Darina Slamenova

Assessment of Antioxidative, Chelating, and DNA-Protective Effects of Selected Essential Oil Components (Eugenol, Carvacrol, Thymol, Borneol, Eucalyptol) of Plants and Intact Rosmarinus officinalis Oil

Selected components of plant essential oils and intact Rosmarinus officinalis oil (RO) were investigated for their antioxidant, iron-chelating, and DNA-protective effects. Antioxidant activities were assessed using four different techniques. DNA-protective effects on human hepatoma HepG2 cells and plasmid DNA were evaluated with the help of the comet assay and the DNA topology test, respectively. It was observed that whereas eugenol, carvacrol, and thymol showed high antioxidative effectiveness in all assays used, RO manifested only antiradical effect and borneol and eucalyptol did not express antioxidant activity at all. DNA-protective ability against hydrogen peroxide (H2O2)-induced DNA lesions was manifested by two antioxidants (carvacrol and thymol) and two compounds that do not show antioxidant effects (RO and borneol). Borneol was able to preserve not only DNA of HepG2 cells but also plasmid DNA against Fe(2+)-induced damage. This paper evaluates the results in the light of experiences of other scientists.

Study of N-nitrosomorpholine-induced DNA strand breaks in Caco-2 cells by the classical and modified comet assay: influence of vitamins E and C.

We have studied the genotoxic effects of the well-known heterocyclic liver carcinogen N-nitrosomorpholine (NMOR), an N-nitroso compound, which was prepared in our laboratory by nitrosation of the secondary amine morpholine with NaNO2. NMOR induced DNA strand breaks in human colon carcinoma Caco-2 cells. The concentration-dependent DNA-damaging effects of NMOR were proved by the comet assay. We further characterized DNA strand breaks induced by NMOR as follows: 1) We pretreated cells with vitamins E and C, which are able to eliminate oxidative DNA damage. 2) We varied the pH of the comet assay (12.1 and 13). In general, alkali-labile sites are stable until pH is raised to 12.5. 3) We used the site-specific repair enzymes exonuclease III and formamidopyrimidine-DNA glycosylase in the modified comet assay. Results showed that NMOR-induced DNA strand breaks have their origin exclusively in alkali-labile sites. Nevertheless, vitamins E and C decreased the level of DNA strand breaks. These results showed that antioxidants may have biological activities other than free radical scavenging that relate to their cancer-prevention properties. Our conceptions about reduction of NMOR-induced DNA lesions in Caco-2 cells by vitamins E and C are presented in this work.

Comparison of biological processes induced in HepG2 cells by tert-butyl hydroperoxide (t-BHP) and hydroperoxide (H2O2): The influence of carvacrol.

This paper presents comparisons of biological impacts of the oxidants H2O2 and t-BHP on human liver cells, and shows modulation of these effects by the phenolic compound carvacrol. To understand better how these oxidants exert their effect on DNA and on the activity of the enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPx), we measured intracellular antioxidant glutathione (iGSH) and intracellular reactive oxidative species (iROS). DNA lesions corresponded to single-strand DNA breaks, alkali-labile lesions and formamido-pyrimidine-DNA-glycosylase (FPG)-sensitive sites. Pre-treatment of cells with carvacrol substantially decreased the number of H2O2-induced DNA lesions, but the number of t-BHP-induced DNA lesions was not reduced. Activities of both SOD and GPx were stimulated significantly by carvacrol and were reduced by the combined effect of carvacrol and oxidants. H2O2 and t-BHP alone influenced the level of antioxidant enzymes differently. While H2O2 did not markedly change the activity of SOD or GPx, lower concentrations of t-BHP stimulated activity of SOD and mainly GPx. The level of iROS was increased by both oxidants and decreased by carvacrol applied either alone or with oxidants. The level of iGSH was not influenced in any of the treatments tested. Our results show that although both oxidants induced oxidative stress and damaged cellular DNA, their influences on other molecular processes were different. The protective effect of carvacrol against DNA-damaging effects of H2O2 was unambiguous, but reduction by carvacrol of the DNA-damaging effect of t-BHP was not observed. These results suggest that the phenolic compound carvacrol contributes to the defence mechanisms of the human organism, but these beneficial effects are dependent on the origin and source of the actual oxidative stress.

EFFECT OF DIETARY INTAKE OF VITAMIN A OR E ON THE LEVEL OF DNA DAMAGE, CHROMOSOMAL ABERRATIONS AND MICRONUCLEI INDUCED IN FRESHLY ISOLATED RAT HEPATOCYTES BY DIFFERENT CARCINOGENS

Hepatocytes freshly isolated from male Wistar rats fed a common diet or a vitamin A- or vitamin E-supplemented diet (each for 21, 28, or 41 days) were assayed for sensitivity to DNA breakage and cytogenetic changes induced by carcinogens. Different indirectly acting carcinogens were assayed. N-nitrosomorpholine (NMOR) was the only agent that induced DNA breaks, chromosomal aberrations, and micronuclei in all experiments. Benzo[a]pyrene (B[a]p) and dimethyldibenzo[c,g]carbazole (diMeDBC) induced only DNA breaks in all experiments. Occasionally, B[a]P induced chromosomal aberrations and micronuclei, and diMeDBC induced micronuclei, but not chromosomal aberrations. These results demonstrated that the tested carcinogens assayed at concentrations highly effective in a hypoxanthine phosphoribosyltransferase/V79 system significantly increased DNA damage, while cytogenetic changes were less frequent. In hepatocytes from rats fed vitamin A, a reduction in the severity of all three end points was observed after NMOR treatment. After B[a]P treatment, we found a reduction in DNA breaks and chromosomal aberrations; after treatment with diMeDBC, we observed a reduction in DNA breaks. Treatment with vitamin E was less effective: it reduced DNA strand breaks induced by B[a]P and partially reduced those induced by diMeDBC and NMOR and the level of micronuclei induced by NMOR and B[a]P. Both vitamins reduced the level of DNA strand breaks induced by the oxidative effect of a visible light-excited photosensitizer.

Effects of Dietary Intake of a Fungal β-D-Glucan Derivative on the Level of DNA Damage Induced in Primary Rat Hepatocytes by Various Carcinogens

Abstract: Water-soluble derivative of chitin-glucan complex used in our study, carboxymethyl chitin-glucan (CM-CG), enables oral administration without harmful side-effects, which can occur upon parenteral administration of the insoluble fungal β-D-glucans. The aim of this study was to determine in ex vivo experiments the effects of dietary CM-CG on the level of DNA lesions in primary rat hepatocytes induced by various indirectly acting carcinogens. Multiorgan carcinogen benzo[a]pyrene (BaP); two hepatocarcinogens, dimethyldibenzocarbazole (diMeDBC) and N-nitrosomorpholine (NMOR); as well as a complex mixture of organic compounds adsorbed on ambient air particles (TP-S) were used for this purpose. The amount of DNA lesions was assessed using the comet assay and the micronucleus test. In addition, the mitotic indexes and the frequencies of necrotic and apoptotic cells were evaluated as well. Our results showed that the diet enriched with CM-CG (200 mg/kg of body weight) during 21 days did not induce any negative effect on DNA nor did the mitotic indexes and the frequencies of necrotic and apoptotic cells differ statistically from the controls. On the other hand, the hepatocytes isolated from CM-CG fed animals were more resistant to the action of all genotoxins used in our study [BaP (5–20 μM), diMeDBC (0.2–2 μM), NMOR (3.4–10.2 mM), TP-S (5–20 μM)]. We can conclude that in addition to the known immunopotentiating activity of β-D-glucans, they can efficiently inhibit the genotoxicity of carcinogens requiring metabolic activation in rat heptocytes.

Induction of DNA-lesions in Freshly Isolated Rat Hepatocytes by Different Genotoxins and Their Reduction by Lignin Given Either as a Dietary Component or in In Vitro Conditions

Abstract: The aim of our investigation was to verify the protective effect of lignin on DNA in rat hepatocytes damaged by 3 different genotoxins attacking DNA in a different manner. Hydrogen peroxide was used for induction of direct single strand breaks of DNA, visible light-excited methylene blue for induction of oxidized DNA lesions and 1,2-dibromo-3-chloropropane for induction of alkali-labile DNA lesions. Hepatocytes were pre-treated with lignin either immediately after isolation, i.e., in in vitro conditions, or the hepatocytes were isolated from rats fed a lignin enriched diet for 21 days (ex vivo conditions). The protective effect of lignin applied to hepatocytes by the first or by the second approach was tested on the level of DNA using classical and modified single cell gel electrophoresis (SCGE). We found that lignin applied by each way significantly reduced the level of direct DNA strand breaks induced by H 2 O 2 , alkali-labile sites of DNA induced by DBCP as well as oxidative DNA lesions induced by visible light-excited methylene blue. These results confirm that lignin represents a very important micronutrient in our vegetable food, protecting DNA against damaging effects of different genotoxicants.

Study of the biological effects of theophylline on V79 cells: Viability, membrane permeability, and metabolic cooperation

The effect of theophylline, a specific inhibitor of phosphodiesterase, on gap junction-mediated intercellular communication between Chinese hamster V79 cells was examined. It was found that addition of theophylline to coculture of 6-thioguanine-resistant (TGr) and 6-thioguanine-sensitive (TGs) V79 cells significantly increased the recovery of TGr cells. This finding indicates an inhibition of metabolic cooperation of V79 cells by theophylline. Theophylline was tested at concentrations <0.3 mg/ml, which were neither cytotoxic (after short or continuous exposure) nor inhibited the synthesis of DNA, RNA, and proteins. At the tested concentrations, no change was found in the membrane permeability of cells. Theophylline did not increase the incorporation of glucose into the cells.

Chromatographic analyses of Lavandula angustifolia and Rosmarinus officinalis extracts and their biological effects in mammalian cells and cell-free systems

Knowledge of biological properties of natural compounds allows to understand their therapeutic value, efficacy and security. We investigated: composition of Lavandula angustifolia (LA) and Rosmarinus officinalis (RO) extracts, their antioxidant capacity, cytotoxicity and genotoxicity, their DNA-protective potential against DNA damage induced in hamster V79 cells by several genotoxins or in plasmid DNA by Fe2+ ions and activity of antioxidant enzymes in cells treated with these extracts. Higher cytotoxicity, observed at higher concentrations of extracts, was accompanied by the increased level of single-strand (ss) DNA breaks as well as formamidopyrimidine DNA glycosylase (Fpg) sensitive sites. LA and RO extracts were able to protect DNA of hamster cells as well as plasmid DNA against ss DNA breaks induced by genotoxins and Fe2+. LA extract mildly increased the activity of superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT), while RO extract decreased the activity of SOD, but increased the activity of CAT and GPx. Cell-free tests confirmed antioxidant activity of both extracts. The biological properties of LA and RO extracts showed that they could have a positive impact on human health.