Juraj Lábaj

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.

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.

Influence of immunization with non-genotoxic PAH-KLH conjugates on the resistance of organisms exposed to benzo(a)pyrene.

Polycyclic aromatic hydrocarbons (PAH) are recognized as common environmental pollutants released into the environment from many natural as well as man-made sources, and some have been classified as potent carcinogens. The main representative of the carcinogenic PAH is benzo(a)pyrene (B(a)P) which is known to induce genotoxic effects in vitro and in vivo, detected as PAH-DNA adducts. Long-term PAH exposure may be accompanied by an immunological response with the formation of antibodies against PAH as well as against PAH-DNA adducts. This paper describes the use of four PAH-keyhole-limpet haemocyanin (KLH) conjugates for the induction of specific and cross-reactive anti-PAH antibodies and focuses on the potential protective effects of anti-PAH antibodies produced after immunization of mice. In the in vitro experiments with HepG-2 cells, the genotoxicity of the PAH-KLH conjugates and the neutralizing effect of induced anti-PAH antibodies were evaluated. The titer of specific anti-PAH antibodies in sera and the amounts of DNA adducts in liver homogenates from immunized mice were investigated in vivo. The results show that anti-PAH antibodies of class IgG were induced during immunization. All the PAH-KLH conjugates tested were non-genotoxic and did not induce detectable DNA adducts in HepG2 cells or in the liver of immunized mice. The results show that only B(a)P-specific and B(a)P cross-reactive antibodies are able to neutralize B(a)P or its activated metabolites, which was revealed by a sudden decrease in the titer of anti-B(a)P antibodies in mouse sera after exposure to B(a)P. Furthermore, the anti-B(a)P antibodies produced by immunization were effective in reducing the amount of DNA adducts in mouse livers after intraperitoneal (i.p.) exposure to B(a)P. The results suggest that immunization with PAH-KLH conjugates can protect organisms against the adverse effects of carcinogenic PAH.

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.

Lignin-stimulated reduction of oxidative DNA lesions in testicular cells and lymphocytes of sprague-dawley rats in vitro and ex vivo.

Abstract: Lignin biopolymers constitute 30% of plant biomass and belong to the most abundant organic polymers on earth. We showed previously that this important component of dietary fiber exhibited a protective effect against the overall DNA damage induced by H2O2 or N-methyl-N′-nitro-N-nitrosoguanidine in hamster lung cells and human foreskin cells cultured in vitro. The objective of the present work was to examine DNA-protective effects of lignin in rat testicular cells and rat peripheral blood lymphocytes using in vitro and ex vivo experiments. H2O2 and visible light-excited methylene blue (MB) were used as DNA-damaging agents. Testicular cells were chosen because the germinal epithelium of testes is one of the most proliferately active tissues potentially susceptible to DNA-damaging effects. As a second target peripheral blood lymphocytes were chosen because dietary lignin or its metabolites circulate in the animal organism probably through the blood system. For the in vitro experiments, isolated cells were preincubated with lignin for 2 h before treatment with one of the oxidative agents. In ex vivo experiments, the cells were exposed to H2O2 or visible light-excited MB after isolation from rats fed either a common diet or a lignin-supplemented diet. The water-soluble, sulfur-free lignin used in experiments was obtained by fractionation of hardwood hydrolysate. The level of direct single-strand DNA breaks in H2O2-treated cells was measured by the classical comet assay, and the level of oxidative DNA lesions in visible light-treated cells was measured by a modified comet assay. We found that lignin reduced DNA lesions induced by H2O2 or visible light-excited MB both in vitro and ex vivo. The major conclusion of our study is that lignin polymer obtained by fractionation of hardwood hydrolysate manifested a specific type of antimutagenic effect.

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.