Tomáš Gichner

Induction of somatic DNA damage as measured by single cell gel electrophoresis and point mutation in leaves of tobacco plants

The induction and measurement of DNA damage in nuclei of plant tissues is a new area of study with the alkaline single cell gel electrophoresis/comet assay. Methods to isolate plant cell nuclei cause high levels of DNA damage which are detected by the comet assay. We developed a method to isolate nuclei from leaf tissue of Nicotiana tabacum (a1+/a1; a2+/a2) in a modified Sörensen buffer that resulted in constant, low tail moment values for the negative controls. After treating intact tobacco plants with 1-8 mM ethyl methanesulfonate (EMS) we obtained a direct concentration-response with an average median tail moment of 65.9+/-4.4 micro(m) for plants exposed to the highest EMS concentration as compared to the median control tail moment value of 4.1+/-0.8. We found that the highest resolution was obtained with electrophoretic conditions of 0.74 V/cm at 300 mA for 20 min. Multiple leaves could be analyzed per plant within each treatment group and the tail moments were not significantly different. Tobacco seedlings were treated with EMS in the same manner as used for the comet assay and mutations were induced in the leaf primordia. The mean mutant frequency for the control was 1.46+/-0.20 mutant sectors/leaf. The mutant frequency increased in a concentration dependent manner; the mutant frequency induced by 8 mM EMS was 37.89+/-2.37 mutant sectors/leaf. The comet tail moment values and the leaf mutant frequency were highly correlated (r=0.98). The genetic response factor was calculated by the ratio of the difference in the response within the linear portion of each concentration-response curve divided by the slope of the curve. The genetic response factor for the tail moment was 7.82 while the value for mutation induction was 7.76. In this paper we describe a sensitive method with high resolution to apply the alkaline comet assay to plant leaves. The comet assay response was compared to that of induced point mutation. With this sensitive method for nuclei isolation from plant leaves, the alkaline SCGE assay could be incorporated into in situ plant environmental monitoring.

Evaluation of DNA damage and mutagenicity induced by lead in tobacco plants

Tobacco (Nicotiana tabacum L. var. xanthi) seedlings were treated with aqueous solutions of lead nitrate (Pb2+) at concentrations ranging from 0.4 mM to 2.4 mM for 24 h and from 25 microM to 200 microM for 7 days. The DNA damage measured by the comet assay was high in the root nuclei, but in the leaf nuclei a slight but significant increase in DNA damage could be demonstrated only after a 7-day treatment with 200 microM Pb2+. In tobacco plants growing for 6 weeks in soil polluted with Pb2+ severe toxic effects, expressed by the decrease in leaf area, and a slight but significant increase in DNA damage were observed. The tobacco plants with increased levels of DNA damage were severely injured and showed stunted growth, distorted leaves and brown root tips. The frequency of somatic mutations in tobacco plants growing in the Pb2+-polluted soil did not significantly increase. Analytical studies by inductively coupled plasma optical emission spectrometry demonstrate that after a 24-h treatment of tobacco with 2.4 mM Pb2+, the accumulation of the heavy metal is 40-fold higher in the roots than in the above-ground biomass. Low Pb2+ accumulation in the above-ground parts may explain the lower levels or the absence of Pb2+-induced DNA damage in leaves.

A comparison of DNA repair using the comet assay in tobacco seedlings after exposure to alkylating agents or ionizing radiation

We employed single cell gel electrophoresis to analyze the kinetics of DNA repair in nuclei isolated from tobacco plants exposed to ethyl methanesulfonate (EMS), N-ethyl-N-nitrosourea (ENU) and gamma-radiation. DNA repair was measured as the reduction of the tail moment values as a function of time after the mutagen treatment ended. DNA damage in leaf nuclei of EMS-or ENU-treated tobacco plants persisted over a 72h recovery period. However, a reduction of the SCGE tail moment values in nuclei isolated from leaves was observed over a 4-week period of recovery. Newly emerged leaves expressed a lower level of DNA damage due to more efficient repair and/or dilution of initial DNA lesions during cell division. After 24h recovery, leaf nuclei from cells exposed to 20 or 40Gy of gamma-radiation expressed complete DNA repair. These data indicate that DNA lesions induced by alkylating agents are not readily repaired and persist beyond 4 weeks. Enzymes necessary to repair gamma-induced DNA lesions are fully functional in non-replicating leaf cells and single and double strand breaks are rapidly repaired.

The mutagenic activity of 1-alkyl-1-nitrosoureas and 1-alkyl-3-nitro-1-nitrosoguanidines

Abstract The mutagenic activities of five 1-alkyl-1-nitrosoureas and six 1-alkyl-3-nitro-1-nitrosoguanidines were tested and compared on Arabidopsis thaliana . All the nitrosoureas tested were active, whereas only methyl and ethyl derivatives of nitrosogunidines were. There was a tendency for the mutagenic effectiveness to decrease with increasing number of C-atoms in the alkl group. The nitrosoamides tested had a much higher mutagenic efficiency than X-rays, i.e. at the same level of induced mutations they caused less sterility. The possible reasons for the activity of nitrosoguanidines in Arabidopsis and their lack of mutagenic activity in barley are briefly discussed.

DNA damage induced by indirect and direct acting mutagens in catalase-deficient transgenic tobacco. Cellular and acellular Comet assays.

We have measured the level of DNA damage induced by treating roots (cellular Comet assay) and isolated root nuclei (acellular Comet assay) of catalase-deficient (CAT1AS) and wild-type (SR1) tobacco with the promutagen o-phenylenediamine (o-PDA) and the direct acting genotoxic agents hydrogen peroxide and ethyl methanesulphonate (EMS). The roots of CAT1AS have about 60% less catalase activity compared to the roots of SR1. The promutagen o-PDA applied on tobacco roots induced significantly higher levels of DNA damage in the CAT1AS transgenic line than in SR1, while after application of o-PDA on isolated root nuclei, no DNA damage could be detected. In the catalase-deficient line CAT1AS about six-fold lower concentrations of H(2)O(2) are sufficient to induce the same levels of DNA damage as in SR1. By contrast, after treatment of isolated root nuclei with H(2)O(2) no difference in the induced levels of DNA damage was observed between CAT1AS and SR1. The DNA damaging effect of EMS was not affected by the presence of catalase in the tobacco roots and the levels of DNA damage measured by the cellular and acellular assay were similar. Comparing the effects of genotoxic agents in both the cellular and acellular Comet assays may help to elucidate their mechanism of action. Differences in both systems may reveal the participation of scavengers and of repair and metabolic enzymes on the activity of the genotoxic agent and the role of the cell wall in preventing the agent from reacting with nuclear DNA.

Comparison of DNA damage in plants as measured by single cell gel electrophoresis and somatic leaf mutations induced by monofunctional alkylating agents

The use of single cell gel electrophoresis (SCGE) has recently been applied to plant systems. We optimized the experimental conditions for SCGE analysis using nuclei isolated from different tissues of intact plants. Concentration–response curves of genomic DNA migration were analyzed in intact plants treated with the monofunctional alkylating agents ethyl methanesulfonate (EMS), methyl methanesulfonate (MMS), N‐ethyl‐N‐nitrosourea (ENU), and N‐methyl‐N‐nitrosourea (MNU). These data were used to calibrate SCGE tail moment values to induced somatic mutation in plant leaves. We used a genotoxicity index to compare genomic DNA damage and the induction of somatic mutation in the leaf tissues. The rank order of the genotoxic potency of these alkylating agents assayed by SCGE was MNU ≫ MMS > ENU > EMS. The rank order for the mutagenic potency of these agents was MNU ≫ ENU ≅ MMS > EMS. The data demonstrate the utility of SCGE analysis in plant systems. The use of SCGE will permit a larger range of plants for use as in situ environmental monitors. Also, this approach may be used to search for crop plant germplasm accessions with enhanced genomic stability. We investigated whether the intragenomic distributions of DNA damage induced by these alkylating agents were uniform and random. When a plot of the ratio of the %tail DNA and tail length versus the concentration of the test mutagen was generated, the induced SCGE data deviated from a random distribution of genomic DNA damage. Environ. Mol. Mutagen. 33:279–286, 1999

Induction of somatic mutations in Tradescantia clone 4430 by three phenylenediamine isomers and the antimutagenic mechanisms of diethylditiocarbamate and ammonium meta-vanadate

Three isomers of the promutagen phenylenediamine at mM concentrations were plant-activated and induced mutation in stamen hairs of Tradescantia clone 4430. The rank order of the mutagenicity of the isomers was: o-phenylenediamine > m-phenylenediamine > p-phenylenediamine with corresponding mutagenic potencies of 5.60, 1.43, and 0.46 mutant stamen hair cells/mumole, respectively. Diethyldithiocarbamate (DEDTC) and ammonium meta-vanadate (vanadate) repressed the mutagenic activity of o-phenylenediamine (o-PDA) in intact plants. Based on inhibition kinetics and reaction rates, the mechanism of DEDTC antimutagenicity was attributed to the inhibition of peroxidases that are required in the plant activation of o-PDA to mutagenic product(s). Spectrophotometric measurements of equimolar concentrations of o-PDA and vanadate demonstrated that the antimutagenic property of vanadate was mainly due to its reactivity with o-PDA.

Differential genotoxicity of ethyl methanesulphonate, N-ethyl-N-nitrosourea and maleic hydrazide in tobacco seedlings based on data of the Comet assay and two recombination assays

The purpose of this study was to determine if mutagen-induced DNA damage is correlated with the frequency of induced recombination events. The alkylating agents ethyl methanesulphonate (EMS) and N-ethyl-N-nitrosourea (ENU), and the plant growth regulator and herbicide maleic hydrazide (MH) were compared in tobacco seedlings for their ability to induce DNA damage measured by the Comet assay, and recombination activity measured by the GUS gene reactivation assay, and by the somatic twin sectors assay. While EMS and ENU induced a dose-dependent increase in DNA damage in leaf nuclei, MH had no significant effect. By contrast, MH induced a 6-fold higher frequency of homologous recombination as expressed by the GUS assay and a 2.8-fold higher frequency of somatic twin sectors than after EMS treatments.

Induction and repair of DNA damage as measured by the Comet assay and the yield of somatic mutations in gamma-irradiated tobacco seedlings

The advantage of using the tobacco (Nicotiana tabacum var. xanthi) mutagenicity assay is the ability to analyze and compare on the same plants under identical treatment conditions both the induced acute DNA damage in somatic cells as measured by the Comet assay and the yield of induced leaf somatic mutations. Gamma-irradiation of tobacco seedlings induced a dose-dependent increase in somatic mutations from 0.5 (control) to 240 per leaf (10Gy). The increased yield of somatic mutations was highly correlated (r = 0.996) with the increased DNA damage measured by the Comet assay immediately after irradiation. With increased dose of gamma-irradiation, the averaged median tail moment values ( +/- S.E.) significantly increased from 1.08 +/- 0.10 (control) to 20.26 +/- 1.61 microm (10Gy). Nuclei isolated from leaves 24h after irradiation expressed tail moment values that were not significantly different from the control (2.08 +/- 0.11). Thus a complete repair of DNA damage induced by gamma-irradiation and measurable by the Comet assay was observed, whereas the yield of somatic mutations increased in relation to the radiation dose. Data on the kinetics of DNA repair and of DNA damage induced by gamma-radiation on isolated tobacco nuclei, and on nuclei isolated from irradiated leaves and roots are presented.

Monitoring the genotoxicity of soil extracts from two heavily polluted sites in Prague using the Tradescantia stamen hair and micronucleus (MNC) assays.

We have taken soil samples from two sites in Prague, the capital of the Czech Republic, that are heavily polluted by motor vehicles. As a negative control, soil samples from a recreational site in Prague with no motor vehicle traffic were used. Soil samples from these sites were extracted with water or 5% dimethylsulphoxide (DMSO) for 24 h and cuttings of Tradescantia clone 4430 were immersed for 12 h at 25 degrees C in the extraction solutions. As a positive control Tradescantia plants have been treated with the promutagenic arylamine o-phenylenediamine at the same treatment conditions. None of the tested soil extractions significantly increased the frequency of somatic mutations in the stamen hair assay. By contrast, a 5% DMSO soil extract from one of the tested sites (entrance of the Letná tunnel) significantly increased the frequency of micronuclei (MNC) in the pollen mother tetrad cells. A repetition of the treatment 14 days later also resulted in an increase in the frequency of MNC, however the increase was not statistically significant. This study was conducted for the International Programme on Plant Bioassays.