DNA Damage In Nuclei Research Articles

The role of catalytic and regulatory domains of human PrimPol in DNA binding and synthesis.

Human PrimPol possesses DNA primase and DNA polymerase activities and restarts stalled replication forks protecting cells against DNA damage in nuclei and mitochondria. The zinc-binding motif (ZnFn) of the C-terminal domain (CTD) of PrimPol is required for DNA primase activity but the mechanism is not clear. In this work, we biochemically demonstrate that PrimPol initiates de novo DNA synthesis in cis-orientation, when the N-terminal catalytic domain (NTD) and the CTD of the same molecule cooperate for substrates binding and catalysis. The modeling studies revealed that PrimPol uses a similar mode of initiating NTP coordination as the human primase. The ZnFn motif residue Arg417 is required for binding the 5'-triphosphate group that stabilizes the PrimPol complex with a DNA template-primer. We found that the NTD alone is able to initiate DNA synthesis, and the CTD stimulates the primase activity of NTD. The regulatory role of the RPA-binding motif in the modulation of PrimPol binding to DNA is also demonstrated.

Linifanib induces apoptosis in human ovarian cancer cells via activation of FOXO3 and reactive oxygen species

Linifanib is known as an inhibitor of receptor tyrosine kinase. Even though it has been widely recognized as efficient inhibitor of receptor tyrosine kinases, anti-carcinogenic effect has not been investigated enough in ovarian cancer. In this study, we investigated the anti-cancer effect of linifanib on human ovary cancer SKOV3 cells. WST-1, cell counting assay, and observation of morphological changes were performed to evaluate the cytotoxic effect of linifanib in SKOV3 cells. We analyzed SKOV3 cells treated with linifanib using Muse cell analyzer. We focused on investigating the effect of linifanib on DNA damage in nucleus. Additionally, intracellular reactive oxygen species (ROS) level was measured through Muse cell analyzer. Western blotting was performed to evaluate the protein expression level related to apoptosis. We found that linifanib inhibited proliferation of SKOV3 cells. Our results showed that linifanib induced apoptosis in SKOV3 cells. Additionally, linifanib induced DNA damage in SKOV3 cells. We found that intracellular ROS level increased after treatment of linifanib in SKOV3 cells. Interestingly, FOXO3 was transferred from cytosol into nucleus after linifanib treatment. Taken together, our results supported that linifanib inhibited the proliferation of human ovary cancer SKOV3 cells, which suggested that linifanib might have the potential to be developed as drugs for ovarian cancer treatment.

Subcellular-Targeted Near-Infrared-Responsive Nanomedicine with Synergistic Chemo-photothermal Therapy against Multidrug Resistant Cancer.

Multidrug resistance (MDR) is a major obstacle to effective cancer treatment. Therefore, developing effective approaches for overcoming the limitation of MDR in cancer therapy is very essential. Chemotherapy combined with photothermal therapy (PTT) is a potential therapeutic option against MDR. Herein, we developed a subcellular-targeted near-infrared (NIR)-responsive nanomedicine (Fe3O4@PDA-TPP/S2-PEG-hyd-DOX, abbreviated as Fe3O4-ATSPD) as a new photothermal agent with improved photothermal stability and efficiency. This system demonstrates high stability in blood circulation and can be accumulated at the tumor site by magnetic targeting enhanced permeability and retention effect (EPR). Near-infrared (NIR) irradiation at the tumor site generates a photothermal effect from the photosensitizer Fe3O4@PDA, leading to a dramatic decrease in mitochondrial membrane potential. Simultaneously, the conjugated drugs released under low pH condition in endosomes or lysosomes cause nucleus DNA damage and cell apoptosis. This subcellular-targeted NIR-responsive nanomedicine with efficient integration of diagnosis and therapy could significantly enhance MDR cancer treatment by combination of chemotherapy and PTT.

Ionizing Radiation Upregulates Glutamine Metabolism and Induces Cell Death via Accumulation of Reactive Oxygen Species.

Glutamine metabolism provides energy to tumor cells and also produces reactive oxygen species (ROS). Excessive accumulation of ROS can damage mitochondria and eventually lead to cell death. xCT (SLC7A11) is responsible for the synthesis of glutathione in order to neutralize ROS. In addition, mitophagy can remove damaged mitochondria to keep the cell alive. Ionizing radiation kills tumor cells by causing the accumulation of ROS, which subsequently induces nuclear DNA damage. With this in mind, we explored the mechanism of intracellular ROS accumulation induced by ionizing radiation and hypothesized new methods to enhance the effect of radiotherapy. We used MCF-7 breast cancer cells and HCT116 colorectal cancer cells in our study. The above-mentioned cells were irradiated with different doses of X-rays or carbon ions. Clone formation assays were used to detect cell proliferation, enzyme-linked immunosorbent assay (ELISA) detected ATP, and glutathione (GSH) production, while the expression of proteins was detected by Western blot and quantitative real-time PCR analysis. The production of ROS was detected by flow cytometry, and immunofluorescence was used to track mitophagy-related processes. Finally, BALB/C tumor-bearing nude mice were irradiated with X-rays in order to further explore the protein expression found in tumors with the use of immunohistochemistry. Ionizing radiation increased the protein expressions of ASCT2, GLS, and GLUD in order to upregulate the glutamine metabolic flux in tumor cells. This caused an increase in ATP secretion. Meanwhile, ionizing radiation inhibited the expression of the xCT (SLC7A11) protein and reduced the generation of glutathione, leading to excessive accumulation of intracellular ROS. The mitophagy inhibitor, or knockdown Parkin gene, is able to enhance the ionizing radiation-induced ROS production and increase nucleus DNA damage. This combined treatment can significantly improve the killing effect of radiation on tumor cells. We concluded that ionizing radiation could upregulate the glutamine metabolic flux and enhance ROS accumulation in mitochondria. Ionizing radiation also decreased the SLC7A11 expression, resulting in reduced GSH generation. Therefore, inhibition of mitophagy can increase ionizing radiation-induced cell death.

Mesenchymal Stem Cells Early Response to Low-Dose Ionizing Radiation

IntroductionMesenchymal stem cells (MSCs) are applied as the therapeutic agents, e.g., in the tumor radiation therapy.Purpose of the StudyTo evaluate the human adipose MSC early response to low-dose ionizing radiation (LDIR).Materials and MethodsWe investigated different LDIR (3, 10, and 50 cGy) effects on reactive oxygen species production, DNA oxidation (marker 8-oxodG), and DNA breaks (marker ɣ H2AX) in the two lines of human adipose MSC. Using reverse transcriptase–polymerase chain reaction, fluorescence-activated cell sorting, and fluorescence microscopy, we determined expression of genes involved in the oxidative stress development (NOX4), antioxidative response (NRF2), antiapoptotic and proapoptotic response (BCL2, BCL2A1, BCL2L1, BIRC2, BIRC3, and BAX1), in the development of the nuclear DNA damage response (DDR) (BRCA1, BRCA2, ATM, and P53). Cell cycle changes were investigated by genes transcription changes (CCND1, CDKN2A, and CDKN1A) and using proliferation markers KI-67 and proliferating cell nuclear antigen (PCNA).ResultsFifteen to 120 min after exposure to LDIR in MSCs, transient oxidative stress and apoptosis of the most damaged cells against the background of the cell cycle arrest were induced. Simultaneously, DDR and an antiapoptotic response were found in other cells of the population. The 10-cGy dose causes the strongest and fastest DDR following cell nuclei DNA damage. The 3-cGy dose induces a less noticeable and prolonged response. The maximal low range dose, 50 cGy, causes a damaging effect on the MSCs.ConclusionTransient oxidative stress and the death of a small fraction of the damaged cells are essential components of the MSC population response to LDIR along with the development of DDR and antiapoptotic response. A scheme describing the early MSC response to LDIR is proposed.

Nuclei DNA Damage Due to Extreme High-Temperature Exposure during Forensic Identification Examination

ccurate personal identification is important in investigations because an error in the identification process may bring fatal consequences during trial. The most common identification process is the Deoxyribonucleic acid [DNA] analysis. Degraded DNA sample due to extremely high-temperature exposure may limit DNA analysis. This study aimed to analyze DNA damage patterns caused by an extremely high temperature using STR (short tandem repeat) CODIS marker. This study was conducted at the Forensic and Medicolegal Department, Laboratorium Balai Besar Kesehatan Surabaya, Ministry of Health of the Republic of Indonesia, Human Genetic Study Group of Universitas Airlangga, and Faculty of Science and Technology of Universitas Brawijaya Malang from July until October 2009. Results of PCR visualization using STR CODIS for costae demonstrated that the THO1 detection (+) in 1,2500C - 40’: 25% and the TPOX detection (+) in 1,0000C - 30’: 50% whereas the results from molar teeth showed that the THOI locus detection (+) in 1,2500C - 30’: 25% and TPOX in 1,0000C - 40’: 50%. Results for PCR visualization using mini-STR CODIS for the costae presented that the mini-THOI in 1,2500C - 20’: 50% (+) while for the molar tooth the mini-THOI in 1,2500C - 30’ : 25% (+) and mini-TPOX in 1,0000C - 40’ : 50% (+). All loci were detected on costae and second molar teeth samples of the control group. Thus, extreme high-temperature exposure significantly decreased the DNA level of second costae and second molar tooth. Sequence patterns of STR loci successfully detected were TPOX, THO1, and CSF1PO.

Chemopreventive Effect of β-Cryptoxanthin on Human Cervical Carcinoma (HeLa) Cells Is Modulated through Oxidative Stress-Induced Apoptosis.

The present study was aimed to assess cellular and molecular events involved in the chemopreventive activities of β-cryptoxanthin derived from mandarin oranges (Citrus unshiu Marc.) on human cervical carcinoma (HeLa) cells. In vitro experiments established that β-cryptoxanthin significantly inhibited the proliferation of HeLa cells with the IC50 value of 4.5 and 3.7 µM after 24 and 48 h of treatments, respectively. β-cryptoxanthin-treated HeLa cells exhibited enhanced levels of oxidative stress correlated with significant downregulation of anti-apoptotic Bcl-2, and upregulation of pro-apoptotic Bax mRNA expression. Moreover, β-cryptoxanthin triggered nuclear condensation and disruption of the integrity of the mitochondrial membrane, upregulated caspase-3, -7, and -9 mRNA, and enhanced activation of caspase-3 proteins, resulting in nuclei DNA damage and apoptosis of HeLa cells. Remarkably, TUNEL assay carried out to detect nuclei DNA damage showed 52% TUNEL-positive cells after treatment with a physiological concentration of β-cryptoxanthin (1.0 μM), which validates its potential as an anticancer drug of natural origin.

Construction of Nucleus-Targeting Iridium Nanocrystals for Photonic Hyperthermia-Synergized Cancer Radiotherapy.

Prominent tumor-cell nucleus targeting of radiosensitizer substantially affects the therapeutic consequence of advanced tumor radiotherapy via lethal nucleus DNA damage. Herein, ultrasmall iridium nanocrystals (Ir NCs, <5 nm) are constructed for efficient tumor-specific photonic hyperthermia-synergized radiotherapy. To endow the NCs with qualified cell nucleus-targeting performance, polyethylene glycol (PEG)-modified Ir NCs are decorated with αv β3 integrin-targeting cyclic arginine-glycine-aspartic (c(RGDyC)), designated as RGD, peptides and human immunodeficiency virus-1 transactivator of transcription protein(TAT), respectively, facilitating the tumor-cell-membrane (with overexpressed αv β3 integrin) and cell-nucleus targeting. The formulated Ir-RGD-TAT (Ir-R/T) NCs are demonstrated to accumulate inside the nucleus of tumor cells and generate effective DNA lesions upon X-ray irradiation. Further in vivo evaluations verify the satisfactory carcinoma destruction performance against 4T1 tumor xenografts. Importantly, the intriguing photonic NIR adsorption of Ir-R/T NCs has enabled the hyperthermia therapeutics accompanied with photoacoustic imaging modalities, achieving clinically promising biocompatible multifunctional radiosensitized nanoplatforms for effective tumor therapeutics.

Hoechst-naphthalimide dyad with dual emissions as specific and ratiometric sensor for nucleus DNA damage

A ratiometric fluorescent sensor (Hoe-NI) was developed by connecting a nucleus targeted Hoechst unit to a naphthalimide dye via “click chemistry”. The sensor achieves high specific nucleus labeling with wash-free staining method in various kinds of living cells. The fluorescence ratio of the two emission bands (450nm for Hoechst and 505nm for naphthalimide) is changed sensitively to the variation of DNA concentrations, which provides the quantitative information in the processes of DNA damage induced by hydroxyl radicals and antitumor drug. Therefore, Hoe-NI is a recommendable sensor for the monitoring of nuclear DNA damage that reveals the health status of cells.

DNA replication after mutagenic treatment in Hordeum vulgare

The temporal and spatial properties of DNA replication in plants related to DNA damage and mutagenesis is poorly understood. Experiments were carried out to explore the relationships between DNA replication, chromatin structure and DNA damage in nuclei from barley root tips. We quantitavely analysed the topological organisation of replication foci using pulse EdU labelling during the S phase and its relationship with the DNA damage induced by mutagenic treatment with maleic hydrazide (MH), nitroso-N-methyl-urea (MNU) and gamma ray. Treatment with mutagens did not change the characteristic S-phase patterns in the nuclei; however, the frequencies of the S-phase-labelled cells after treatment differed from those observed in the control cells. The analyses of DNA replication in barley nuclei were extended to the micronuclei induced by mutagens. Replication in the chromatin of the micronuclei was rare. The results of simultanous TUNEL reaction to identify cells with DNA strand breaks and the labelling of the S-phase cells with EdU revealed the possibility of DNA replication occurring in damaged nuclei. For the first time, the intensity of EdU fluorescence to study the rate of DNA replication was analysed.

Potato virus X induces DNA damage in leaf nuclei of the host plant Nicotiana tabacum L. var. xanthi

We employed the comet assay (single cell gel electrophoresis) to evaluate induced DNA damage in nuclei isolated from tobacco leaves (Nicotiana tabacum var. xanthi) inoculated with Potato virus X (PVX). The highest DNA damage, expressed by the tail moment value, was observed in the inoculated leaves and decreased in the 1st to 4th systemic leaves. DNA damage increased with the time after the inoculation (from day 3 to day 21) and was higher in nuclei isolated from a part of the leaf at the petiole compared to nuclei isolated from the leaf tip. A Pearson moment correlation (r = 0.94) between the induced DNA damage and the PVX titres expressed by ELISA absorbance values was observed. The PVX infection did not induce a significant increase in the rate of somatic mutations evaluated by appearance of dark green, yellow, and double green/yellow sectors on the heterozygous pale green leaves of N. tabacum var. xanthi.

Copper-induced oxidative damage, antioxidant response and genotoxicity in Lycopersicum esculentum Mill. and Cucumis sativus L.

Adequate copper (Cu(2+)) concentrations are required for plants; however, at higher concentrations it can also cause multiple toxic effects. In the present study, lipid peroxidation, hydrogen peroxide levels as well as ascorbate peroxidase (APX: EC 1/11/1/11) and catalase (CAT: EC 1.11.1.6) activities were determined in Lycopersicum esculentum Mill. and Cucumis sativus L. seedlings after 7-day exposure to copper sulfate. In addition, DNA damage in these two crops was assessed by measuring micronucleus (MN) frequency and tail moments (TM) as determined by Comet assay. Inhibitory copper concentrations (EC(50): 30 and 5.5ppm for L. esculentum and C. sativus, respectively) were determined according to dose-dependent root inhibition curves, and EC(50) and 2×EC(50) were applied. Malondialdehyde (MDA) and H(2)O(2) levels significantly increased in all groups studied. CAT activity increased in treatment groups of C. sativus. APX activity increased in L. esculentum seedlings due to 2×EC(50) treatment. Reductions in mitotic indices (MI) represented Cu(2+)dependent root growth inhibition in all treatment groups studied. According to TMs and MN frequencies, copper exposure induced significant DNA damage (p<0.05) in all study groups, whereas the DNA damage induced was dose dependent in C. sativus roots. In conclusion, Cu(2+)induced oxidative damage, elevations in H(2)O(2) levels and alterations in APX and CAT activities, as well as significant DNA damage in nuclei of both study groups. To our knowledge, this is the first comparative and comprehensive study demonstrating the effects of copper on two different plant species at relevant cytotoxic concentrations at both biochemical and genotoxicity levels with multiple end points.

Assessing the genotoxicity of urban air pollutants using two in situ plant bioassays

Genotoxicity of urban air has been analysed almost exclusively in airborne particulates. We monitored the genotoxic effects of airborne pollutants in the urban air of Perugia (Central Italy). Two plant bioindicators with different genetic endpoints were used: micronuclei in meiotic pollen mother cells using Tradescantia-micronucleus bioassay (Trad-MCN) and DNA damage in nuclei of Nicotiana tabacum leaves using comet assay ( Nicotiana-comet). Buds of Tradescantia clone # 4430 and young N. tabacum cv. Xanthi plants were exposed for 24 h at three sites with different pollution levels. One control site (indoor control) was also used. The two bioassays showed different sensitivities toward urban pollutants: Trad-MCN assay was the most sensitive, but DNA damage in N. tabacum showed a better correlation with the pollutant concentrations. In situ biomonitoring of airborne genotoxins using higher plants combined with chemical analysis is thus recommended for characterizing genotoxicity of urban air.

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 in the root cells of Allium cepa seeds growing in soil of high background radiation areas of Ramsar – Iran

Plants are unique in their ability to serve as in situ monitors for environmental genotoxins. We have used the alkaline comet assay for detecting induced DNA damage in Allium cepa to estimate the impact of high levels of natural radiation in the soils of inhabited zones of Ramsar. The average specific activity of natural radionuclides measured in the soil samples for 226Ra was 12,766 Bq kg −1 whereas in the control soils was in the range of 34–60 Bq kg −1. A positive strong significant correlation of the DNA damage in nuclei of the root cells of A. cepa seeds germinated in the soil of high background radiation areas with 226Ra specific activity of the soil samples was observed. The results showed high genotoxicity of radioactively contaminated soils. Also the linear increase in the DNA damage indicates that activation of repair enzymes is not triggered by exposure to radiation in HBRA.

The Expression of Bcl-XL, Bcl-XS and p27Kip1 in Topotecan-Induced Apoptosis in Hepatoblastoma HepG2 Cell Line

Background: To assess the efficacy of topotecan, a topoisomerase I specific inhibitor in S-phase, the reagent-induced apoptosis and cytotoxicity as well as related proteins expression, had been preliminarily investigated in human hepatoblastoma HepG2 cells. Methods: Microculture tetrazolium assay (MTT), HE staining, transmission electron microscopy (TEM), flow cytometry (FCM), quantitative immunocytochemistry (QI), gene tranfection and RNAi technology were employed to carry out the exploration. Results: Topotecan could potently kill HepG2 cells via inducing apoptosis and demonstrated strong cytotoxicity in a time, dose-dependent manner with IC50 of about 95 μ g/L. According to morphologic observation and FCM analyses, it was confirmed that the drug treatment, causing significant S-phase arrest, could trigger a typical interphase apoptosis, the main traits of which were identified as chromatin pycnosis and cytoplasm condensation. It was shown that the expression of Bcl-XL was simultaneously down-regulated with the up-regulation of Bcl-XS in cytoplasm, which was possibly a key downstream event following the topotecan-induced DNA damage in nucleus. The expression level of p27Kip1, a negative regulator in cell cycle at G1/S transient, was also elevated. Transfection of pcDNA 3.1-p27Kip1 into HepG2 cells could abrogate the cytotoxicity in a degree while silence of p27Kip1 with siRNA in drug treatments could significantly increased the chemosensitivity, strongly indicating that the up-regulation of p27Kip1 was not an apoptosis-promoting, but a self-rescue response against drug by moderate G0/G1 arrest. Conclusion: Topotecan had potent cytotoxicity against HepG2 cells by triggering an interphase apoptosis possibly mediated by increasing the ratio of Bcl-XS/Bcl-XL. Up-regulation of p27Kip1in TPT treatments could be a protective response for self-rescue and silence of the gene markedly augmented TPT cytotoxicity. Therefore, the experiment in vitro could provide a new idea for the clinical chemotherapy based on the combination of traditional drugs with the specific-siRNA targeted on the protective response gene.

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 Střimice which is highly polluted with heavy metals and on nonpolluted soil from the recreational site Jezeří, 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.

Comet 분석을 통한 방사선처리 고추세포의 핵 DNA 손상평가

방사선에 노출된 고추유묘의 잎으로부터 세포핵을 분리하여 단세포전기영동방법인 comet 분석을 통하여 핵 DNA의 손상정도를 조사하였다. Comet 분석에서 꼬리부분의 길이 (T)와 머리부분의 길이 (H)를 측정하여 T/H 비율을 조사하였다. 무처리세포는 T/H 비율이 1.28이었으나 50 및 100 Gy의 방사선을 처리한 세포는 각각 3.54 및 3.39로 방사선처리에 의해 상당량의 핵 DNA가 손상을 입은 것으로 나타났다. Comet의 head-DNA량은 무처리가 76.8%였으나 50 및 100 Gy를 처리한 세포는 각각 55.9% 및 59.5%를 보였다. 고선량의 방사선을 처리하기 전에 미리 20 Gy 이하의 저선량 방사선을 종자에 전처리하였을 경우 종자의 발아 및 생장에 대한 영향은 없었지만, 후속 고선량에 대한 핵 DNA의 손상은 경감되는 경향을 보였다. We employed single cell gel electrophoresis method (comet assay) to analyze the degree of nucleus-DNA damage in the leaves of red pepper (Capsicum annuum L.) seedlings exposed to <TEX>$^{60}$</TEX> CO v-radiation stress. Nucleus-DNA damage was measured as the ratio of tail length (T) to head length (H) in individual comet image isolated from pepper leaf cell. The T/H ratio of control-cells and treated-cells at 50 or 100 Gy were 1.28 and 3.54 or 3.39, respectively, suggesting that nuclei of pepper cells were severely damaged in the integrity of DNA strand by the treatment of enhanced v-radiation. The percentage of head-DNA in control-cells was 76.8%, whereas those of 50 and 100 Gy treated-cells were 55.9% and 59.9%, respectively. Pretreatment of low dose (4 to 20 Gy) radiation to seeds decreased DNA-damage in the leaves of seedlings treated with high dose radiation at 50 or 100 Gy. In this experiment, we developed a sensitive, reliable and rapid method for evaluating genotoxic effect in the nuclei of plant cells by employing comet assay.

Superoxide dismutase의 활성차이에 따른 식물세포의 paraquat에 대한 반응과 핵 DNA 손상 검정

Superoxide dismutase의 활성차이에 따른 식물세포의 paraquat에 대한 반응과 핵 DNA 손상 검정

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.