Darina Slameňová

Protective effects of fungal (1→3)-β-d-glucan derivatives against oxidative DNA lesions in V79 hamster lung cells

beta-Glucans belong to the class of substances known as biological response modifiers with a broad range of activity. We have investigated two types of glucans: (1-->3)-beta-D glucan from the bakers yeast Saccharomyces cerevisiae and beta-glucan-chitin complex from the mycelium of filamentous fungus Aspergillus niger. Since these fibrillar beta-glucans are insoluble in water, their water-soluble derivatives--carboxymethyl glucan (CM-G), sulfoethyl glucan (SE-G), and carboxymethyl chitin-glucan (CM-CG) were prepared and tested. The aim of the present work was to investigate the protective effect of the prepared glucan derivatives against oxidative DNA damage induced by H2O2 and visible light-excited Methylene Blue in V79 hamster lung cells. The level of DNA damage (DNA strand breaks) was measured using the single cell gel electrophoresis, so called comet assay. Our findings demonstrate that all three tested glucans reduce oxidative DNA damage. The ability to reduce genotoxic activity increased in the order: CM-G<SE-G<CM-CG. We suggest that the analyzed glucans exhibit protective effects against oxidative damage to DNA as a consequence of scavenging of both *OH radicals and singlet oxygen.

Investigation of anti-oxidative, cytotoxic, DNA-damaging and DNA-protective effects of plant volatiles eugenol and borneol in human-derived HepG2, Caco-2 and VH10 cell lines

Plant volatiles, which can get into the human organism in food, medicines, or cosmetic preparations, frequently manifest antibacterial, antifungal, antiviral and other effects. We studied anti-oxidative, cytotoxic, genotoxic and possible DNA-protective effects of eugenol and borneol. Anti-oxidative activities of aqueous and ethanolic solutions of these two volatile compounds of plants were determined by a spectrophotometric method by the use of the stable DPPH radical. Borneol did not show any anti-oxidative activity even at the highest concentrations soluble in water or ethanol (<1000mM), while eugenol did manifest anti-oxidative activity, and at much lower concentrations (5-100 microM). The cytotoxicity of eugenol and borneol as well as their DNA-damaging effects and their influence on sensitivity of cells against the DNA-damaging effects of H(2)O(2) were investigated in three different cell lines, i.e. malignant HepG2 hepatoma cells, malignant Caco-2 colon cells, and nonmalignant human VH10 fibroblasts. The trypan-blue exclusion assay showed that in the three cell lines the cytotoxicity of eugenol was significantly higher than that of borneol. Single-cell gel electrophoresis revealed that borneol did not cause any DNA strand-breaks at the concentrations studied, but showed that all concentrations of eugenol (<600 microM) significantly increased the level of DNA breaks in human VH10 fibroblasts and to a lower degree in Caco-2 colon cells. The DNA-damaging effects of eugenol were not observed in metabolically active HepG2 hepatoma cells. Borneol and eugenol differed also with respect to their DNA-protective effects. While borneol protected HepG2 and, to a lesser extent, VH10 cells (but not Caco-2) against H(2)O(2)-induced DNA damage, eugenol either did not change the cellular sensitivity to H(2)O(2) (HepG2 cells) or it even increased the sensitivity (Caco-2 and VH10 cells). These results do not indicate any correlation between the DNA-protective and the anti-oxidative capacities of eugenol and borneol.

Rosemary-stimulated reduction of DNA strand breaks and FPG-sensitive sites in mammalian cells treated with H2O2 or visible light-excited Methylene Blue.

In this study possible protective effects of rosemary against oxidative DNA damage induced by H2O2- and visible light-excited Methylene Blue in colon cancer cells CaCo-2 and hamster lung cells V79 were investigated. The level of DNA damage (DNA strand breaks) was measured using the classical and modified single cell gel electrophoresis, so-called comet assay. Our findings showed that an ethanol extract from rosemary reduced the genotoxic activity of both agents after a long-term (24 h; 0.3 microg/ml) or short-term (2 h; 30 microg/ml) pre-incubation of cells. We suggest that the extract of rosemary exhibits a protective effect against oxidative damage to DNA as a consequence of scavenging of both *OH radicals and singlet oxygen ((1)O2).

The effects of sodium azide on mammalian cells cultivated in vitro

Sodium azide acted cytostatically to cytotoxically on 2 lines of mammalian cells. After application of the substance in an acid environment the highest cytostatic effect was noted. The results of the DNA-synthesis inhibition test suggest that sodium azide does not damage the DNA of the observed fibroblasts with any of the tested modes of application. In Chinese hamster cells neither 20-h treatment in medium nor 60-min treatment in an acid environment gave rise to significantly increased occurrence of 6-TG-resistant mutations. The results of the DNA-synthesis inhibition test, as well as the mutagenicity testing, do not suggest the possibility that treatment with sodium azide might induce DNA damage in the observed human and Chinese hamster cells. The cytostatic effect of sodium azide on the fibroblasts studied is probably not accompanied by a genotoxic effect.

Effects of borneol on the level of DNA damage induced in primary rat hepatocytes and testicular cells by hydrogen peroxide.

The aim of this paper was to evaluate genotoxic effects of borneol and its ability to change DNA-damaging effects of H2O2 in rat hepatocytes and testicular cells. Both in vitro and ex vivo approaches were used in the case of hepatocytes. Testicular cells were tested only ex vivo, i.e. shortly after isolation from rats supplemented by borneol. Cytotoxicity of borneol increased in in vitro conditions in a concentration-dependent manner and it was associated with DNA-damaging effects at toxic concentrations. While non-toxic concentrations of borneol applied in vitro protected cells against H2O2-induced DNA damage and interfered only partly with rejoining of H2O2-induced DNA strand breaks, cytotoxic concentrations of borneol manifested synergy with H2O2, i.e. enhanced DNA-damaging effects of H2O2. On the other side, borneol given to rats in drinking water decreased the level of DNA damage induced by H2O2 in both hepatocytes and testicular cells. Our results show that though at higher concentrations (2-h treatment with >2 mM borneol >0.3084 mg/ml) borneol acts cytotoxically and genotoxically on primary hepatocytes cultured in vitro, if given to rats during 7 days in a daily concentration of 17.14 or 34.28 mg/kg it reduces genotoxicity of H2O2 in both hepatocytes and testicular cells.

Cytotoxicity and genotoxicity testing of sodium fluoride on Chinese hamster V79 cells and human EUE cells

The cytotoxic effects of sodium fluoride (NaF) on hamster V79 cells and human EUE cells were studied by measuring the cloning efficiency and DNA, RNA and protein synthesis in cells cultured in the presence of NaF. Potential mutagenicity of NaF was followed on the basis of induced 6-thioguanine-resistant mutants in treated Chinese hamster V79 cells. The results showed that the addition of 10-150 micrograms of NaF per ml of culture medium induced 10-75% cytotoxic effect on hamster V79 cells but had no toxic effect on human EUE cells. NaF was cytotoxic to human EUE cells at considerably higher concentrations (200-600 micrograms/ml). Growth of both cell types with 100 and 200 micrograms of NaF per ml caused inhibition of 14C-thymidine, 14C-uridine and 14C-L-leucine incorporation. This means that NaF inhibits macromolecular synthesis whereby damaging effects were less drastic in human EUE cells. The results of detailed mutagenicity testing on hamster V79 cells showed that NaF did not show any mutagenic effect after long-term (24-h) incubation of hamster cells in the presence of 10-400 micrograms of NaF per ml of culture medium.

Borneol administration protects primary rat hepatocytes against exogenous oxidative DNA damage

Experimental evidences suggest that most essential oils possess a wide range of biological and pharmacological activities that may protect tissues against oxidative damage. In this study, we investigated DNA-protective effect of borneol, a component of many essential oils, against oxidative DNA damage induced in primary cultures of rat hepatocytes. Borneol was added to drinking water of Sprague-Dawley rats and DNA resistance against oxidative agents was compared in hepatocytes originated from control and borneol-treated rats. Oxidative stress induced by visible light-excited methylene blue (MB/VL) or 2,3-dimethoxy-1,4-naphthoquionone (DMNQ) resulted in increased levels of DNA lesions measured by the modified single cell gel electrophoresis. Borneol (17 or 34 mg/kg body weight) added to drinking water of rats for 7 days reduced the level of oxidative DNA lesions induced in their hepatocytes by MB/VL or DMNQ. To explain the increased resistance of DNA towards oxidative stress, we measured the base-excision repair (BER) capacity in liver cell extracts of control and borneol-supplemented rats on DNA substrate of HepG2 cells containing oxidative damage. Our results showed that administration of borneol in drinking water had no effect on incision activity of hepatocytes isolated from supplemented rats. The spectrophotometric assessment of enzymatic antioxidants superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities and the flow cytometric assessment of total intracellular glutathione (iGSH) in primary hepatocytes of borneol-supplemented rats showed no changes in SOD and GPx activities but higher iGSH content particularly in hepatocytes of higher borneol dose (34 mg/kg) supplemented rats in comparison to control animals. Despite the fact that borneol had no effect either on BER of oxidative DNA damage or on the levels of antioxidant enzymes and manifested no reducing power and radicals scavenging activity, it increased significantly the level of non-enzymatic antioxidant iGSH which could reduce the oxidative DNA lesions induced by MB/VL or DMNQ.

Reduction of carcinogenesis by bio-based lignin derivatives

Abstract The potential medicinal application of lignin component of biomass derived from chemical treatment of wood for paper production was examined with regard to its ability to bind N -nitrosoamines and bile acids. Correlation between adsorption affinity of lignins towards N -nitrosodiethylamine and their cross-linking density was revealed. In contrast to lignin, all the tested carbohydrate preparations were poor adsorbents. It was revealed that the most effective lignin adsorbents—modified kraft and prehydrolysis lignins inhibit mutagenicity and SOS response induced by 4-nitroquinoline- N -oxide. Moreover, these lignin preparations exhibited a protective effect on deoxyribonucleic acid (DNA) in hamster V79 cells and in human VH10 and Caco-2 colon carcinoma cells exposed to hydrogen peroxide (H 2 O 2 ) treatment due to their antioxidant nature. In the case of N -methyl- N ′-nitro- N -nitrosoguanidine—treatment lignins reduced alkylation of DNA due to their high affinity for adsorption of mutagenic N -nitroso compounds. This dual ability of lignin tested to decrease genotoxic activity of chemicals seems to be very promising for their application as natural antimutagenic and anticarcinogenic agents.

Lignin antioxidants for preventing oxidation damage of DNA and for stabilizing polymeric composites

Abstract The antioxidative behavior of various lignin preparations derived from chemical wood treatment for paper production was examined. All lignin samples exhibited higher antioxidant activity than Trolox. The protective effect of lignin antioxidants against H2O2-induced oxidative damage of DNA in human carcinoma cells and male rats was evaluated. Moreover, lignin was tested as a stabilizer in the processing of polypropylene composites, as well as in thermo-oxidative aging of styrene-butadiene vulcanizates. The results indicate that the lignin preparations show great potential as antioxidants in human diets and polymer blends. Lignin has the potential to protect living organisms against cancer diseases and contributes to the protection of polymers against degradation.

Changes in the radioresistance of bacteria after the inhibition of proteosynthesis in the preirradiation phase

The strain ofEscherichia coli WP2 (tryv) was irradiated with UV light, at a dosage of 240 erg/mm. Proteosynthesis was inhibited by the elimination of the essential amino acid from the cultivation medium. Changes in radioresistance were followed during 45 minutes of starvation and during the subsequent 45 minutes of restitution after the addition of the essential amino acid. The radioresistance of the cells showed a linear increase immediately after the removal of the essential amino acids, proportional to the duration of the inhibition of proteosynthesis. The increase in radioresistance was shown to be reversible. After the addition of the essential amino acid there was an immediate decrease in radioresistance which was most marked in the first 15 minutes.