THE ROLE OF NATURAL BIOPOLYMERS IN GENOTOXICITY OF MUTAGENS / CARCINOGENS ELIMINATION

Nowadays naturally occuring 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 β-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 β-glucan) belonging to the most abundant class of natural biopolymers may contribute to cancer prevention and therapy.


INTRODUCTION
A rational use of chemopreventive agents is based not only on the assessment of their efficacy and safety but also on understanding of their mechanisms of action.A detailed classification is proposed which covers variety of mechanisms interfering with different phases of mutagenesis and carcinogenesis.Several mechanisms, such as inhibition of genotoxic effects, antioxidant activity and scavenging of free radicals, inhibition of cell proliferation and signal transduction modulation can be involved [1][2][3] .To such compounds also belong microbial skeletal polysaccharides which possess marked immunological properties ranging from non-specific stimulation of host immune system, resulting in antitumor, antiviral and anti-infective effects, to antioxidant, antimutagenic or hematopoetic activity [4][5][6][7] .In the recent years much evidence has been collected indicating that microbial polysaccharides play a role of signalling molecules for innate immune system 8 where they are recognized by genetically predetermined pattern recognition receptors (PRRs) located on the surface of immunocompetent and also other type of cells (e.g., epitelial) [9][10][11] .Among the yeast polysaccharides, specific PRRs have been idenfied and described for (1→3)β-D-glucans 9,12 .
β-Glucans isolated from fungi, bacteria and lichens belong to the class of substances known as biological response modifiers (BRMs), which modify the host's biological response by stimulation of the immune system 13 .
Recet decades have brought increased attention to the research of BRMs.Introduction of regulatory peptides and biomodulators in combination with chemotherapy (biotherapy) was a significant contribution to antineoplastic therapy.Among the microbial polysaccharides mainly derivatives of β-glucans isolated from the cell walls of yeast and fungi have been studied regarding their anticancer activities.The efficiency of chemotherapy of Lewis lung carcinoma with cyclophosphamide was affected by administration of the yeast carboxymethyl glucan -a well-known macrophage simulator 14 .Soluble β-glucan enhanced killing of retinal carcinoma micrometastases 15 .
As some polysaccharides isolated from the yeast cell walls belong to the class of substances known as BRMs with a broad range of activity, and the principal strategy of new anticancer drug modulators evaluation involves also antimutagenicity/anticarcinogenicity studies, the main concern of the present study has been given to examination of the carboxymethyl glucan (CMG) eligibility to exert antigenotoxic effect after its application on four genetic model organisms.

MATERIAL AND METHODS
Material: Carboxymethyl glucan (CMG) is a derivative of β-D-glucan which was isolated from the cell walls of baker's yeast S. cerevisiae by extraction with diluted alkali (6% NaOH at 60 o C) followed by treatment with diluted acid (4% phosporic acid extraction at room temperature) 16 .Insoluble β-D-glucan was solubilized by carboxymethylation described in detail by Machová et al. 16 .The degree of substitution (DS) determined by potentiometric titration was 0.8 and molecular weight determined by HPCL was 250 kD.
Results are means of five experiments.For statistic analysis Student's t-test was used.
Chlamydomonas reinhardtii bioprotectivity assay: Algal (recombination-repair-deficient) strain uvs10 of the unicellular green alga Chlamydomonas reinhardtii, isolated at the Department of Genetics, Faculty of Science, Comenius University, Bratislava, Slovakia 18 was treated with MMS (0.1-0.5 %) and simultaneously with MMS and CMG (10 -6 M) for 30 min in the dark, and then plated on agar dishes 19 .Survival was evaluated by microscopic method which enabled to distinguish algal cells died due to cytotoxic and due to genotoxic (lethal mutations) effect of MMS.Results are means of five experiments.For statistic analysis Wilcoxon's two sample test was used.

Simultaneous phytotoxicity and anticlastogenicity assay:
This assay was carried out on plant species Vicia sativa (L.) according to Murín 20 .After 24 h of soaking at 25 o C in the tested CMG (10 -5 M) and maleic hydrazide (MH), which was used as a positive control 21 , the seeds of V. sativa were allowed to germinate on Petri dishes (diameter = 18.5 cm) with filter paper soaked with the same concentrations of tested CMG and MH as those used for soaking.Phytotoxicity was assayed after 72 h of the dark cultivation in the thermostat at 25 o C. The seedlings roots of V. sativa were measured, and the growth inhibition percentages were assessed.The seedlings in which the root growth was inhibited at least by 25 %, 50 % and 75 % were fixed and used for chromosome and genome mutability evaluation.The roots were fixed and permanent slides were prepared by the Feulgen method.Chromosome aberrations were determined at least in 500 ana-telophases.For statistic analysis the Student's t-test was used.
Salmonella/microsome assay (Ames assay): For the potential CMG antigenotoxicity assessment the Ames assay was performed according to the published procedure by Maron and Ames 22 .The Salmonella typhimurium tester strains TA97, TA98 TA100, TA102 were obtained typhimurium TA97 (Fig. 3), but his + revertants decreasing was statistically significant only at the highest concentration of CMG.

DISCUSSION
The conventional treatment of surgery, radiation, and chemotherapy has been the cornerstone of cancer treatment over the past 50 years.Today, the clinical success of these treatments has reached a plateau.There is an urgent need to break through this cure plateau by trying fresh approaches.Acceptance and utilization of BRMs, including CMG, is one of them.As β-D-glucan isolated from S. cerevisiae is water insoluble, has higher molecular weight, and is resistant against alkali-acid treatments, it was processed by carboxymethylation 15 on purpose to change these properties and facilitate its potential utilization in medicine.

RESULTS
In this study potential antigenotoxic properties of CMG were investigated using yeast, algae, plant and bacteria as model genetic systems.
CMG significantly reduced the frequency of ofloxacininduced revertants at the ilv1 locus in the toxicity and antimutagenicity assay in S. cerevisiae D7 (Fig. 1).It can be suggested that the antimutagenic effect of CMG against ofloxacin may be based on its ability to scavenge reactive oxygen species.
Results obtained after simultaneous treatment of algal recombination-repair-deficient strain uvs10 with MMS and CMG documented that CMG exerted bioprotective effect (Fig. 2) because it reduced cytoxicity and mutagenicity (lethal mutations) of MMS.
Data obtained from the simultaneous phytotoxicity and anticlastogenicity assay revealed that 10 -6 M CMG significantly reduced clastogenic effect of MH applied on V. sativa seeds (Table 1).
However, in the Ames assay CMG did not exert statistically significant antimutagenic/anticarcinogenic effect (so that data are not shown), despite of the fact that it reduced 9-aminoacridine-induced mutagenicity in S.

The role of natural biopolymers in genotoxicity of mutagens/carcinogens elimination
For in vitro screening of antimutagens/anticarcinogens various prokaryotic and eukaryotic model organism, which enable to monitor different genetic endpoints, have been used 23 .Reduction of the genotoxic effect of MMS applied on recombination-repair-deficient strain of Chlamydomonas reinhardtii by CMG could be either a result of their interaction resulting in the MMS inactivation (CMG acting as desmutagen), or result of stimulation of non-damaged repair mechanism(s) (e.g., excision repair) by CMG (CMG acting as bioantimutagen).But, anti genotoxicity explanation due to acting in a desmutagenic manner, is more probable.Moreover, its antioxidative capacity based on efficient free-radical scavenging was also revealed by other authors 6,7,24 .In our experiments on yeast performed with ofloxacin, which is known to be inhibitor of DNA gyrase 25 and producer of reactive oxygen species (ROS) [26][27][28] , a decreased number of revertant colonies (Fig. 1) may be based on ability of CMG to prevent the oxidative damage to DNA induced by ROS which play important role in the multistage carcinogenesis and mutagenesis.Thus, CMG possesses high antioxidative activity as well as expressive antimutagenic/bioprotective effects, exerted through combined application with other biologically active compound (mutagen/carcinogen). CMG as a representative of fungal polysaccharides is of pharmacological importance due to its potential employment in cancer prevention and therapy.

Table 1 .
The anticlastogenic effect of CMG against MH-induced clastogenicity in Vicia sativa L.