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.     

Adaptive response in different mitotic cycles after irradiation

The frequency of cells with chromosome aberrations and the number of aberrations per cell have been studied by metaphase analysis in the nonirradiated progeny of irradiated human blood lymphocytes. DNA fragmentation (DNA double-stranded breaks) has been investigated by DNA comet assay. To study the adaptive response (AR), PHA-stimulated lymphocytes were irradiated by the adaptive dose (0.05 Gy) in 24 h and by challenge dose (1 Gy) in 48 h after stimulation. The first through fourth mitoses were identified by 5-bromodeoxyuridine. It was found that the frequency of chromosome aberrations and double-strand breaks were increased in all mitotic cycles after the challenge irradiation. In most individuals, the adaptive response is induced by adaptive and challenge irradiations in the first and the second mitotic cycles (48 and 72 h after stimulation, respectively); however, it is absent in the third and the fourth mitoses. In the first mitosis (1Gy in 48 h after stimulation), only chromatid aberrations are observed; chromosome aberrations were registered in subsequent mitoses. DNA comet assay showed that the adaptive response was obvious at 48–72 h, but not 96 h, after stimulation. It can be concluded that the nonirradiated progeny of irradiated lymphocytes have genomic instability. The adaptive response is manifested up to the third mitosis and is explained by the decreasing number of chromatid and chromosome aberrations and DNA fragmentation. We suppose that double-stranded DNA breaks may be damage signals for the induction of adaptive response.     

Synthesis of xylanolytic enzymes and other carbohydrases by the fungus Penicillium canescens: Transformants with an altered copy number of the gene xlnR and an encoding transcriptional activator

A fragment of Penicillium canescens genomic DNA carrying the xlnR gene coding for a translational activator of xylanolytic genes was isolated. It was demonstrated that a loss of this function in genetically modified transformants resulted in a drastic decrease in the production of P. canescens major xylanases and had a negative effect on the syntheses of several other extracellular xylanases. An increase in the dose of the xlnR gene elevated the xylanolytic activity as well as the activities of a number of other auxiliary enzymes involved in xylan degradation. The activities of two P. canescens major secreted enzymes—β-galactosidase and α-L-arabinofuranosidase—appeared weakly dependent on the translational activator xlnR.     

The molecular and cellular consequences of the chernobyl accident

In this paper the results of research at 5–10 and 24 years after the Chernobyl accident are summarized. These results include the investigation of genomic instability, formation of the adaptive response, genome damage, and oxidative status. The studies were performed on cells in culture, mice, children and adults who lived in the contaminated areas, and liquidators of the consequences of the Chernobyl accident. Inhibition of cell proliferative activity, late cell death, and the increase in micronucleus and giant cell frequency were observed after the exposure of cells in culture in the accident zone followed by their culturing in laboratory conditions. In the progeny of the exposed cells, the effect of enhanced radiosensitivity was detected. Thus, it can be assumed that exposure of parental cells in culture in the area of the accident induced genomic instability that resulted in the development of various abnormalities in progeny cells. At the organism level, the Chernobyl zone exposure of mice caused an increase in radiosensitivity; as well, a decrease in the endotheliocyte density in the cerebral cortex and other brain tissues was observed. In the blood lymphocytes of children stimulated by PHA, a more than two times increase in micronucleus cell frequency was detected. A reduced number of individuals with significant adaptive response was found in both the juvenile and adult groups. In all investigated populations, an increased number of individuals with enhanced radiosensitivity were observed in response to low-dose radiation exposure. At 24 years after the accident liquidators were subjected to examinations, which revealed an increased frequency of cells with micronuclei and chromosome-type aberrations in blood lymphocytes, an elevated level of DNA double strand breaks, and a reduced level of reactive oxygen species compared to those of the control group. This means that the genomic instability that was accumulated by the residents of the contaminated regions and liquidators as a result of the accident leads to damage of the genetic apparatus, an increase in radiosensitivity, and hypoxia as late consequences that all are risk factors and increase the probability of the development of tumor and non-tumor diseases. The development of the above-mentioned pathological processes may occur in the distant future.

     

Cloning of Penicillium canescens Endo-1,4-β-xylanase Gene and Construction of Multicopy Strains

The complete gene xylA that encodes endo-1,4--xylanase secreted byPenicillium canescens was cloned and sequenced. The coding region of the gene is separated by eight introns. The protein comprises 302 amino acids of the mature protein and 25 amino acids of the signal peptide. The xylanase of P. canescens belongs to the glycosyl hydrolase family 10. Nucleotide sequences for binding catabolite repression protein CREA and transactivator protein were detected in the promoter region. A set of multicopy strains displaying a seven to eightfold increase in xylanase yield was obtained. The fraction of xylanase in most productive strains amounted to 30–50% of the total secreted protein.     

Role of the carbamoylation reaction in the biological activity of methyl nitrosourea

Study of the mutagenic action of methyl nitrosourea (MNU) on the CHO-AT3-2 Chinese hamster cell at 2 regimes of cell treatment (a short-term regime and prolonged 1-h treatment) revealed that increase in the duration of treatment enhanced both cell lethality and clastogenic and mutagenic effects at the TK locus and did not influence the mutation frequency at the OUAr locus. On the basis of kinetic considerations it can be concluded that the base-pair substitution-type mutants (e.g., OUAr) appear as a result of DNA alkylation and the mutants at loci with a wide spectrum of registered mutants (the TK locus) are related to a greater extent to the carbamoylating activity of MNU. This conclusion is confirmed by measurements of the effects of sequential treatment with MNU (7 min) and KNCO (1 h). A synergistic increase in lethality, clastogenicity and mutagenicity at the TK locus was found in experiments with the combined treatment of cells with ethyl methanesulfonate (EMS) and KNCO. Besides, pretreatment of cells with potassium cyanate and subsequent exposure to MNU, EMS and benzopyrene (BP) produced synergistic effects in all the tests: lethality, clastogenicity and mutation frequency at the OUAr and TK loci. Posttreatment of cells with KNCO also led to a synergistic increase in the effects of MNU, EMS and BP treatment in several tests, but not in the OUAr locus. The possible mechanism and levels of interactions between alkylation and carbamoylation and the possibility that potassium cyanate causes supramolecular lesions are discussed.     

Mechanism of adaptive response: Estimation of human lymphocyte adaptive response to radiation using various criteria

Blood lymphocytes of 15 healthy donors have been investigated for their ability to reduce radiosensitivity after low-dose irradiation-radio-induced adaptive response (AR). The frequency of unstable chromosome aberrations was used to evaluate cell radiosensitivity after the irradiation of cells in low adaptive (5 cGy) and high challenge (1 Gy) doses in comparison with the effect of challenge irradiation only. Three indexes have been used, i.e., (A) the frequency of cells with aberrations per total analyzed cell, (B) the number of chromosome aberrations per one cell, (C) and the number of chromosome aberrations per one aberrant cell. It was found that the donors can be divided in the four following groups: 1. AR was not estimated any of the indexes used; 2. AR was estimated with indexes A and B, but not C; 3. AR was shown by indexes B and C; 4. AR was evident with all three indexes. The generally accepted AR repair model only explains the appearance of group-3 and-4 donors, but not group-2. For the purpose of understanding the AR mechanisms and the difference in AR estimations with various criteria, the metaphase distribution by the number of chromosome aberrations has been analyzed for each donor. It was shown that, in group-2 donors, the number of cells without aberrations after adaptive and challenge irradiations was significantly higher than after irradiation with a challenge dose only. Thus, in this group, AR is formed as a result of the changed frequency of cells in the 0 class (population shift). A similar shift is observed in the metaphase distribution in the donors of group 4, but not in group 3. The data obtained show that AR is probably a result of several processes, including the activation of the reparation of premutational genome damages, population shifts evident in the frequency of undamaged cells, and, possibly, the activation of apoptotic cell death. The complex character of AR is reflected to different degrees in each criterion of radiosensitivity.     

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.     

Effects of Treatment with a Composite Preparation (2-Chloroethylphosphonic Acid and Methacide) or Butylated Hydroxyanisole on Ethylene Release in Apples

We studied the effect of a Russian composite preparation (2-chloroethylphosphonic acid and methacide) and butylated hydroxyanisole on ethylene release in whole fruit and peel disks of two apple cultivars, Antonovka obyknovennaya (Antonovka) and Simirenko's rennet (Simirenko). Treatment with the composite preparation was followed by an increase in ethylene release from both the whole apples and peel disks. The development of microbial infection (fruit rot) in the whole apples became less pronounced after the treatment. Treatment of whole apples with the antioxidant butylated hydroxyanisole (BHA) increased the intensity of ethylene release during the first subsequent days; thereafter, ethylene release decreased and was 10–15% lower than in the control on days 10–12. In model experiments, BHA decreased ethylene release from apple peel disks below control levels as early as on the first day after treatment. Antonovka apples gave quick responses to the treatment. In the late-ripening Simirenko apples, the response persisted for a longer period. Our results suggest that treatment with physiologically active preparations affects the ethylene release, ripening, and preservation of apples in storage.