Reduction of DNA-damaging effects of anti-HIV drug 3'-azido-3'-dideoxythymidine on human cells by ursolic acid and lignin biopolymer.

In this study we verified our assumption that the genotoxicity of the effective anti-HIV drug 3'-azido-3'-dideoxythymidine (AZT) on human cells could be reduced by non-toxic concentrations of two antioxidants that occur frequently in nature (ursolic acid and lignin biopolymer). Cytotoxicity of these natural compounds, well-known by their antimutagenic effects, was evaluated by the trypan blue exclusion technique. Genotoxic activity of AZT was measured on the basis of AZT-induced single and double strand breaks to DNA in two histopathologically different types of human cells, hepatoma cells HepG2 and colonic cells Caco-2. Induction of DNA strand breaks was measured by the comet assay processed in parallel at pH > or = 13.0 (standard alkaline technique which enables to recognize single strand DNA breaks of different origin) and at pH = 9.0 (neutral technique which enables to recognize double strand DNA breaks). As the level of AZT-induced double strand DNA breaks was rather low, protective effects of the antioxidants tested were evaluated only against AZT-induced single strand DNA breaks by the standard alkaline comet assay. Our findings showed that 1 h pre-incubation of cells with ursolic acid or lignin preceding to 3 h treatment of cells with AZT (3 mg/ml) significantly decreased in both cell types the level of AZT-induced single strand DNA breaks. Pre-incubation of HepG2 or Caco-2 cells with a mixture of both natural antioxidants did not increase the effects of individual treatments. This study confirms that AZT is genotoxic toward both used cell types of human origin and that ursolic acid and biopolymer lignin can protect the cells studied against genotoxic effect of AZT.

Repair of oxidative DNA lesions in blood lymphocytes isolated from Sprague-Dawley rats; the influence of dietary intake of lignin.

Living organisms possess a variety of self-protective mechanisms which decrease the free radical attack on DNA and so reduce the risk of cancer. Protection of DNA by endogenous antioxidant systems may be significantly increased by numerous exogenously administered antioxidants. Many of them represent important dietary factors. Biopolymer lignin with its phenolic structure can be included into this group of micronutrients. The aim of the present work was to investigate: 1. the effect of biopolymer lignin, given to Sprague-Dawley (SD) rats in diet, on the level of oxidative DNA lesions induced by oxidative stress in freshly isolated peripheral blood lymphocytes in vitro and 2. the influence of lignin on kinetics of rejoining of DNA strand breaks induced in lymphocytes under these conditions. As model oxidative agents were used H2O2 and visible light in the presence of the photosensitizer Methylene Blue. We found out that dietary intake of lignin caused a significant decrease of H2O2-induced DNA strand breaks and visible light-induced oxidative DNA lesions in freshly isolated rat lymphocytes, but it did not influence the kinetics of rejoining of DNA strand breaks.

Lignin reduces ofloxacin-induced mutagenicity in Euglena assay.

The possible protective effect of sulphur-free beech lignin polymer on the mutagenicity of ofloxacin in Euglena gracilis was studied. The generation of oxygen species by ofloxacin and their possible interaction with lignin was verified by physico-chemical measurements. The UV absorbance spectra of ofloxacin with and without lignin showed no interaction between these two compounds. The production of superoxide anion radical (O2-) by ofloxacin was significantly reduced in the presence of lignin (AIR = 0.57 +/- 0.03, p < 0.01). Lignin, at concentrations of 125 and 250 micrograms/ml decreased the E. gracilis bleaching activity of ofloxacin to 39.9% and 2.8%, respectively. A lignin concentration of 500 micrograms/ml eliminated the bleaching activity of ofloxacin very efficiently. Our results are consistent with the concept that lignin biopolymer has the capability of reducing genotoxic activity by scavenging reactive oxygen species.

Oxidative/antioxidative effects of different lignin preparations on DNA in hamster V79 cells.

Endogenous oxidative damage to DNA is thought to be an important etiologic factor in the development of chronic diseases such as cancer. Many products of the vegetable kingdom have been suggested to limit oxidative damage to DNA in humans. To this group belong lignins, polyphenols present in all plants (including edible plants). The aim of this study was to examine oxidative/antioxidative effects of different lignin preparations on mammalian DNA. In addition to a water-soluble sulfur-free lignin 1 which was obtained by fractionation of hardwood hydrolysate, we investigated lignin 2 (obtained by oxidation of lignin 1), lignin 3 (prepared by the extraction of lignin 2 with a mixture ethanol-water 3:1), lignin 4 (Na-salt of lignin 3) and lignin 5 (prepared by extraction of lignin 2 with diethylether). Our results showed that only the original lignin 1 did not increase substantially the level of DNA damage. Lignins 2, 3, 4 and 5 increased both the level of frank DNA strand breaks + alkali-labile sites and the level of FPG-sensitive sites representing oxidative damage to DNA. Lignin 1 was further tested for its antioxidative activity against DNA base modifications generated by visible light+photosensitizer. Obtained results confirmed the oxygen species-scavenging activity of lignin 1.

Detection of lignin biopolymer- and vitamin E-stimulated reduction of DNA strand breaks in H2O2- and MNNG-treated mammalian cells by the comet assay.

In this study the possible protective effects of water-soluble sulfur-free lignin biopolymer and vitamin E (alpha-tocopherol) on DNA in human VH10 cells and hamster V79 cells exposed to H2O2 and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) were investigated. The level of DNA damage (DNA strand breaks) was measured using single-cell gel electrophoresis, i.e., comet assay. Lignin biopolymer and vitamin E exhibited a protective effect against the overall DNA damage induced after H2O2 treatment. If H2O2-treated human cells were incubated for 90 minutes to ligate frank breaks of DNA, two lesion-specific enzymes, endonuclease III and formamidopyrimidine DNA glycosylase (FPG), significantly increased the level of DNA strand breaks originating from oxidized pyrimidines and purines. Preincubation of cells with lignin or vitamin E reduced mainly the level of oxidized pyrimidines. Reduction of oxidized purines was less evident. In addition, lignin biopolymer exhibited a protective effect against MNNG-induced DNA damage, whereas vitamin E exhibited a protective effect only against H2O2-induced DNA damage. These findings suggest that the antioxidant nature of lignin biopolymer enables a reduction of the level of frank breaks and of oxidized DNA bases in H2O2-treated cells, and its adsorptive capacity enables binding of nitroso compounds and reduction of alkylation in MNNG-treated cells.

Growth of Saccharomyces cerevisiae, Rhodotorula rubra and Bullera alba in the presence of beechwood prehydrolyzate-based lignin fractions.

The growth of yeast strains Saccharomyces cerevisiae, Rhodotorula rubra and Bullera alba isolated from natural lake microflora was examined in the presence of prehydrolysis lignin and/or its water-soluble derivative. The stimulation effect of the water-soluble lignin derivative was higher in comparison with that of unmodified lignin. The structural changes of the lignin macromolecule by the yeasts determined by IR spectroscopy indicate oxidative degradation and demeth(ox)ylation similar to that found with lignin-degrading hyphal fungi. The results indicate a partial utilization of lignin by the yeasts as carbon source.

Antimicrobial properties of aromatic compounds of plant origin.

The antimicrobial action of 11 compounds involving guaiacyl- and syringyl-like structures (low-molecular-weight part of lignin), gallic acid and its derivatives, cinnamic acid and its derivatives, veratric acid, anisic acid and crotonic acid (a total of 25 compounds) against bacteria, yeast-like organisms and protozoa was examined. Aromatic compounds modified in the C-side chain and aldehydes were effective preferentially against Trichomonas vaginalis, whereas against bacteria and yeast-like organisms eugenol was the most effective inhibitor.

Antibiotic properties of lignin components.

Inhibitory effects of compounds with guaiacyl and syringyl structure, representing the structure of native lignin, were studied on model cultures of bacteria, yeasts, yeast-like microorganisms and moulds. Isoeugenol exhibited the most pronounced inhibitory effect on growth of the studied microorganisms.