shown that loss or reduction in their expression causes increased DNA damage and micronucleus formation in the presence of radiation. The fact that GLA exposure is known to enhance the efficacy of radiation treatment raised the question whether the Ku proteins could be involved in this effect as seen for other molecules such as roscovitine and flavopiridol. GLA altered the mRNA expression of E2F1, cyclin D1, and bax, but no changes were found for bcl2, Ku70, and Ku80. Alterations in protein expression were observed for bax, Ku80, and E2F1. The 45% decrease in E2F1 expression was proportional to decreased cell proliferation (44%). Morphological analysis found a 25% decrease in mitotic activity in the GLA-treated cells, which was accompanied by a 49% decrease in S-phase by FACS analysis. A 39% increase in the number of micronuclei detected by Hoechst fluorescence points to GLA`s effects on cell division even at concentrations that do not produce significant increases in apoptosis. Most important was the finding that Ku80 expression, a critical protein involved in DNA repair as a heterodimer with Ku70, was decreased by 71%. It is probable that reduced Ku80 is responsible for the increase in micronucleus formation in GLA-treated cells in a similar manner to that found in Ku80 null cells exposed to radiation. The decreased expression of Ku80 and E2F1 could make cells more susceptible to radiotherapy and chemotherapy. (C) 2009 IUBMB

Chemoprotective effect of the tetrahydrofuran lignan grandisin in the in-vivo rodent micronucleus assay

Objectives The chemoprotective effect of the tetrahydrofuran lignan grandisin against DNA damage induced by cyclophosphamide (200 mg/kg) has been evaluated using the in vitro rodent micronucleus assay. Methods The effects of a daily oral administration of grandisin (2, 4, or 8 mg/kg) for five days before exposure to cyclophosphamide on the frequency of micronucleus in the bone marrow of normal mice exposed and unexposed to cyclophosphamide were investigated (n = 5 per group). Electrochemical measurements were applied to investigate whether the antimutagenic effects of grandisin could be, at least in part, a consequence of its or its metabolite`s antioxidant properties. Key findings Grandisin did not show mutagenic effects on the bone marrow cells of exposed mice. On the other hand, the oral administration of grandisin (2, 4, or 8 mg/kg) per day reduced dose-dependently the frequency of micronucleus, induced by cyclophosphamide, in all groups studied. Cyclic voltammograms showed two peaks for a grandisin metabolite, which were absent for grandisin. Conclusions Under the conditions tested herein, this study has shown that mice treated with grandisin presented, in a dose-dependent manner, a protective effect against cyclophosphamide-induced mutagenicity. This effect could be, at least in part, associated to grandisin bioactivation. These data open new perspectives for further investigation into the toxicology and applied pharmacology of grandisin.

Genotoxic effects of X-rays on keratinized mucosa cells during panoramic dental radiography

Objectives: The aim of this study was to evaluate the genotoxic effects of X-rays on epithelial gingival cells during panoramic dental radiography using a differentiated protocol for the micronucleus test. Methods: 40 healthy individuals who underwent this procedure for diagnostic purposes on request from their dentists agreed to participate in this study. All of them answered a questionnaire before the examination. Epithelial gingival cells were obtained from the keratinized mucosa of the upper dental arcade by gentle scraping with a cervical brush immediately before exposure and 10 days later. Cytological preparations were stained according to the Feulgen-Rossenbeck reaction, counterstained with fast green 1% for 1 min and analysed under a light microscope. Micronuclei, nuclear projections (broken eggs) and degenerative nuclear alterations (pyknosis, karyolysis, karyorrhexis and condensed chromatin) were scored. Results: The frequency of micronuclei was significantly higher after exposure (P < 0.05), as were frequencies of nuclear alterations indicate of apoptosis (P < 0.001). Conclusions: These results indicate that X-ray radiation emitted during panoramic dental radiography induces a genotoxic effect on epithelial gingival cells that increases the frequency of chromosomal damage and nuclear alterations indicative of apoptosis.

Genotoxicity and composition of particulate matter from biomass burning in the eastern Brazilian Amazon region

In the present study Tradescantia pallida micronucleus (Trad-MCN) bioassay was used to assess the genotoxicity of particulate matter with a mass median aerodynamic diameter less than 10 pm (PM(10)) in Tangara da Serra (MT), a Brazilian Amazon region that suffers the impact of biomass burning. The levels of PM (coarse and fine size fractions) and black carbon (BC) collected were also measured. Furthermore, the alkanes and polycyclic aromatic hydrocarbons (PAHs) were identified and quantified in the samples taken during the burning period by gas chromatography with flame ionization detection (GC-FID). The PM and BC results for both fractions indicate a strong correlation (p < 0.001). The analysis of alkanes indicates an anthropic influence. Retene was the most abundant PAH found, an indicator of biomass burning, and 12 other PAHs considered to be potentially mutagenic and/or carcinogenic were identified in this sample. The Trad-MCN bioassay showed a significant increase in micronucleus frequency during the period of most intense burning, possibly related to the mutagenic PAHs that were found in such extracts. This study demonstrated that Trad-MCN was sensitive and efficient in evaluating the genotoxicity of organic compounds from biomass burning. It further emphasizes the importance of performing chemical analysis, because changes in chemical composition generally have a negative effect on many living organisms. This bioassay (ex situ), using T. pallida with chemical analysis, is thus recommended for characterizing the genotoxicity of air pollution. Crown Copyright (C) 2011 Published by Elsevier Inc. All rights reserved.

Establishment of the comet assay in the freshwater snail Biomphalaria glabrata (Say, 1818)

The single cell gel eletrophoresis or the comet assay was established in the freshwater snail Biomphalaria glabrata. For detecting DNA damage in circulating hemocytes, adult snails were irradiated with single doses of 2.5. 5, 10 and 20 Gy of Co-60 gamma radiation. Genotoxic effect of ionizing radiation was detected at all doses as a dose-related increase in DNA migration. Comet assay in B. glabrata demonstrated to be a simple, fast and reliable tool in the evaluation of genotoxic effects of environmental mutagens. (c) 2008 Elsevier B.V. All rights reserved.

Cytotoxic and genotoxic effects of tambjamine D, an alkaloid isolated from the nudibranch Tambja eliora, on Chinese hamster lung fibroblasts

Marine organisms have been shown to be potential sources of bioactive compounds with pharmaceutical applications. Previous chemical investigation of the nudibranch Tambja eliora led to the isolation of the alkaloid tambjamine D. Tambjamines have been isolated from marine sources and belong to the family of 4-methoxypyrrolic-derived natural products, which display promising immunosuppressive and cytotoxic properties. Their ability to intercalate DNA and their pro-oxidant activity may be related to some of the biological effects of the 4-methoxypyrrolic alkaloids. The aim of the present investigation was to determine the cytotoxic, pro-oxidant and genotoxic properties of tambjamine D in V79 Chinese hamster lung fibroblast cells. Tambjamine D displayed a potent cytotoxic effect in V79 cells (IC50 1.2 mu g/mL) evaluated by the MTT assay. Based on the MTT result, V79 cells were treated with different concentrations of tambjamine D (0.6. 1.2. 2.4 and 4.8 mu g/mL). After 24 h, tambjamine D reduced the number of viable cells in a concentration-dependent way at all concentrations tested. assessed by the trypan blue dye exclusion test. The hemolytic assay showed that the cytotoxic activity of tambjamine D was not related to membrane disruption (EC50 > 100 mu g/mL). Tambjamine D increased the number of apoptotic cells in a concentration-dependent manner at all concentrations tested according to acridine orange/ethidium bromide staining, showing that the alkaloid cytotoxic effect was related to the induction of apoptosis. MTT reduction was stimulated by tambjamine D, which may indicate the generation of reactive oxygen species. Accordingly, treatment of cells with tambjamine D increased nitrite/nitrate at all concentrations and TBARS production starting at the concentration corresponding to the IC50. Tambjamine D, also, induced DNA strand breaks and increased the micronucleus cell frequency as evaluated by comet and micronucleus tests, respectively, at all concentrations evaluated. showing a genotoxic risk induced by tambjamine D. (C) 2008 Elsevier Ireland Ltd. All rights reserved.