Gentiana asclepiadea and Armoracia rusticana can modulate the adaptive response induced by zeocin in human lymphocytes.

Zeocin is a member of bleomycin/phleomycin family of antibiotics isolated from Streptomyces verticullus. This unique radiomimetic antibiotic is known to bind to DNA and induce oxidative stress in different organisms producing predominantly single- and double- strand breaks, as well as a DNA base loss resulting in apurinic/apyrimidinic (AP) sites. The aim of this study was to induce an adaptive response (AR) by zeocin in freshly isolated human lymphocytes from blood and to observe whether plant extracts could modulate this response. The AR was evaluated by the comet assay. The optimal conditions for the AR induction and modulation were determined as: 2 h-intertreatment time (in PBS, at 4°C) given after a priming dose (50 µg/ml) of zeocin treatment. Genotoxic impact of zeocin to lymphocytes was modulated by plant extracts isolated from Gentiana asclepiadea (methanolic and aqueous haulm extracts, 0.25 mg/ml) and Armoracia rusticana (methanolic root extract, 0.025 mg/ml). These extracts enhanced the AR and also decreased DNA damage caused by zeocin (after 0, 1 and 4 h-recovery time after the test dose of zeocin application) to more than 50%. These results support important position of plants containing many biologically active compounds in the field of pharmacology and medicine.

Biological activity of plant extract isolated from Papaver rhoeas on human lymfoblastoid cell line.

Varied medicinal plants are known as a source of natural phytochemicals with antioxidant activities that can protect organisms from oxidative stress and from various chronic diseases. Papaver rhoeas has a long history of medicinal usage, especially for ailments in adults and children. The possible cytotoxicity, genotoxicity and potential antioxidant effect of plant extract isolated from flowers of Papaver rhoeas was investigated in human lymfoblastoid cell line (TK6). Antioxidant activity of this extract was determined using the DPPH assay. The plant extract exhibited dose dependent free radical scavenging ability. The growth activity assay was used for determination of cytotoxicity. To assess potential genotoxicity the comet assay was used. The lower extract concentrations (0.25 and 0.5 mg/ml) neither exerted cytotoxic, nor genotoxic effects in TK6 cells but they stimulated cell proliferation. The concentration 25 mg/ml scavenged almost 85% of DPPH free radical. On the other hand, this concentration had strong cytotoxic and genotoxic effect on TK6 cells. The balance between beneficial and harmful effects should be always considered when choosing the effective dose.

Yeast cell wall polysaccharides as antioxidants and antimutagens: can they fight cancer?

Polysaccharides represent the major part of the yeast cell wall dry weight and build the skeletal carcass defining cell wall stability and cell morphology (beta-D-glucans) or constitute amorphous matrix and cell surface fibrous material (mannans and mannoproteins). It is known that yeast cell wall beta-D-glucans reveal immunomodulating properties, which allows for their application in anti-infective and antitumor therapy. Recent data also suggest that polysaccharides reveal antioxidant activity that can result in their protective function as antioxidants, antimutagens, and antigenotoxic agents. The paper provides a review of our continuing research involving water-soluble derivatives of beta-D-glucan isolated from the baker's yeast Saccharomyces serevisiae and of a glucomannan isolated from the industrial yeast Candida utilis. The results are confronted with the available literature data. The derivatives of beta-D-glucan demonstrated potent inhibitory effect on lipid peroxidation comparable to that of the known antioxidants and exerted DNA protection from oxidative damage. The free radical scavenging activity was confirmed by spin-trap electron paramagnetic resonance. Antimutagenic and antigenotoxic activity of the yeast polysaccharides was demonstrated using yeast, bacterial, and algal models. The derivatives of beta-D-glucan exerted potent enhancement of tumor necrosis factor alpha (TNF-alpha) released from murine macrophages and revealed synergistic effect with cyclophosphamide in the treatment of Lewis lung carcinoma and two types of lymphosarcoma in murine models. The results indicate significant protective antioxidant, antimutagenic, and antigenotoxic activities of the yeast polysaccharides and imply their potential application in anticancer prevention/therapy.

Flavonoids potentiate the efficacy of cytarabine through modulation of drug-induced apoptosis.

Recent studies have provided strong evidence for potential beneficial effects of flavonoids in chemoprevention or in combination with chemotherapeutics in tumor cells treatment. The aim of this work was to compare the antioxidant properties of four flavonoids with emphasis on association of these antioxidant properties with their effects on the therapeutic efficacy of cytarabine (AraC) using L1210 leukemia cells. The results of antiproliferative studies showed that antiproliferative potential of flavonoids tested decreased in the order: isorhamnetin > kaempferol > myricetin > rutin, while their antioxidant properties decreased in the order: rutin > myricetin > kaempferol > isorhamnetin. Combinational treatment of isorhamnetin, kaempferol and myricetin with AraC led to synergism in their antiproliferative activities (CIs < 1). Rutin exhibited antagonism with AraC (CIs > 1). Apoptotic DNA fragmentation and flow cytometry analyses revealed that synergism in antiproliferative activities of compounds tested might be due to potentiation of AraC-induced apoptosis. In conclusion, our results clearly indicate that isorhamnetin, kaempferol and myricetin despite their antioxidant properties might be used to increase the sensitivity of leukemia cells to AraC treatment.

Natural microbial polysaccharide sulphoethyl glucan as antigenotoxic and cancer preventing agent.

Naturally occurring polysaccharides isolated from the yeasts are the substances with versatile intriguing biomodulatory activities. One of the novel derivatives prepared from the (1 --> 3)-beta-D-glucan isolated from the cell walls of baker's yeast Saccharomyces cerevisiae is sulfoethyl glucan (SEG). Its DNA-protective, antimutagenic, anticlastogenic and cytotoxic/cytostatic enhancing effect was evaluated using five eukaryotic systems. SEG showed bioprotective effect in recombination- repair-deficient strain of alga Chlamydomonas reinhardtii against methyl methanesulfonate-induced genotoxicity, antimutagenic effect against ofloxacin-induced genetic changes in yeast Saccharomyces cerevisiae assay and anticlastogenic activity in plants Vicia sativa and Vicia faba assays against maleic hydrazide-induced clastogenicity. In the combined application with cytostatic drug vumon, SEG exerted enhancement of the drug's cytotoxic/cytostatic effect in the cell revitalization assay using mouse leukemia cells. The study sheds light on the possible mechanisms of actions and utilization of this microbial polysaccharide derivative in the cancer prevention and therapy.

DNA-protective activities of hyperforin and aristoforin.

The aim of this study was to explain the molecular mechanisms of action of hyperforin, a phluoroglucinol derivative found in Hypericum perforatum L. and its more stable derivative aristoforin. DNA-topology assay revealed partial DNA-protective activities of hyperforin and aristoforin against Fe(2+)-induced DNA breaks. In order to assess molecular mechanisms underlying DNA-protective activity, the potential antioxidant activity of hyperforin and aristoforin was investigated using DPPH and OH scavenging assays, reducing power assay and Fe(2+)-chelating assay. We also studied interaction of hyperforin and aristoforin with DNA using established protocols for fluorescence titration. The ability of the studied compounds to relax topoisomerase I with electrophoretic techniques was investigated. The reduction in the fluorescence of hyperforin indicated an interaction between hyperforin and DNA with a binding constant of 0.2×10(8)M(-1). We suggest that a mechanism of hyperforin/aristoforin DNA-protective abilities is based on free radicals (mainly OH) scavenging activity.

Unicellular green alga Chlamydomonas reinhardtii as an activation system for 2-aminofluorene.

Despite the promutagenic/procarcinogenic potential, polycyclic aromatic amines are widely spread in the environment. Biotransformation of the polycyclic aromatic amine 2-aminofluorene (2-AF) was proved in mammals and higher plants. The algal cell/microbe coincubation assay is an additional system that complemented those proved in mammals and higher plants, useful for detection and conversion of environmental promutagens, mainly in aquatic environments. The unicellular green algae may be a good activating system in coincubation assays in that the algal cells exist as a natural system. To increase the effectiveness of this metabolizing system, different modifications of the standard experimental procedure were conducted. Algae can accumulate and metabolize promutagenic pollutants, some of which may differ from those activated by the animal microsome metabolizing system (S9 mix) and by the plant cell/microbe coincubation assay. 2-AF was activated in the algal cell/ microbe coincubation assay in which wild-type Chlamydomonas reinhardtii cells were used as an activating system and the bacteria Salmonella typhimurium TA98, YG1024, and yeast Saccharomyces cerevisiae D7 as the genetic indicator organisms. It was converted to the mutagenic product(s) for the strain YG1024, but the strain TA98 did not exhibit any increase in the mutant yield of His+ revertants. Consequently, metabolites from 2-AF are substrates for O-acetyltransferase. A direct comparison of algal 2-AF activation with mammalian activation system (S9 mix) proved the higher activity of mammalian microsome system (S9 mix). After the combination of both activation systems, a slight synergetic effect was found. Although the genetic endpoints induced by 2-AF using both modifications of the algal cell/S. cerevisiae coincubation assay and those obtained in intact yeast cells were similar at the equitoxic concentrations, 2-AF activation by the algal supernatant slightly increased the genetic endpoints studied.

DNA-protective activity of new ribonucleotide reductase inhibitors.

The DNA-protective activity of hydroxyurea (HU) and novel ribonucleotide reductase (RR) inhibitors amidox (AX), didox (DX) and trimidox (TX) was examined using hydrogen peroxide as the DNA-damaging agent. The exposure of superspiralized plasmid DNA molecules (pBR 322) to H2O2 under precisely defined in vitro conditions initiates a change in DNA topology (DNA from I relaxes to DNA form II). This electrophoretically monitored change in the plasmid DNA topology is related to the induction of ss-DNA breaks and corresponds with DNA exposition to free radicals. The inhibition of DNA relaxation (the prevention of DNA damage induced by hydrogen peroxide) depended on the free radical scavenging capacity of the drugs investigated. HU exerted DNA protective activity at a concentration of 4 mM, AX at concentration of 1 microM, TX at a concentration of 5 microM and DX at a concentration of 25 microM (the free radical scavenging activity increases from HU to AX in following manner: HU << DX < TX < AX). It can be concluded that the new synthetic RR-inhibitor AX which is being investigated at the preclinical level as a potential anti-cancer drug possess the highest capacity for scavenging of free radicals.

New DNA repair-deficient mutants of Chlamydomonas reinhardtii.

Two new UV-sensitive mutants of Chlamydomonas reinhardtii, uvs12 and uvs13, were characterized. Genetic analysis proved that they were non-allelic. They complemented the other repair-deficient mutants uvs8, uvs9, uvs10 and uvs11. While uvs12 may have an impaired excision-repair pathway, uvs13 is, by its UV sensitivity under non-photoreactivating conditions, very similar to uvsE1 and uvs10, but differs in the effect of caffeine on its survival. After UV, survival of some repair-deficient mutants was, under photoreactivating conditions, much lower than that of phr1, while survival of other repair-deficient mutants did not differ from that of a wild-type strain. A lower UV survival of some dark-repair-defective mutants of Chlamydomonas reinhardtii, under photoreactivating conditions, can perhaps be used as an additional criterion for mutants defective in the excision-repair pathway.

Interactions between photolyase and dark repair processes in Chlamydomonas reinhardtii.

The participation of DNA photolyase in dark repair processes has been reported in some heterotrophic organisms. To assess the role of photolyase in dark repair in photoautotrophs, double mutants of Chlamydomonas reinhardtii deficient in dark repair and photoreactivation were constructed and assayed for UV sensitivity in different posttreatment light conditions (with or without subsequent photoreactivation). We found that a functional PHR1 gene enhanced dark survival in the excision deficient (uvs9, uvs12) and in the recombination deficient (uvs10) genetic backgrounds but failed to do so in the strain deficient in a repair pathway other than excision and recombination (uvs13). Therefore we can conclude that photolyase may stimulate dark repair processes in C. reinhardtii also via pathway(s) other than nucleotide excision repair. The fact that some of the double mutants deficient in dark repair and photoreactivation survived better in the light than in the dark supports the idea that additional photorepair might be active and may enhance survival in a specific genetic background.

A Chlamydomonas reinhardtii UV-sensitive mutant uvs15 is impaired in a gene involved in several repair pathways.

In this report, three DNA repair-deficient mutants of Chlamydomonas reinhardtii (uvs13, uvs14, uvs15) were characterized by using genetic, mutational and biochemical analyses. The mutant strain uvs15 belongs to the most sensitive repair-deficient mutants following exposure to all agents used. It is deficient in the nuclear excision-repair pathway, whereas uvs13 and uvs14 are not blocked in removal of pyrimidine dimers. Mutation study also revealed differences among strains. The mutant uvs15 does not mutate after UV and X-ray irradiation, and there is very low mutation rate after MNNG. These findings might indicate the involvement of UVS15 gene product in regulation of several repair pathways. Contrary to this, uvs14 showed higher mutation frequency, both spontaneous and induced after UV and MNNG treatments. Tetrad dissection proved that the uvs13 and uvs14 genes are located on the right arm of the linkage group I in the vicinity of the previously mapped uvs10 gene. Both mutants belong to the same repair pathway, which is different from that of uvs10 and uvs15.

Mutagenic activity of phosmet, the active component of the organophosphorus insecticide Decemtione EK 20 in Salmonella and Saccharomyces assays.

Phosmet, the active component of the organophosphorus insecticide Decemtione EK 20, was shown to be mutagenic in the standard Ames Salmonella/mammalian microsome assay in the absence and presence of metabolic activation. It appears to be a direct mutagen inducing base substitution mutations (TA100) as well as a weak frameshift mutagen (TA97). This compound was genotoxic in the Saccharomyces cerevisiae D7 strain. It significantly increased reverse mutation, mitotic crossing-over and slightly, but not significantly, increased gene conversion at the highest concentration used.

Metabolic activation of meta-phenylenediamine by the alga Chlamydomonas reinhardtii.

Promutagens/procarcinogens arylamines are widely distributed in the environment. While it is accepted that these compounds can be metabolized to ultimate mutagens in mammals and higher plants, in aquatic plants they have not yet been explored. Intact wild-type and repair-deficient strains of Chlamydomonas reinhardtii and Saccharomyces cerevisiae D7 strain were assayed for their ability to activate meta-phenylenediamine (m-PDA) to an ultimate mutagen. The different responses of the algal wild-type strain and repair-deficient strains to the toxic and mutagenic effects of m-PDA were observed. Recombination repair played an important role in repair of damage induced to C. reinhardtii DNA by this arylamine. The examined isomer of phenylenediamine induced mutations in both algal and yeast cells. m-PDA was activated in the algal cell/microbe coincubation assay in which algal cells were used as an activating system and bacteria Salmonella typhimurium and yeast Saccharomyces cerevisiae as the genetic indicator organisms. This new assay is, in addition to the animal microsome metabolizing system and the plant cell/microbe coincubation assay, suitable for the detection of environmental promutagens and their conversion to mutagens mainly in aquatic environments.

General characteristics, molecular and genetic analysis of two new UV-sensitive mutants of Chlamydomonas reinhardtii.

Two new UV-sensitive mutants of Chlamydomonas, UVS10 and UVS11, were isolated. Both behave as single nuclear mutations. UVS10 was mapped to linkage group I. UVS11 is a separate, unlinked mutation but has not yet been located to a specific linkage group. Both mutants are proficient in the excision of pyrimidine dimers from nuclear DNA. The survival of UV-irradiated UVS11 is increased when plated in the presence of 1.5 mM caffeine, similar to wild-type. Caffeine has no effect on the survival of UV-irradiated UVS10. UV-irradiated UVS11 frequently divides at least once before dying, in contrast to UVS10 or wild-type. UVS11 also exhibits a much increased frequency of mutation to streptomycin resistance after UV irradiation.

Effects of supercypermethrin, a synthetic developmental pyrethroid, on four biological test systems.

The genotoxic potential of the insecticide supercypermethrin, a second-generation pyrethroid, was studied on four different test systems. It was non-mutagenic to Salmonella typhimurium strains TA1535, TA100, TA1538, TA98 and TA97 in the presence and absence of S9 mixture. It induced gene conversion at the tryptophan locus and induced point mutations at the isoleucine locus in Saccharomyces cerevisiae cells. A slight increase in the frequency of aberrant anaphases and telophases in root tips of Hordeum vulgare and Vicia faba was observed, but no genotoxic effects were detected in Drosophila melanogaster.

The mutagenic effect of the new insecticide and acaricide pyridathion.

The potential mutagenic effect of the new organophosphate insecticide and acaricide, pyridathion, was tested on Escherichia coli, strains WP2, WP2 uvrA, and on Salmonella typhimurium, strains TA100 and TA98, both with and without metabolic activation. The compound was tested at 1, 10 and 100 micrograms/ml. The analysis of chromosomal aberrations in mouse bone marrow was performed after a single peroral application of pyridathion at doses of 0.6, 1.11, 6.0, 12.0 and 24.0 mg . kg-1 b.w., i.p. application at 6.0 mg . kg-1 b.w., and repeated application (5 times) p.o. at 1.0, 2.5 and 5.0 mg . kg-1 b.w. Analysis of rat bone marrow was performed after a 3-month peroral application of pyridathion at 0.07, 0.175 and 0.35 mg . kg-1 b.w. An analysis of chromosomal aberrations in human peripheral lymphocytes in vitro was performed 24 h after the application of pyridathion into the culture in concentrations of 9.2 X 10(-3), 9.2 X 10(-4) and 9.2 X 10(-5) moles. The insecticide did not exert any mutagenic effect on the bacteria. There was no significant increase in the frequency of chromosomal abnormalities in bone marrow of mice of rats, or in human peripheral lymphocytes in vitro, compared with controls.

Genotoxicity evaluation of Rastim 30 DKV, the plant growth regulator, on five test systems.

The possible mutagenic activity of Rastim 30 DKV, a new plant growth regulator, was studied on five model test systems. It did not increase the frequency of His+ revertants in Salmonella typhimurium strains TA97, TA98, TA100, TA1535, TA1538 in the absence and the presence of S9 mix. It slightly increased rates of genetic changes in Saccharomyces cerevisiae, mainly convertants at the tryptophan locus. No clastogenic effect was observed after Vicia faba root-tip meristem treatment, and at the lowest concentration used its mitotic activity was significantly increased. No chlorophyll mutants after the treatment of two cultivars of barley were observed. Though no sex-linked recessive lethals were scored in Drosophila melanogaster males, the rates of aneuploids induced in their germ cells were significantly increased.

Bioactivation of promutagens by the unicellular green alga Chlamydomonas reinhardtii.

The aromatic amine 2-aminofluorene (2-AF) was activated by the intact Chlamydomonas reinhardtii cells to a mutagen that exhibited toxic and mutagenic effects comparable to those of the direct-acting mutagen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). There were different responses of the wildtype and repair-deficient strains to the toxic and mutagenic effect of 2-AF. The recombination repair plays a major role in repair of damages induced in the C. reinhardtii DNA by the aromatic amine promutagen 2-AF and the direct-acting mutagen MNNG. The 2-AF activation has also been analyzed by algal cells/microbe coincubation assay. This new assay is used in addition to animal microsome-metabolizing system (S9 fraction) and plant cell/microbe coincubation assay. This additional system is suitable for detection of environmental promutagens and their conversion to mutagens, mainly in aquatic environments.

Comparison of the phenylenediamine isomers bioactivation by the green alga Chlamydomonas reinhardtii.

The correlation between the chemical structure of arylamines meta-, orto-, para-phenylenediamine (m-PDA, o-PDA, p-PDA), and their mutagenic activity is known. It is accepted that these promutagenic compounds are metabolized to ultimate mutagens in mammals and higher plants. In our previous work, we used the alga Chlamydomonas reinhardtii as the activating organism and the bacteria Salmonella typhimurium and yeast Saccharomyces cerevisiae as the genetic indicators for m-PDA activation. In the present work, we used the same activation system for o-PDA and p-PDA activation. Different responses of the yeast and algal wild-type strain and of the repair-deficient strains to the toxic and mutagenic effects of o-PDA and p -PDA were observed. p-PDA had the most toxic effect on both intact yeast and algal cells and in the algal cell/microbe coincubation assays. Concerning repair-deficient algal strains, the recombination-deficient strain was the most sensitive to both compounds tested, indicating that the recombination process played an important role in the DNA repair of arylamines. The rank order of the PDA isomers mutagenicity (including m-PDA) was o-PDA > m-PDA > p-PDA for revertants in intact yeast and forward mutants in algae; m-PDA > o-PDA > p-PDA in the algal cell/S. typhimurium long-term coincubation assay, the algal cell/S. cerevisiae coincubation assay, and the intact S. cerevisiae assay for gene convertants as well.

Comparative DNA protectivity and antimutagenicity studies using DNA-topology and Ames assays.

Two experimental techniques, the DNA-topology assay and the Ames assay, were proved to be suitable for monitoring compounds with a genotoxic potential and/or with an antimutagenic effect. Both procedures were used in assaying the acid-mine water (AMW) containing toxic metals and sulfoethyl chitin-glucan (SE-Ch-G), a derivative of chitin-glucan, in which bioprotective activities were detected earlier. It was shown that after toxic metal concentrations were decreased due to AMW dilution to the limits that correspond with those set by the Slovak Technical Norm (STN) for drinking water, AMW was not genotoxic in the Ames assay. As it is possible to detect any single-strand DNA (ssDNA) break in the DNA-topology assay, the SE-Ch-G protective effect against the ssDNA breaks induced by Fe(2+) in the DNA-topology assay was recorded. SE-Ch-G exhibited the antimutagenic potential after its application simultaneously with diagnostic mutagens in the Ames assay. These results demonstrate the complementarity of both experimental systems.