On Antimutagenesis Mechanism in Plants

The effect of nitrosylmethylurea (NMU) on the mitotic index and the frequency of cells with aberrations, as well as the effects of pre- and posttreatment with antioxidant ambiol on the NMU effects were studied on seedlings of common winter wheat Triticum aestivum, cultivar Moskovskaya 39. Both pre- and posttreatment with ambiol resulted in antimutagenic effect but after posttreatment, the effect was lower. Irrespective of type of seedling treatment with ambiol and the time of their fixation (45, 48, and 51 h), when mitotic index is plotted versus frequency of cells with aberrations, all experimental points fall on the same regression line with coefficient of correlation of −0.82 (P < 0.001). This implies that the same mechanism underlies antimutagenic effect irrespective of when the antimutagen was applied, before or after the knockout mutagen dose. This also suggests that the antimutagenic effect is independent of the degree of the mutagen-induced damage, because by the time of posttreatment, the volume of genome damage is already determined and the antimutagen fails to change it. Finally, this suggests that irrespective of time of antimutagen treatment, the mutation frequency is reduced by the mechanism of stimulated repopulation.__________Translated from Genetika, Vol. 41, No. 5, 2005, pp. 676–679.Original Russian Text Copyright © 2005 by Serebryanyi, Zoz, Morozova.     

Effect of antioxidant-ambiol on the radiation adaptive response

For studying of the mechanism of adaptive response of plants the seeds of soft wheat Triticum aestivum cultivar Moscovskaya 39 were irradiated in doses 0.25, 50 and 0.25 + 50 Gy and the frequency of cells with aberrations and the mitotic activity in the meristem of seedlings were scored. The pre- and post-treatments of seeds with antioxidant--ambiol were also used. It was found that the exposure of seeds to 0.25 Gy reduce the effects of challenge dose of 50 Gy: the mitotic index increases and the frequency of cells with aberrations decreases--the adaptive response appear. It was also found that the pretreatment with ambiol reduce the effects of the irradiation in the dose of 50 Gy. Post-treatment was less efficiently. Both treatments raise the adaptive response. The correlation between the frequency of aberrant cells and the mitotic index was found and, regardless of the type of treatment all points of experiment fall on the common regression line with the regression coefficient -0.85 (p < 0.01). These facts serve as evidence (1) that the radioprotective effect by the pre- and post-treatment occurs by a common mechanism and (2) that the in the exhausted concentration antioxidant does not change the extent of genome damage inflicted by irradiation. The evidence is consistent with the hypothesis that a nonspecific inducible process of stimulated repopulation was a mechanism of adaptive response of plants.     

Stimulated Repopulation as a Basis of Antimutagenesis and the Adaptive Response in Plants

Wheat seeds were used to study (1) modification of the radiation adaptive response (AR) with antioxidant anphen and (2) modification of the clustogenic effect of N-nitroso-N-methylurea (NMU) with various agents. Pretreatment with anphen enhanced AR. Each pretreatment (irradiation with 0.25 Gy, treatment with anphen, treatment with anphen followed by irradiation with 0.25 Gy) decreased aberration frequency. This parameter proved to be in a linear dependence with mitotic index (MI) with correlation coefficient –0.978; the regression line passed through the point corresponding to spontaneous MI and spontaneous aberration frequency. Upon treatment with NMU, the antimutagenic effect was observed for various pretreatments (a low concentration of NMU, antioxidant phenoxan, irradiation with 0.25 Gy). Again, MI and aberration frequency were in inverse proportion with correlation coefficient –0.99, and the regression line passed through the point with spontaneous MI and spontaneous aberration frequency. The same dependence was observed for previously published data on modification of radiation AR with phenoxan. The results were hard to explain in terms of the repair-associated mechanism of AR and the antimutagenic effect. Hence, a nonspecific inducible process of stimulated repopulation was assumed to be a common mechanism of AR and the antimutagenic effect in plants.     

Stimulated repopulation as a basis of antimutagenesis and the adaptive response in plants

Wheat seeds were used to study (1) modification of the radiation adaptive response (AR) with antioxidant anphen and (2) modification of the clustogenic effect of N-methyl-N-nitrosourea (MNU) with various agents. Pretreatment with anphen enhanced AR. Each pretreatment (irradiation with 0.25 Gy, treatment with anphen, treatment with anphen followed by irradiation with 0.25 Gy) decreased aberration frequency. This parameter proved to be in a linear dependence with mitotic index (MI) with correlation coefficient -0.978; the regression line passed through the point corresponding to spontaneous MI and spontaneous aberration frequency. Upon treatment with MNU, the antimutagenic effect was observed for various pretreatments (a low concentration of MNU, antioxidant phenoxan, irradiation with 0.25 Gy). Again, MI and aberration frequency were in inverse proportion with correlation coefficient -0.99, and the regression line passed through the point with spontaneous MI and spontaneous aberration frequency. The same dependence was observed for previously published data on modification of radiation AR with phenoxan. The results were hard to explain in terms of the repair-associated mechanism of AR and the antimutagenic effect. Hence, a nonspecific inducible process of stimulated repopulation was assumed to be a common mechanism of AR and the antimutagenic effect in plants.     

Anticlastogenic effect of Vitamin C on cisplatin induced chromosome aberrations in human lymphocyte cultures.

Vitamin C (ascorbic acid) is an antioxidant that can scavenge free radicals and protect cellular macromolecules, including DNA, from oxidative damage induced by different agents. The protective effect of Vitamin C on cisplatin induced chromosome aberrations has been determined in the human peripheral lymphocyte chromosome aberration test in vitro. The results of treatments with Vitamin C indicated that it statistically significantly decreases the number of chromosome aberrations and number of metaphases with aberrations induced with cisplatin, but it can not completely protect cells from damage. The test concentrations of Vitamin C (10 and 100 microg/ml) had a limited antimutagen effect on cisplatin (0.5 microg/ml), which can cause genetic damage through free radical mechanisms. The antimutagen effect included the anticlastogenic effect of Vitamin C and its ability to decrease the number of aneuploid mitoses. Vitamin C showed the most efficient anticlastogenic effect during simultaneous treatment with cisplatin. Also, Vitamin C reduced cell toxicity of cisplatin during simultaneous treatment.     

Antimutagenesis by factors affecting DNA repair in bacteria

The term 'antimutagen' was originally used to describe an agent that reduces the apparent yield of spontaneous and/or induced mutations, regardless of the mechanisms involved. The 'antimutagens' include 'desmutagens' and 'bio-antimutagens'. In this article, our attention was focused on the bio-antimutagens affecting DNA repair in bacteria. Cobaltous chloride reduced the frequency of mutations in Escherichia coli induced by MNNG. The possibility that metal compound inhibits the growth of mutagen-treated cells was examined. The results clearly showed that the antimutagen surely reduces the mutation rate. The target of cobaltous chloride was found to be cellular factors including Rec A. Vanillin and cinnamaldehyde had strong antimutagenic activities against UV, 4NQO and AF-2. They stimulated Rec A-dependent recombination repair functions in the mutagen-treated cells. Among plant materials, tannins possess antimutagenic activity against UV-induced mutations in E. coli. It has been found that tannic acid stimulates the excision repair encoded by the uvrA gene thereby reducing the yield of mutants. Substances which are antimutagenic in bacterial systems also had antimutagenic activity in cultured mammalian cell systems. Vanillin reduced the frequency of mutagen-induced chromosomal aberrations.     

Anticlastogenic effect of Vitamin C on cisplatin induced chromosome aberrations in human lymphocyte cultures

Vitamin C (ascorbic acid) is an antioxidant that can scavenge free radicals and protect cellular macromolecules, including DNA, from oxidative damage induced by different agents. The protective effect of Vitamin C on cisplatin induced chromosome aberrations has been determined in the human peripheral lymphocyte chromosome aberration test in vitro. The results of treatments with Vitamin C indicated that it statistically significantly decreases the number of chromosome aberrations and number of metaphases with aberrations induced with cisplatin, but it can not completely protect cells from damage. The test concentrations of Vitamin C (10 and 100 μg/ml) had a limited antimutagen effect on cisplatin (0.5 μg/ml), which can cause genetic damage through free radical mechanisms. The antimutagen effect included the anticlastogenic effect of Vitamin C and its ability to decrease the number of aneuploid mitoses. Vitamin C showed the most efficient anticlastogenic effect during simultaneous treatment with cisplatin. Also, Vitamin C reduced cell toxicity of cisplatin during simultaneous treatment.     

Antimutagenic mechanisms of Phyllanthus orbicularis when hydrogen peroxide is tested using Salmonella assay

Phyllanthus orbicularis is a medicinal plant, endemic to Cuba, whose aqueous extract has proven antiviral properties and antimutagenic activities against aromatic amines and hydrogen peroxide. In addition, this plant extract presents antioxidant activity. In this paper, using the Salmonella assay with the experimental approaches of co-incubation, pre- and post-treatments, it is shown that the P. orbicularis extract protects bacterial cells from oxidative damage and mutation by some intracellular mechanism, irrespective of its antioxidant activity.     

Chemosignals from isolated females have antimutagenic effect in dividing the cells of bone marrow from male mice of the CBA strain

A level of X-ray induced mitotic disturbances in the cells of the bone marrow of male mice was studied under the modifying influence of chemosignals from isolated adult female mice of the CBA strain. It has been shown that the frequency of chromosomal aberrations in irradiated (4 Gr) males after exposing them for 24 hours on bedding soiled with female chemosignals is lower than in irradiated males in cages with clean bedding. The mechanisms and importance of the antimutagenic effect of female house mouse chemosignals are discussed.     

Acetylsalicylic acid exhibits anticlastogenic effects on cultured human lymphocytes exposed to doxorubicin

Acetylsalicylic acid (ASA) is a non-steroidal anti-inflammatory drug (NSAID) with many pharmacological properties, such as anti-inflammatory, antipyretic and analgesic. Many studies have suggested the possible efficiency of ASA and other NSAIDs in preventing cancer. ASA could also have antimutagenic and antioxidant properties. The aim of this study was to investigate the possible clastogenic and anticlastogenic effects of different concentrations of ASA on doxorubicin-induced chromosomal aberrations in human lymphocytes. Human blood samples were obtained from six healthy, non-smoking volunteers; and the chromosomal aberration assay was carried out using conventional techniques. The parameters analyzed were mitotic index, total number of chromosomal aberrations and percentage of aberrant metaphases. The concentrations of ASA (25, 50 or 100 microg/mL) tested in combination with DXR (0.2 microg/mL) were established on the basis of the results of the mitotic index. The treatment with ASA alone was neither cytotoxic nor clastogenic (p>0.01). In lymphocyte cultures treated with different combinations of ASA and DXR, a significant decrease in the total number of chromosome aberrations was observed compared with DXR alone (p<0.01). This protective effect of ASA on DXR-induced chromosomal damage was obtained for all combinations, and it was most evident when ASA was at 25.0 microg/mL. In our experiments, ASA may have acted as an antioxidant and inhibited the chromosomal damage induced by the free radicals generated by DXR. The identification of compounds that could counteract the free radicals produced by doxorubicin could be of possible benefits against the potential harmful effects of anthracyclines. The results of this study show that there is a relevant need for more investigations in order to elucidate the mechanisms underlying the anticlastogenic effect of ASA.     

Suppression of 6-TG-resistant mutations in V79 cells and recessive spot formations in mice by vanillin

The antimutagenic effects of vanillin, anisaldehyde, cinnamaldehyde and coumarin were investigated in cultured Chinese hamster V79 cells in vitro. The frequencies of 6-TG-resistant mutations induced by UV or X-rays were decreased by treatment with each compound during the expression time. These decreases were not due to cytotoxic effects on cellular growth or killing effects on damaged cells.The antimutagenic effect of vanillin was also investigated in vivo in the mouse spot test using male PW and female C57BL/10 mice. Female mice were injected intraperitoneally with ethylnitrosourea (ENU) on the 10th day of pregnancy and received 3 successive oral administrations of vanillin. Administration of vanillin decreased the ENU-induced frequency of recessive carrier pups. These results indicate that vanillin acts as an antimutagen in mammalian cells both in vitro and in vivo.     

cis-Dichlorodiammine platinum(II) induced aberrations in mouse bone-marrow chromosomes

The clastogenic effect of cis-dichlorodiammine platinum(II) (cis-platin) on mouse bone-marrow chromosomes has been studied. Cis-platin was injected at 3 different doses. Cells were fixed at different time intervals after treatment. Different types of aberrations together with the percent of mitotic index and frequency of abnormal metaphases were studied. The aberrations observed were primarily chromatid breaks, although isochromatid breaks, interchanges, and multiple breaks were also observed. A dose- and time-dependent effect was observed for both inhibition of mitotic index and frequency of abnormal metaphases. Trypsin-Giemsa staining of bone-marrow metaphase chromosomes from normal mice was compared with the bands of metaphase chromosomes obtained after Giemsa staining of chromosomes from platinum-treated mice and they were observed to be identical. Bands were present up to 120 h and aberrations were also induced in such plates.     

Antimutagenic effect of alpha-tocopherol on the gene mutation frequency in Salmonella]

The work presents the data on the antimutagenic effect of alpha-tocopherol on the frequency of N-nitroso-N-methyl urea induced gene mutations in Salmonella in vitro and in vivo. In vitro tests have revealed the dependence of decreasing the rate of induced mutations both on combination of treatments with mutagen/antimutagen and on the dose of the mutagen. In vivo tests have demonstrated that the mutagenic effect of alpha-tocopherol depends on the duration of its action on organisms.     

Tea tannin components modify the induction of sister-chromatid exchanges and chromosome aberrations in mutagen-treated cultured mammalian cells and mice

The modifying effects of tannin components extracted from green tea and black tea on mutagen-induced SCEs and chromosome aberrations were studied. These tannin components did not affect spontaneous SCEs and chromosome aberrations in cultured Chinese hamster cells. The frequency of SCEs and chromosome aberrations induced by mitomycin C (MMC) or UV was enhanced by the posttreatment with tea tannin components. When cells were post-treated with tea tannin components in the presence of metabolic enzymes of rat liver (S9 mix), the modifying effects on the induction of SCEs and chromosome aberrations by mutagens were complicated. MMC- and UV-induced SCEs and chromosome aberrations were suppressed by the posttreatment with tea tannin components at low concentrations (less than or equal to 6.7 micrograms/ml) with S9 mix. At a high concentration of tea tannin components (20 micrograms/ml) with S9 mix, a co-mutagenic effect was observed. The modifying effects of tea tannin components were shown to occur in the G1 phase of the cell cycle. In cells from a patient with xeroderma pigmentosum (XP) and a normal human embryo, MMC-induced SCEs were suppressed by the posttreatment with tea tannin components in the presence of S9 mix, and enhanced in the absence of S9 mix. On the other hand, tea tannin components modified SCE frequencies in UV-irradiated normal human cells but not in UV-irradiated XP cells. Our results suggested that tea tannin components themselves inhibited DNA-excision repair and resulted in a co-mutagenic effect, while in the presence of S9 mix metabolites of tea tannin components promoted DNA-excision repair activity and resulted in an antimutagenic effect. MMC-induced chromosome aberrations in mouse bone marrow cells were suppressed by the pretreatment with green tea and black tea tannin mixture.     

The effect of sodium fluoride on DNA synthesis, mitotic indices and chromosomal aberrations in human leukocytes treated with trenimon in vitro

Human leukocyte cultures were set up with Ham's F-10 medium and stimulated with PHA-M. Treatment of the cells in G₁ from 15–20 h with 0.5 × 10⁻⁶ M Trenimon resulted in a considerable cell cycle delay, as measured by [³H]-TdR autoradiography and determination of mitotic indices. Under these conditions only few cells incorporated the tracer at the same time as most cells did in untreated cultures. However, this did not lead to a mitotic activity at the same time as obtained in controls. Most of the treated cells started their DNA synthesis and mitotic activities with a delay of around 20 h, as compared with the controls. Continuous treatment of the cells with 10⁻³ M NaF had no effect on [³H]TdR labelling or mitotic indices in otherwise untreated cultures, but led to an impressive effect on DNA synthesis in Trenimon-treated cultures, without a considerable effect on the mitotic indices. This finding could beexplained as due to a lower alkylation in cellular DNA in the presence of NaF. More cells can start with their DNA synthesis, although they are, like Trenimontreated cultures, incapable of completing it normally. Analyses of the effect of NaF on chromosomes aberrations induced by Trenimon revealed that pre-, simultaneous and post-treatments significantly enhanced the frequency of undamaged mitoses. Continuos fluoride treatment also protected the cells from Trenimon-induced damage, but the effect was not significant, possibly because of heavily damaged mitoses which appeared under these conditions. We interpret our findings as an indication of a real anti-mutagenic activity of NaF.     

The induction of latent liver damage by cyclophosphamide

The effect of the cyclophosphamide on the changes of the mitotic index (MI), metaphase-prophase ratio (M/P) and chromosomal aberrations in postmetaphase in regenerating rat liver was studied. Cyclophosphamide was injected in the single doses of 100 and 200 mg/kg i.p. 2 hours before partial hepatectomy (PHE). The results have shown that cyclophosphamide caused latent liver damage which was manifested after PHE in reduced MI and increased frequency of chromosomal aberrations. The extent of the changes induced by cyclophosphamide corresponds to that due to the effect of 3 or 6 Gy of X-rays, respectively.     

A comparative study of the antimutagenic effects of antioxidants on chemical mutagenesis in Drosophila melanogaster

The 1,4-dihydropyridine derivative 2,6-dimethyl-3,5-diethoxycarbonyl-4-(Na carboxylate)-1,4-dihydropyridine (1,4-DHP) was studied for antimutagenic effects in the dominant lethal test and in the sex-linked recessive lethal test of Drosophila melanogaster. The observed effects were compared with those of the radioprotectors cysteine and cysteamine and with those of the phenolic antioxidant butylated hydroxytoluene (BHT). In a wide range of concentrations, including low ones, 1,4-DHP reduces the frequency of EMS-induced genetic damage (point mutations and chromosome breakage). A reduction of the mutation rate induced by EMS in adults could be observed independently of the developmental stages (larvae or imago) pretreated with 1,4-DHP. The protective effect of this new antimutagen against the alkylating agent depended on both the 1,4-DHP dose and the level of the EMS-induced mutation rate. The effect of 1,4-DHP was more pronounced than that of the studied radioprotectors. It is concluded that dihydropyridine-type compounds are able to protect eukaryote germs cells from genetic damage produced by direct-acting mutagens such as EMS.     

The effect of diepoxybutane on chromosomes of Vicia faba

Summary The frequency of aberrations induced by DEB depends on the temperature and concentration of the agent. The effect of DEB is delayed. Pretreatment with EDTA increased the frequency of aberrations. The frequency of aberrations was also increased after simultaneous treatment with DEB and chloramphenicol or streptomycin. Pre- and posttreatment with protein synthesis inhibitors had a reverse effect, the frequency of aberrations was decreased.     

Evaluation of the clastogenicity and anticlastogenicity of the carotenoid bixin in human lymphocyte cultures

Carotenoids are regarded as effective antioxidants, antimutagenic and anticarcinogenic agents. Annatto, a red-yellow extract obtained from seeds of Bixa orellana L. is a mixture of several carotenoids and one of them bixin (BXN), is known as its major coloring compound. Studies on BXN clastogenicity and anticlastogenicity in cultured human lymphocytes have not been reported so far. Therefore, the present study was undertaken to investigate the ability of BXN to induce chromosomal aberrations in human lymphocytes in vitro and to examine the possible anticlastogenic effect of this carotenoid in chromosomal damage induced by the clastogen cisplatin (cDDP). Human blood samples were obtained from six healthy, non-smoking volunteers; two females and four males aged 18-35 years. The concentrations of BXN (1.0; 2.5; 5.0 or 10 microg/mL) tested in combination with cDDP were established on the basis of mitotic index (MI) measurements. The data showed that BXN was not cytotoxic or clastogenic, when compared to untreated control. A marked decrease in the MI values compared to the untreated control and an increased percentage of aberrant metaphases was seen in all cultures treated with cDDP. The carotenoid efficiency in reducing the inhibitory effect of cDDP on lymphocyte MI is concentration-dependent. Cultures simultaneously treated with BXN and cDDP showed a statistically significant reduction in total chromosomal aberrations and aberrant metaphases. In our experiments, BXN may have acted as an antioxidant by intercepting free radicals generated by cDDP. The data obtained in the present study suggest that dietary carotenoids may act as protective agents against clastogenic effects of antitumor agents. However, extensive studies are necessary to elucidate the mechanism of action of BXN before its therapeutic use.     

Protective roles of exogenous polyamines on chromosomal aberrations in <Emphasis Type="Italic">Hordeum vulgare</Emphasis> exposed to salinity

The effects of exogenous polyamines (PAs): spermine (Spm), spermidine (Spd), cadaverine (Cad) and putrescine (Put) on mitotic activity and chromosomal aberrations in root meristem cells of Hordeum vulgare L. (barley) seeds exposed to salinity were analyzed. The PAs significantly inhibited cell division in distilled water. Furthermore, most of these PAs (except for Spd) caused a significant increase in the frequency of chromosomal aberrations as compared to control group. Seeds treated with Put caused the highest percentage of mitotic abnormalities in total. The negative effect of salinity on mitotic index and the frequency of chromosomal aberrations increased with increasing salt concentration. PAs studied could not be successful in ameliorating of the negative effect of salinity on mitotic activity. Particularly, exposure to Cad and 0.40 M NaCl caused a complete block of cell division in total. However, most of the PA studied showed a perfectly performance in alleviating the detrimental effects of increasing salinity on chromosomal aberrations.