Antioxidative and antimutagenic activity of yeast cell wall mannans in vitro

Antioxidative and antimutagenic effect of yeast cell wall mannans, in particular, extracellular glucomannan (EC-GM) and glucomannan (GM-C.u.) both from Candida utilis, mannan from Saccharomyces cerevisiae (M-S.c.) and mannan from Candida albicans (M-C.a.) was evaluated. Luminol-dependent photochemical method using trolox as a standard showed that EC-GM, GM-C.u., M-S.c. and M-C.a. have relatively good antioxidative properties. EC-GM exhibited the highest antioxidative activity, followed by GM-C.u. and M-S.c. M-C.a. showed the least antioxidative activity. These mannans were experimentally confirmed to exhibit different, statistically significant antimutagenic activity in reducing damage of chloroplast DNA of the flagellate Euglena gracilis induced by ofloxacin and acridine orange (AO). We suggest that the antimutagenic effect of EC-GM, GM-C.u., M-S.c. and M-C.a. against ofloxacin is based on their ability to scavenge reactive oxygen radicals. With AO, the reduction of the chloroplast DNA lession could be a result of the absorptive capacity of the mannans. The important characteristics of mannans isolated from the yeast cell walls, such as good water solubility, relatively small molecular weight (15-30kDa), and antimutagenic effect exerted through different mode of action, appear to be a promising features for their prospective use as a natural protective (antimutagenic) agents.     

Triterpenoid cynarasaponins from Cynara cardunculus L. reduce chemically induced mutagenesis in vitro

Saponins, steroid or triterpene glycosides, are known to have a broad spectrum of biological and pharmacological activities. Three different triterpenoid saponins, marked here as 1s, 2s and 3s, from involucral bracts of Cynara cardunculus L. were isolated and their antimutagenic effect was assessed. Using spectrophotometric method it was shown that all three substances, 1s, 2s and 3s, possess very good absorptive capability. The antimutagenic effect of these substances was estimated against acridine orange (AO)- and ofloxacin-induced damage of chloroplast DNA in Euglena gracilis assay. These cynarasaponins were experimentally confirmed to exhibit different, statistically significant activity in reducing damage of chloroplast DNA of the flagellate E. gracilis induced by AO and ofloxacin (p_t<0.05–0.01). Our findings suggest that the antimutagenic effect of 1s, 2s and 3s against AO-induced chloroplast DNA impairment could be a result of their absorptive capacity. As far as ofloxacin is concerned, a possible mechanism of the reduction of the chloroplast DNA lesion was not elucidated so far. To our knowledge, these data demonstrate for the first time the antimutagnic activity of saponins isolated from involucral bracts of C. cardunculus exerted through different mode of action.     

The antigenotoxic potential of dietary flavonoids

Human exposure to genotoxic agents has dramatically increased. Both endogenous (reactive species generated during physiological and pathological processes) and exogenous (UV light, ionizing radiation, alkylating agents, antimetabolites and topoisomerase inhibitors, air, water and food pollutants) factors can impair genomic stability. The cumulative DNA damage causes mutations involved in the development of cancer and other disorders (neuromuscular and neurodegenerative diseases, immune deficiencies, infertility, cardiovascular diseases, metabolic syndrome and aging). Dietary flavonoids have protective effects against DNA damage induced by different genotoxic agents such as mycotoxins, food processing-derived contaminants (polycyclic aromatic hydrocarbons, N-nitrosamines), cytostatic agents, other medications (estrogenic and androgenic hormones), nicotine, metal ions (Cd²⁺, Cr⁶⁺), radiopharmaceuticals and ionizing radiation. Dietary flavonoids exert their genoprotection by reducing oxidative stress and modulation of enzymes responsible for bioactivation of genotoxic agents and detoxification of their reactive metabolites. Data on structure–activity relationship is sometimes contradictory. Free hydroxyl groups on the B ring (catechol moiety) and C-3 position of the C ring are important structural features for the antigenotoxic activity. As dietary flavonoids are extensively metabolized, more in vivo studies are needed for a better characterization of their antigenotoxic potential.     

Inhibitory effects of chlorophyllin, hemin and tetrakis(4-benzoic acid)porphyrin on oxidative DNA damage and mouse skin inflammation induced by 12-O-tetradecanoylphorbol-13-acetate as a possible anti-tumor promoting mechanism

Reactive oxygen species (ROS) from both endogenous and exogenous sources can cause oxidative DNA damage and dysregulated cell signaling, which are involved in the multistage process of carcinogenesis such as tumor initiation, promotion and progression. A number of structurally different anticarcinogenic agents inhibit inflammation and tumor promotion as they reduce ROS production and oxidative DNA damage. Evidence suggests that porphyrins can interfere with the actions of various carcinogens and mutagens by forming face-to-face complexes and their antimutagenic or antigenotoxic effects may also be attributed to their antioxidant activities. However, little is known regarding the anti-tumor promoting potential and mechanism of the porphyrin compounds. Based on our previous results on the inhibitory effects of chlorophyllin (CHL), hemin and tetrakis(4-benzoic acid)porphyrin (TBAP) against two-stage mouse skin carcinogenesis, we have investigated their anti-tumor promoting mechanisms. In the present work, CHL, hemin and TBAP reduced superoxide anion generation by 12-O-tetradecanoylphorbol-13-acetate (TPA) in differentiated HL-60 cells and the production of hydroxyl radicals by Fenton reaction. Porphyrins exert a dose-related inhibition of his(+) reversion in Salmonella typhimurium TA102 induced by tert-butylhydroperoxide (t-BOOH). DNA strand breaks by ROS derived from H(2)O(2)/Cu(II) and the formation of 8-hydroxydeoxyguanosine (8-OH-dG) in calf thymus DNA treated with H(2)O(2)/UV also were inhibited markedly by porphyrins in a concentration-dependent manner. Furthermore, CHL, hemin and TBAP decreased myeloperoxidase (MPO) activity and H(2)O(2) formation as well as epidermal ornithine decarboxylase (ODC) activity in mouse skin treated with TPA. These results demonstrate that the antioxidative properties of porphyrins are important for inhibiting TPA-induced tumor promotion.     

Yeast mannan structure necessary for co-aggregation with Lactobacillus plantarum ML11-11

Lactic acid bacteria (LAB) Lactobacillus plantarum ML11-11, an isolate from Fukuyama pot vinegar, and yeast Saccharomyces cerevisiae form significant mixed-species biofilm with direct cell-cell contact. Co-aggregation of L. plantarum ML11-11 and S. cerevisiae cells, mediated by the interaction between surface protein(s) on L. plantarum ML11-11 cells and surface mannan of S. cerevisiae cells, contributes significantly to mixed-species biofilm formation. In this study, co-aggregation activities of yeast mutants that were deleted of genes related to mannan biosynthesis were investigated to clarify the mannan structures essential for interaction with L. plantarum ML11-11. Among the 12 deletion mutants which had various incomplete mannan structures, only the mnn2 mutant lost the co-aggregation activity. In the mnn2 mutant, the gene coding the activity of attaching first branching mannose residue to mannan main chain is deleted and therefore the mnn2 mutant has unbranched mannan. From this result, it is clarified that the specific structure, consisted of mannan main chain to which are attached side chains containing one or more mannose residues, is critical for co-aggregation with L. plantarum ML11-11.     

Protective effects of acerola juice on genotoxicity induced by iron in vivo

Metal ions such as iron can induce DNA damage by inducing reactive oxygen species (ROS) and oxidative stress. Vitamin C is one of the most widely consumed antioxidants worldwide, present in many fruits and vegetables, especially inMalpighia glabra L., popularly known as acerola, native to Brazil. Acerola is considered a functional fruit due to its high antioxidant properties and phenolic contents, and therefore is consumed to prevent diseases or as adjuvant in treatment strategies. Here, the influence of ripe and unripe acerola juices on iron genotoxicity was analyzed in vivo using the comet assay and micronucleus test. The comet assay results showed that acerola juice exerted no genotoxic or antigenotoxic activity. Neither ripe nor unripe acerola juices were mutagenic to animals treated with juices, in micronucleus test. However, when compared to iron group, the pre-treatment with acerola juices exerted antimutagenic activity, decreasing significantly micronucleus mean values in bone marrow. Stage of ripeness did not influence the interaction of acerola compounds with DNA, and both ripe and unripe acerola juices exerted protective effect over DNA damage generated by iron.     

In Vitro Genotoxic and Antigenotoxic Effects of Ten Novel Synthesized 4-Thiazolidinone Derivatives

Heterocyclic compounds are found in a variety of drug molecules, and bioactive natural products. 4-Thiazolidinones (4-TZDs), which represent an important class of heterocyclic compounds, are of great interest today with their diverse bioactivities. In this study, ten novel 4-TZD derivatives ( C1 – C10 ) were synthesized, characterized by spectroscopic techniques, and their genotoxic, and antigenotoxic properties were investigated in vitro using the Ames Salmonella/microsome mutagenicity assay in the concentration range of 0.2–1.0 mM/plate. The results revealed that none of the compounds were mutagenic on the three different Salmonella typhimurium strains up to the highest concentration tested. Furthermore, in our study, C1 , C4 , C6 , and C9 showed significant, ranging from moderate to strong, antigenotoxic effects against mutagen-induced DNA damage at relatively higher doses. Among these, C4 had the best potential to inhibit the number of revertant colonies induced by 9-aminoacridine (9-AA), with a maximum inhibition rate of 47.9 % for 1.0 mM/plate. As a result, preliminary knowledge about the safety of the use of ten novel synthesized 4-TZD compounds likely to exhibit many bioactivities was obtained in this study. In addition, the significant in vitro antimutagenic activity of some derivatives increases the importance of studies for the development of new pharmacological agents for cancer prevention.     

Inhibitory effects of chlorophyllin, hemin and tetrakis(4-benzoic acid)porphyrin on oxidative DNA damage and mouse skin inflammation induced by 12-O-tetradecanoylphorbol-13-acetate as a possible anti-tumor promoting mechanism

Reactive oxygen species (ROS) from both endogenous and exogenous sources can cause oxidative DNA damage and dysregulated cell signaling, which are involved in the multistage process of carcinogenesis such as tumor initiation, promotion and progression. A number of structurally different anticarcinogenic agents inhibit inflammation and tumor promotion as they reduce ROS production and oxidative DNA damage. Evidence suggests that porphyrins can interfere with the actions of various carcinogens and mutagens by forming face-to-face complexes and their antimutagenic or antigenotoxic effects may also be attributed to their antioxidant activities. However, little is known regarding the anti-tumor promoting potential and mechanism of the porphyrin compounds. Based on our previous results on the inhibitory effects of chlorophyllin (CHL), hemin and tetrakis(4-benzoic acid)porphyrin (TBAP) against two-stage mouse skin carcinogenesis, we have investigated their anti-tumor promoting mechanisms. In the present work, CHL, hemin and TBAP reduced superoxide anion generation by 12-O-tetradecanoylphorbol-13-acetate (TPA) in differentiated HL-60 cells and the production of hydroxyl radicals by Fenton reaction. Porphyrins exert a dose-related inhibition of his⁺ reversion in Salmonella typhimurium TA102 induced by tert-butylhydroperoxide (t-BOOH). DNA strand breaks by ROS derived from H₂O₂/Cu(II) and the formation of 8-hydroxydeoxyguanosine (8-OH-dG) in calf thymus DNA treated with H₂O₂/UV also were inhibited markedly by porphyrins in a concentration-dependent manner. Furthermore, CHL, hemin and TBAP decreased myeloperoxidase (MPO) activity and H₂O₂ formation as well as epidermal ornithine decarboxylase (ODC) activity in mouse skin treated with TPA. These results demonstrate that the antioxidative properties of porphyrins are important for inhibiting TPA-induced tumor promotion.     

Secretion of cell-wall glycoproteins by yeast protoplasts. Effect of 2-deoxy-d-glucose and cycloheximide

The effect of 2-deoxy-d-glucose and cycloheximide on the synthesis and secretion of the cell-wall constituents protein and mannan in yeast protoplasts was examined in detail. Although the 2-deoxy-d-glucose hardly influenced protein synthesis, a significant parallel inhibition of carbohydrate and protein secretion into the medium was observed. The mechanism of this inhibition is considered as an interference of metabolites of 2-deoxy-d-glucose with the synthesis of yeast mannan. Cycloheximide, which is an effective inhibitor of protein synthesis in yeast (Kerridge, 1958), inhibited the secretion of non-diffusible carbohydrate in yeast protoplasts, but on the other hand had no effect on the activity of particulate yeast mannan synthetase. Our results clearly show that blocking the synthesis of either part of the mannan-protein complex prevents the extracellular appearance of the other component. The nature of this phenomenon is discussed.     

Damage-induced recombination in the yeast Saccharomyces cerevisiae

Prokaryotic and eukaryotic cells have developed a network of DNA repair systems that restore genomic integrity following DNA damage from endogenous and exogenous genotoxic sources. One of the mechanisms used to repair damaged chromosomes is genetic recombination, in which information present as a second chromosomal copy is used to repair a damaged region of the genome. In this review, I summarized what is known about the molecular and cellular mechanisms by which various DNA-damaging agents induce recombination in yeast. The yeast Saccharomyces cerevisiae has served as an excellent model organism to study the induction of recombination. It has helped to define the basic phenomenology and to isolate the genes involved in the process. Given the evolutionary conservation of the various DNA repair systems in eukaryotes, it is likely that the knowledge gathered about induced recombination in yeast is applicable to mammalian cells and thus to humans. Many carcinogens are known to induce recombination and to cause chromosomal rearrangements. An understanding of the mechanisms, by which genotoxic agents cause increased levels of recombination will have important consequences for the treatment of cancer, and for the assessment of risks arising from exposure to genotoxic agents in humans.     

Characterization and antimutagenic activity of soybean saponins

An extract was prepared from a commercial soybean-processing by-product (soybean molasses) and was fractionated into purified chemical components. In previous work, this extract (phytochemical concentrate, PCC) repressed induced genomic DNA damage, whole cell clastogenicity and point mutation in cultured mammalian cells. In the current study, a chemical fraction was isolated from PCC using preparative high-performance liquid chromatography (HPLC). This fraction, PCC100, repressed 2-acetoxyacetylaminofluorene (2AAAF)-induced DNA damage in Chinese hamster ovary (CHO) cells as measured by single cell gel electrophoresis (alkaline Comet assay). Using liquid chromatography-electrospray ionization-mass spectroscopy and 1H and 13C nuclear magnetic resonance (NMR) spectroscopy, PCC100 was shown to consist of a mixture of group B soyasaponins and 2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one (DDMP) soyasaponins. These include soyasaponins I, II, III, IV, V, Be, betag, betaa, gammag and gammaa. Purified soyasapogenol B aglycone prepared from fraction PCC100 demonstrated significant antigenotoxic activity against 2AAAF. To our knowledge, these data demonstrate for the first time the antimutagenic activity of soybean saponins in mammalian cells.     

Neoglycoconjugates of mannan with bovine serum albumin and their interaction with lectin concanavalin A.

Neoglycoconjugates were prepared from mannan isolated from yeast Saccharomyces cerevisiae and activated by periodate oxidation to create aldehyde groups. Various degrees of oxidation introduced 11-28 aldehyde groups per mannan molecule and simultaneously resulted in a molar mass decrease from 46 to 44.5-31 kDa. The activated mannans were subsequently conjugated with bovine serum albumin forming neoglycoconjugates. Some parameters of these mannan-bovine serum albumin conjugates were characterized: saccharide content 25-30% w/w, molar mass within the range 169-246 kDa, and polydispersion (M(w)/M(n)) from 2.8 to 3.6. The interaction of these conjugates with lectin concanavalin A was studied using three different methods: (i) quantitative precipitation in solution; (ii) sorption to concanavalin A immobilized on bead cellulose; and (iii) kinetic measurement of the interaction by surface plasmon resonance. Quantitative precipitation assay showed only negligible differences in the precipitation course of original mannan and the corresponding mannan-bovine serum albumin conjugates. Both the sorption method (equilibrium method) and the surface plasmon resonance measurement (kinetic method) demonstrates that the values of dissociation constant K(D) of all synthetic neoglycoconjugates were within the range 10(-7) - 10(-8) mol x L(-1) (close to K(D) = 10(-8) mol x L(-1) determined by the sorption method for the original mannan). In conclusion, characterization of synthetic neoglycoconjugates confirmed that the method used for their preparation retained the ability of mannan moiety to interact with concanavalin A.     

The role of natural biopolymers in genotoxicity of mutagens/carcinogens elimination

Nowadays naturally occurring compounds with the potential antimutagenic and anticarcinogenic effects are of great importance for their prospective use in cancer chemoprevention and treatment. The new water soluble derivative of microbial polysaccharide beta-D-glucan-carboxymethyl glucan (CMG) belongs to such a category of natural substances. CMG isolated from the cell wall of baker's yeast Saccharomyces cerevisiae is included into the class of biopolymers known as biological response modifiers (BRMs) with a broad range of activities, above all ones interfering with cancer therapy. It was demonstrated on four experimental model systems that biological and consequential medicinal importance of CMG is based on the combined application with another active compound. In the Saccharomyces cerevisiae antimutagenicity assay CMG significantly reduced ofloxacin-induced mutagenicity in the yeast strain D7. CMG exerted bioprotective (anti-toxic and antimutagenic) effect after its simultaneos application with methyl methanesulphonate on the repair-deficient strain uvs10 of the unicellular green alga Chlamydomonas reinhardtii. In the Vicia sativa simultaneous phytotoxicity and anticlastogenicity assay CMG exerted statistically significant anticlastogenic efect against maleic hydrazide-induced clastogenicity in Vicia sativa L. Only in the Salmonella/microsome assay CMG did not exert statistically significant antigenotoxic effect, despite of the fact that it reduced 9-aminoacridine-induced mutagenicity in S. typhimurium TA97, but his(+) revertants decreasing was statistically significant only at the highest CMG concentration used. The data presented unambiguously documented that even biopolysaccharides (e.g., derivatives of beta-glucan) belonging to the most abundant class of natural biopolymers may contribute to cancer prevention and therapy.     

Identification of two Saccharomyces cerevisiae cell wall mannan chemotypes

We have obtained evidence for two structurally and antigenically different Saccharomyces cerevisiae cell wall mannans. One, which occurs widely and is found in S. cerevisiae strain 238C, is already known to be a neutral mannan which yields mannose, mannobiose, mannotriose, and mannotetraose on acetolysis of the (1 --> 6)-linked backbone. The other, which was found in S. cerevisiae brewer's strains, is a phosphomannan with a structure very similar to that of Kloeckera brevis mannan. S. cerevisiae (brewer's yeast strain) was agglutinated by antiserum prepared against Kloeckera brevis cells. The mannan, isolated from a proteolytic digest of the cell wall of the former, did not react with S. cerevisiae 238C antiserum, whereas it cross-reacted strongly with K. brevis antiserum. Controlled acetolysis cleaved the (1 --> 6)-linkages in the polysaccharide backbone and released mannose, mannobiose, mannotriose, and mannotriose phosphate. Mild acid treatment of the phosphomannan hydrolyzed the phosphodiester linkage, yielding phosphomonoester mannan and mannose. The resulting phosphomonoester mannan reacted with antiserum prepared against K. brevis possessing monoester phosphate groups on the cell surface. alpha-d-Mannose-1-phosphate completely inhibited the precipitin reaction between brewer's yeast mannan and the homologous antiserum. Flocculent and nonflocculent strains of this yeast were shown to have similar structural and immunological properties.     

Antimutagenic and anticarcinogenic effects of Phyllanthus amarus

This study aimed to examine the antimutagenic and anticarcinogenic potential of Phyllanthus amarus Schum. et Thonn. using the bacterial preincubation mutation assay and an in-vivo alkaline elution method for DNA single-strand breaks in hamster liver cells. The aqueous extract of the entire plant showed an antimutagenic effect against induction by 2-aminofluorene (AF2), 2-aminoanthracene (2AA) and 4-nitroquinolone-1-oxide (4-NQO) in Salmonella typhimurium strains TA98 and TA100, and in Escherichia coli WP2 uvrA/pKM101. All the results were dose-dependent; however, inhibition of N-ethyl-N-nitrosoguanidine (ENNG)-induced mutagenesis was observed only with S. typhimurium TA100. The extract also exhibited activity against 2-nitrofluorene (2NF) and sodium azide-induced mutagenesis with S. typhimurium TA98 and TA100, respectively. Based on the alkaline elution method, the plant extract prevented in vivo DNA single-strand breaks caused by dimethylnitrosamine (DMN) in hamster liver cells. When the extract was administered 30 min prior to the administration of DMN, the elution rate constant decreased more than 2.5 times, compared to that of control. These results indicate that P. amarus possesses antimutagenic and antigenotoxic properties.     

Chemical structure of the cell-wall mannan of Candida albicans serotype A and its difference in yeast and hyphal forms

The structure of the cell-wall mannan from the J-1012 (serotype A) strain of the polymorphic yeast Candida albicans was determined by acetolysis under mild conditions followed by HPLC and sequential NMR experiments. The serotype A mannan contained beta-1,2-linked mannose residues attached to alpha-1,3-linked mannose residues and alpha-1,6-linked branching mannose residues. Using a beta-1,2-mannosyltransferase, we synthesized a three-beta-1,2-linkage-containing mannoheptaose and used it as a reference oligosaccharide for 1H-NMR assignment. On the basis of the results obtained, we derived an additivity rule for the 1H-NMR chemical shifts of the beta-1,2-linked mannose residues. The morphological transformation of Candida cells from the yeast form to the hyphal form induced a significant decrease in the phosphodiesterified acid-labile beta-1,2-linked manno-oligosaccharides, whereas the amount of acid-stable beta-1,2 linkage-containing side chains did not change. These results suggest that the Candida mannan in candidiasis patients contains beta-1,2-linked mannose residues and that they behave as a target of the immune system.     

Fluorescent colloidal gold: a cytochemical marker for fluorescent and electron microscopy.

The gold method was further developed for fluorescent microscopy. Gold granules (12 nm in size) were labelled with rhodamine conjugates of Concanavalin A and avidin. The fluorescent markers were used to mark cell wall mannan on the yeast Saccharomyces cerevisiae either by the one-step, or by the two-step method via a biotinyl derivative of ConA. By fluorescence or transmission electron microscopy, the two-step method was found to achieve a higher density of marking.     

Inactivation of the cyclin-dependent kinase Cdc28 abrogates cell cycle arrest induced by DNA damage and disassembly of mitotic spindles in Saccharomyces cerevisiae.

Eukaryotic cells may halt cell cycle progression following exposure to certain exogenous agents that damage cellular structures such as DNA or microtubules. This phenomenon has been attributed to functions of cellular control mechanisms termed checkpoints. Studies with the fission yeast Schizosaccharomyces pombe and mammalian cells have led to the conclusion that cell cycle arrest in response to inhibition of DNA replication or DNA damage is a result of down-regulation of the cyclin-dependent kinases (CDKs). Based on these studies, it has been proposed that inhibition of the CDK activity may constitute a general mechanism for checkpoint controls. Observations made with the budding yeast Saccharomyces cerevisiae, however, appear to disagree with this model. It has been shown that high levels of mitotic CDK activity are present in the budding yeast cells arrested in G2/mitosis as the result of DNA damage or replication inhibition. In this report, we show that a novel mutant allele of the CDC28 gene, encoding the budding yeast CDK, allowed cell cycle passage through mitosis and nuclear division in the presence of DNA damage and the microtubule toxin nocodazole at a restrictive temperature. Unlike the checkpoint-defective mutations in CDKs of fission yeast and mammalian cells, the cdc28 mutation that we identified was recessive and resulted in a loss of the CDK activity, including the Clb2-, Clb5-, and Clb6-associated, but not the Clb3-associated, CDK activities. Examination of several known alleles of cdc28 revealed that they were also, albeit partially, defective in cell cycle arrest in response to UV-generated DNA damage. These findings suggest that Cdc28 kinase in budding yeast may be required for cell cycle arrest resulting from DNA damage and disassembly of mitotic spindles.