Genomic damage induced in tobacco plants by chlorobenzoic acids--metabolic products of polychlorinated biphenyls.

Tobacco seedlings (Nicotiana tabacum var. xanthi) were treated for 24 h with mono-(2- and 3-CBA), di-(2,5- and 3,4-CBA), and tri-(2,4,6- and 2,3,5-CBA)-chlorobenzoic acids (CBAs) and with the mixture of polychlorinated biphenyls--Delor 103, or cultivated for 1 or 2 weeks in soil polluted with the CBAs. DNA damage in nuclei of leaves and roots was evaluated by the comet assay. A significant increase in DNA damage was observed only at concentrations of CBAs that caused withering of leaves or had lethal effects within 2-4 weeks after the treatments. As the application of CBAs did not induce somatic mutations, the induced DNA migration is probably caused by necrotic DNA fragmentation and not by DNA damage resulting in genetic alteration. In contrast, the application of the monofunctional alkylating agent ethyl methanesulphonate as a positive control resulted in a dose-response increase of DNA damage and an increase of somatic mutations. Thus, the EMS-produced DNA migration is probably associated with genotoxin-induced DNA fragmentation. The data demonstrate that the comet assay in plants should be conducted together with toxicity studies to distinguish between necrotic and genotoxin-induced DNA fragmentation. The content of 2,5-CBA in tobacco seedlings was measured by reverse-phase high pressure liquid chromatography.

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.

Monitoring toxicity, DNA damage, and somatic mutations in tobacco plants growing in soil heavily polluted with polychlorinated biphenyls.

Heterozygous tobacco (Nicotiana tabacum var. xanthi) plants were cultivated in soil from a dump site highly polluted with polychlorinated biphenyls (PCBs) at Lhenice in South Bohemia, Czech Republic. The total amount of PCBs in the polluted soil, measured by gas chromatography varied from 165 to 265mgkg(-1) of soil. In tobacco plants cultivated for 8 weeks in the polluted soil the amount of PCB in the leaves varied from 11 to 28 and in the roots from 104 to 308mgkg(-1) dry mass. The average leaf area of tobacco plants growing in the PCB-polluted soil was significantly reduced and the DNA damage in leaf nuclei, measured by the comet assay, was slightly but significantly increased compared with controls. The tobacco plants with increased DNA damage showed reduced growth and had distorted leaves. No increase in the frequency of somatic mutations was detected in tobacco plants growing in the PCB-polluted soil.

DNA staining with the fluorochromes EtBr, DAPI and YOYO-1 in the comet assay with tobacco plants after treatment with ethyl methanesulphonate, hyperthermia and DNase-I.

We applied the alkaline version of the single-cell gel electrophoresis (comet) assay to roots and leaves of tobacco (Nicotiana tabacum var. xanthi) seedlings or isolated leaf nuclei treated with: (1) the alkylating agent ethyl methanesulphonate, (2) necrotic heat treatments at 50 degrees C, and (3) DNase-I. All three treatments induced a dose-dependent increase in DNA migration, expressed as percentage of tail DNA. A comparison of the fluorochrome DNA dyes ethidium bromide, DAPI and YOYO-1 demonstrated that for the alkaline version of the comet assay in plants, the commonly used fluorescent dye ethidium bromide can be used with the same efficiency as DAPI or YOYO-1.

Toxicity and DNA damage in tobacco and potato plants growing on soil polluted with heavy metals.

Heterezygous tobacco (Nicotiana tabacum var. xanthi) and potato (Solanum tuberosum var. Korela) plants were cultivated on soil from the site Strimice which is highly polluted with heavy metals and on nonpolluted soil from the recreational site Jezerí, both in North Bohemia, Czech Republic. The total content, the content of bioavailable, easily mobile, and potentially mobile components of heavy metals (Cd, Cu, Pb, and Zn) in the tested soils, and the accumulation of these metals in the above-ground biomass and roots of tested plants were analyzed by flame atomic absorption spectrometry or flameless atomic absorption spectrometry. The average tobacco leaf area and potato plant height were significantly reduced in plants growing on the polluted soil. We have measured the DNA damage in nuclei of leaves of both plant species using the Comet assay. A small but significant increase in DNA damage was noted in plants growing on the polluted soil versus controls. As the tobacco and potato plants with increased DNA damage were severely injured (inhibited growth, distorted leaves), this increase may be associated with necrotic or apoptotic DNA fragmentation. No increase in the frequency of somatic mutation was detected in tobacco plants growing on the polluted soil. Thus, the polluted soil probably induced toxic but not genotoxic effects on tobacco and potato plants.

Evaluation of the nuclear DNA Diffusion Assay to detect apoptosis and necrosis.

We applied the nuclear DNA Diffusion Assay, described as an accurate tool to estimate apoptotic and necrotic cells [N.P. Singh, A simple method for accurate estimation of apoptotic cells, Exp. Cell Res. 256 (2000) 328-337] to tobacco root and leaf cells. In this assay, isolated nuclei are embedded in an agarose microgel on a microscope slide and low molecular-weight DNA fragments diffuse into the microgel. Exposure of the roots to hydrogen peroxide significantly increased the average nuclear area of isolated nuclei. After 4 and 24 h of recovery, all DNA damage was repaired. The data clearly demonstrate that the manifestation of diffused nuclei upon exposure to hydrogen peroxide is not the result of non-repairable apoptotic or necrotic DNA fragmentation, but represents repairable genotoxin-induced DNA damage. In contrast, treatment with the alkylating agent ethyl methanesulphonate (EMS) followed by 24 h of recovery produced a significant increase in the average nuclear area. The contribution of apoptosis to this increase cannot be excluded. Heat treatment of leaves at 50 degrees C for 1-15 min leading to necrosis, and treatment of isolated nuclei with DNase-I, which digests DNA to nucleosome-sized fragments as during apoptosis, also led to a dose-dependent increase in the nuclear area. The use of different fluorochromes (ethidium bromide, DAPI or YOYO-1) for DNA staining yielded similar results in the DNA Diffusion Assay. As all types and sizes of diffused nuclei were observed after EMS and hydrogen peroxide treatments, we were unable to differentiate, on the basis of the structure of the nuclei, between apoptotic or necrotic DNA fragmentation and other types of genotoxin-induced DNA damage in plants.

Cadmium induces DNA damage in tobacco roots, but no DNA damage, somatic mutations or homologous recombination in tobacco leaves.

The heavy metal cadmium (Cd(2+)) applied on tobacco roots in the form of cadmium chloride, induced significantly higher levels of DNA damage as measured by the cellular Comet assay than did treatment of isolated root nuclei, analyzed by use of the acellular Comet assay. DNA damage induced by Cd(2+) in roots of a transgenic catalase-deficient tobacco line (CAT1AS) was higher than in wild-type tobacco (SR1) roots. In contrast to treatment with the positive control ethyl methanesulphonate, Cd(2+) induced no significant DNA damage in leaf nuclei, and neither somatic mutations, nor homologous recombination as measured by the GUS genereactivation assay, were observed in leaves. Analysis of the accumulation of cadmium by inductively coupled plasma optical emission spectrometry demonstrates that roots accumulate almost 50-fold more cadmium than above-ground parts of the tobacco seedlings. This may explain the absence of Cd(2+) genotoxicity in leaves.

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.

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.

Monitoring DNA damage in wood small-reed (Calamagrostis epigejos) plants growing in a sediment reservoir with substrates from uranium mining.

For most plant species growing in polluted areas genotoxicity assays are not available. We have studied the possibility of using the alkaline protocol of the Comet assay as a method for detecting induced DNA damage in a grass Calamagrostis epigejos, growing wild in highly polluted areas. To calibrate the Comet assay for C. epigejos, two model mutagens were applied: the monofunctional alkylating agent ethyl methanesulfonate (EMS) and gamma-rays. With increasing concentrations of EMS (0 to 10 mM, 18 h treatment at 26 degrees C) applied on excised leaves, the DNA damage, as expressed by the tail moment (TM) values, increased from 4.7 +/- 0.9 to 60.8 +/- 2.7 microns. After gamma irradiation (0 to 30 Gy) the TM value increased from 4.2 +/- 0.2 to 48.1 +/- 1.7 microns. A 24 to 72 h recovery of leaves after EMS treatment in an EMS-free medium did not result in a significant change in the induced EMS damage. By contrast, a 24 h recovery after gamma-irradiation led to a complete repair of DNA damage measurable by the Comet assay. We have measured the DNA damage in nuclei of leaves of C. epigejos plants growing in the area of a sediment reservoir with substrates from uranium mining, where the ore was exploited through leaching with sulfuric acid. The average specific activity of natural radionuclides measured in the substrate was for 226Ra = 11,818 Bq kg-1, for 232Th = 66 Bq kg-1 and for 40K = 75 Bq kg-1. No significant increase in the DNA damage in plants growing on the sediment substrate above the DNA damage in control plants was detected by the Comet assay.

DNA damage induced by hydrogen peroxide in cultured tobacco cells is dependent on the cell growth stage.

The level of hydrogen peroxide (H(2)O(2))-induced genomic DNA damage measured by the Comet assay in tobacco suspension cells (TX1) increased as a function of the age of the culture. After treatment of TX1 cells with 15 mM H(2)O(2), the average (+/-S.E.) median tail moment value was only 4.85+/-1.00 microm in nuclei isolated from 2-day-old cells compared to 72.33+/-1.40 microm in nuclei isolated from 12-day-old cells. By contrast, nuclei first isolated from 2 and 12-day-old cells and then treated with H(2)O(2), expressed the same level of DNA damage. The activity of catalases was markedly higher in 2-day-old TX1 cells compared to 12-day-old cells. The results indicate that the reaction of the H(2)O(2) with nuclear DNA is modified by the presence of the plant cell wall, and enzymes and macromolecules present in the cytosol, and is not connected with changes in the nuclear DNA sensitivity during cell suspension growth.