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.

The selected plant extracts exhibited antibacterial activity. The strongest effect was manifested by extracts prepared from Gymnocladus dioicus, Amelanchier ovalis, Exochorda racemosa, Holodiscus discolor, Philadelphus microphyllus, Philadelphus coronarius and Pelargonium tabulare. The percentage inhibition of bacterial growth was 0–41.8%. In addition it was found that extracts isolated from Amelanchier ovalis, Exochorda racemosa and Pelargonium tabulare were specifically effective only against the bacterial strains tested. Copyright © 2000 John Wiley & Sons, Ltd.

7H-dibenzo[c,g]carbazole (DBC) is a potent liver and skin carcinogen, while its synthetic methyl derivative N-methyldibenzo[c,g]carbazole (MeDBC) is tissue specific sarcomagen. It is supposed that sarcomagenic activity of DBC depends on biotransformation at ring-carbon atoms, as with PAH, whereas the heterocyclic nitrogen plays an important role in liver carcinogenicity. The objective of this study was to elucidate the role of cytochrome P4501A1 in metabolic activation of sarcomagenic derivatives of DBC and to characterize the DNA damage profiles induced by DBC and MeDBC in relation to the mode of metabolic activation. The genetically engineered V79MZh1A1 cell line with stable expression of cDNA of human cytochrome P4501A1, the parental V79MZ cell line lacking any cytochrome P450 activity and human hepatocarcinoma Hep G2 cells were used as a model cells. Dose-dependent decrease in colony forming ability (CFA) was found in the V79MZh1A1 cell line after treatment of cells with DBC and MeDBC; however, no change in CFA was induced in parental V79MZ cells. These results were in a good correlation with DNA damaging effects of these two derivatives measured by the alkaline DNA unwinding (ADU) and the modified single cell gel electrophoresis (SCGE) techniques. Differences in DNA damage profiles induced by DBC and MeDBC were found in V79MZh1A1 and Hep G2 cells. These differences were probably the result of different reactive metabolite formation depending on chemical structure of the molecule and ways of biotransformation. This study showed that the cytochrome P4501A1 took part in activation of sarcomagenic DBC derivatives. Moreover, V79 cell lines with stable expression of different cytochromes P450 in combination with DNA repair endonucleases should be a useful tool for characterization of the role of individual cytochromes in metabolic activation pathways of DBC and MeDBC.

The possible protective effects of natural and synthetic antioxidants on DNA in hamster V79 cells exposed to hydrogen peroxide (H2O2) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) were investigated. The levels of DNA damage were measured using single cell gel electrophoresis. It was assessed that natural antioxidants lignin biopolymer and vitamin E (alpha-tocoferol) and a synthetic antioxidant stobadine (STB) exhibited a protective effect against the overall DNA damage induced after H2O2-treatment. In addition, it was observed that lignin NG. These findings suggest that the antioxidant nature of lignin biopolymer enables a rbiopolymer and stobadine (but not vitamin E) also protected against DNA damaged by MNeduction of the levels of frank breaks and of oxidized DNA bases in H2O2-treated cells and its adsorptive capacity enables binding of N-nitroso compounds and reduction of alkylation in MNNG-treated cells. The reduced level of DNA damage in STB-pre-treated samples could be due to its antioxidant properties in the case of H2O2-treatment and to a direct reduction of MNNG activity in the case of the alkylating agent.

We characterized MNNG- and H2O2-induced DNA strand breaks in hamster V79 cells and UV- induced DNA strand breaks in human VH10 cells by two modifications of the comet assay (SCGE) using (a) lesion-specific enzymes endonuclease III (endo III) and formamidopyrimidine-DNA-glycosylase (FPG) as well as enzyme exonuclease III (exo III) recognizing alkali-labile sites and (b) pH-variations of the comet assay. SCGE at pH=13 showed that DNA of H2O2-treated V79 cells incubated for 90 min contains both endo III- and FPG-sensitive sites but that the occurrence of endo III- and FPG-sensitive sites is uncertain in MNNG-treated V79 cells. SCGE processed in parallel at pH 13.0 and pH 12.1 showed that: (1) strand breaks detected in DNA of MNNG- and MMS- treated V79 cells originated from alkali-labile sites; (2) strand breaks detected immediately after the treatment of cells with H2O2 represent mainly breaks stable at both pH values; (3) the same level of DNA strand breaks accumulated in UV-irradiated human VH10 cells during incubation with DNA repair inhibitors can be revealed at both pH values. SCGE at pH 12.1 showed that the level of DNA strand breaks in MNNG- treated V79 cells was increased mainly after digestion with exoIII and that the level of DNA strand breaks in H2O2-treated and 90 min-incubated V79 cells was increased by both endo III and FPG to the same extent as at pH 13.0. Digestion by both endo III and FPG together led to an additive effect.

Mutagenicity of N-cyclohexylthiophthalimide (Duslin P) was tested first by the Ames test in the bacteria, Salmonella typhimurium. The negative results of the Ames test suggested that this compound does not induce mutations in the genome of S. typhimurium under the conditions used. To estimate the cytotoxicity of Duslin P to human cells, we measured cellular DNA and protein as well as cell proliferation, i.e., the mitotic index of treated and control cells. The genotoxic effects were assayed by two biochemical methods developed for detection of single-strand breaks of DNA in mammalian cells, i.e., by the alkaline single cell gel electrophoresis (comet assay) and by the DNA unwinding method, respectively. The DNA unwinding method showed that this compound did not induce DNA damage at concentrations < 7 micrograms/ml. Alkaline single cell gel electrophoresis revealed approximately double the level of DNA damage (in comparison to untreated control DNA) at a concentration of 2 micrograms/ml, which reduced proliferation to approximately 30%, and triple the level of DNA damage at higher concentrations (6 and 7 micrograms/ml), which inhibited completely both DNA synthesis and proteosynthesis. Cells with moderately damaged DNA were more common than cells with heavily damaged DNA. Parallel experiments with the strong mutagen and carcinogen MNNG showed that MNNG induced in cells a high level of DNA damage at concentrations which did not reduce the mitotic index or proteosynthesis, while DNA synthesis inhibited only partially. After treatment with MNNG, cells with heavily damaged DNA were more common than cells with moderately damaged DNA. Duslin P-treated VH10 cells were also tested cytogenetically, confirming that Duslin P induced neither chromosomal aberrations nor aneuploidy. We conclude that Duslin P has no mutagenic effect on bacteria, does not induce chromosomal aberrations and CREST positive or CREST negative micronuclei in human cells and induces only a small increase of DNA damage in human cells which is consistent with DNA fragmentation due to cell death.

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.

In our previous work concerning the biologic effects of theophylline, we found that cells incubated during 48 h at low concentrations of theophylline (0.3 mg/ml of medium) manifested short-term deviations in the rate of DNA replication; however, this short-term inhibition of DNA replication did not reduce either the growth rate or the colony-forming ability of cells. In the present study, we concentrated on cytotoxic and DNA-damaging effects of MNNG on V79 cells precultured with sublethal concentration of methylxanthine theophylline. Cytotoxicity was evaluated on the basis of growth rate of treated cells as well as by colony-forming ability (plating efficiency) test and by trypan blue exclusion test. The level of DNA lesions (strand breaks) induced by MNNG was measured by alkaline DNA unwinding and by the comet assay. In an effort to explain higher cytotoxic effects of MNNG on precultured cells, we studied rejoining of damaged parental DNA after 4 h incubation post-MNNG-treatment as well. We found differences as against the controls in theophylline-precultured cells after treatment with the mutagen and carcinogen MNNG. The higher cytotoxic effect of MNNG in precultured cells was accompanied by a higher level of ss breaks of DNA and by more unrepaired lesions which remained after 4 h in parental DNA. Our results demonstrate that theophylline belongs to the group of agents inhibiting repair of potentially lethal DNA lesions.

Human cells (VH10 or Hep G2) and hamster cells V79 were exposed to different concentrations of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and the level of DNA lesions was evaluated by the DNA unwinding technique, alkaline elution of DNA and the comet assay. All three methods were able to detect the effects of MNNG but with a clear difference in sensitivity. At low concentrations of MNNG the most sensitive method appeared to be the comet assay. After the short-term treatment the comet assay was able to detect the lesions induced by MNNG at approx. 0.1 microgram/ml, alkaline elution of DNA at 1 microgram/ml and DNA unwinding at 1-2 micrograms/ml. MNNG treated VH10 cells, human lymphocytes and V79 cells were also tested cytogenetically, confirming that MNNG induced chromosomal aberrations at concentrations > 1 microgram/ml in VH10 cells (short-term treatment): > 0.2 microgram/ml in V79 cells (long-term treatment) and > 8 micrograms/ml in human lymphocytes (long-term treatment). In some experiments we tried to increase the level of MNNG-induced DNA breaks with help of DNA repair inhibitors cytosine arabinoside (Ara C) and hydroxyurea (HU) which were applied either after or during MNNG treatment. Our results showed that the level of MNNG-induced lesions was increased by simultaneous treatment of cells with MNNG and Ara C and HU. 2 x 10(-5) M Ara C and 2 x 10(-3) MHU were as effective as 10-times higher concentrations of inhibitors. Ara C and HU increased the level of MNNG-induced DNA breaks mainly in combination with lower concentrations of MNNG (< 2 micrograms/ml). Rejoining of DNA breaks was observed in human cells VH10 and Hep G2 as well as in Chinese hamster cells V79 damaged by both lower and higher MNNG-concentrations. All methods showed that MNNG-induced DNA breaks had been gradually rejoined.

Nine substituted tetrazolo-[1,5-c]quinazolines have been tested for cytotoxic effect and structure activity relationships. The most active derivatives were non-substituted in the aromatic ring or substituted by chloro- or bromo- group, and in the pyrimidine ring of quinazoline skeleton by phenyl or morpholine group, respectively. 9-Chloro-5-morpholino-tetrazolo-[1,5-c] quinazoline (CMTQ) at the three highest tested concentrations (344, 176, and 86 mu mol.l(-1)) induced total inhibition of cell proliferation after 24 hours of cultivation. The concentration of 34.4 mu mol.l(-1) caused a delayed cytotoxic effect. While during the first 48 h one part of the cell population proliferated, during the next 24 h 49.7% of the population degenerated. Lower concentrations induced toxicity that was concentration- and time-dependent. Six concentrations of CMTQ induced one-phase unbalanced growth characterized by a 1.05-1.40-fold increase in total cell proteins. In the dying cells the integrity of the cytoplasmic membrane showed damages along with an increase in cell volume before death.

N-cyclohexylthiophthalimide, commercial name Duslin P, at concentrations 0.5-2 micrograms/ml inhibited proliferation of V79 cells and at concentrations > 2 micrograms/ml acted cytotoxically. Inhibition of cumulative DNA synthesis correlated well with the deleterious effects of Duslin P on growth activity and plating efficiency. DNA synthesis was not renewed even 6 h after the treatment of cells. Alkaline elution of DNA of V79 cells treated with Duslin P did not confirm our expectation that this chemical compound has a DNA-damaging effect. Duslin P strongly inhibited protein synthesis at concentrations > 2 micrograms/ml. We suggest that the cytotoxic effects of Duslin P are not accompanied by any genotoxic effects.

Three techniques: single cell gel electrophoresis (SCGE), alkaline elution of DNA (AE), and alkaline DNA unwinding (ADU) were chosen to compare the sensitivity among these methods in detection of DNA damage and repair in human diploid VH10 cell line after short-term exposure to hydrogen peroxide. Using SCGE technique a dose-dependent increase in DNA migration was found in cells exposed to hydrogen peroxide in concentration range from 10 micromol/l to 100 micromol/l. Alkaline DNA unwinding method detected increased level of single strand breaks (ssb) in concentration range from 25 micromol/l to 100 micromol/l of H2O2, and alkaline elution of DNA estimated increased DNA elution rate from concentration 50 micromol/l of H2O2. In a time course study to evaluate the kinetics of DNA repair, both SCGE and ADU techniques showed that the repair of DNA strand breaks is very rapid; the level of ssb in treated cells has returned to near the background level within two hours. After this time damage remaining in the DNA was in the form of oxidised bases as revealed the incubation of treated cells with specific DNA repair endonuclease, formamidopyrimidine-DNA glycosylase.

9-Bromo-5-morpholino-tetrazolo[1,5-c]quinazoline (BMTQ) at the two highest tested concentrations (74.6; 29.8 mumol@l) induced retarded cytotoxic effect. After 24 hours of culturing 23.1-98.8% of the cell population proliferated but after 48 and 72 hours 6.4-80.4% of the cell population degenerated. Other concentrations induced toxicity that was concentration-and time-dependent. The cytolytic concentrations of BMTQ induced integrity damage of cytoplasmatic membrane. The inhibition of cell cycle and the elevated content of proteins in the cell exposed to the cytotoxic concentrations of BMTQ suggest that the cells synthesize protein without entering into mitosis and that dying cells are in the S-phase before death. BMTQ induced 1.75-3.01 times increase of the level of ssDNA in comparison with the control.