Electronic Library of
Volume 46 / Supplement / 1999
MAY 29 - JUNE 2, 1999
Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen AB21 9SB
NEOPLASMA, 46, Supplement, 1999, pp. 3-5
G. Frenzilli, V. Scarcelli, F. Taddei, M. Nigro
Dipartimento di Morfologia Umana e Biologia Applicata, Universita di Pisa, via Volta 4, 56100, Pisa, Italy
The aim of this work was to develop a protocol of comet assay to be used on aquatic sentinel species. For this purpose fish erythrocytes (Gobidae) and two different tissue cell types from mussels (haemolymph cells and gill cells from Mytilus galloprovincialis) were used. The comet assay was carried out in two different versions: a) mild alkaline at pH=12.1, to detect DNA single strand breaks (ssb); and b) alkaline at pH>13, able also to evaluate alkali-labile sites. While fish erythrocytes showed a statistically higher amount of DNA damage when the assay was carried out at pH>13 compared to pH=12.1, no difference in DNA migration was found between slides obtained at pH=12.1 and at pH>13 in mussel haemocytes and gill cells. Two different voltage protocols (low voltage and longer times; high voltage and shorter times) were also looked at. The second one was chosen for biomonitoring Orbetello Lagoon. Spatial and temporal variability of DNA damage were investigated in 6 sampling sites characterised by different degrees of ecological disturbance.
Key words: Fish erythrocytes, haemolymph cells, gill cells, comet assay
NEOPLASMA, 46, Supplement, 1999, pp. 6-7
Laboratoire des Sciences de l’Environnement, ENTPE, rue Maurice Audin, 69518 Vaulx-en-Velin, France
Equipe d’Ecotoxicologie Aquatique, INRA-SCRIBE, Campus de Beaulieu, Rennes, France
NEOPLASMA, 46, Supplement, 1999, pp. 8
Department of Environment, Technology and Social Studies, Roskilde University, P.O. Box 260, DK-4000 Roskilde, Denmark. E-mail: firstname.lastname@example.org
Monitoring of DNA damage was carried out on mussels sampled from coastal waters affected by genotoxic waste water. The comet assay was used on haemolymph cells and on gill cells. The preliminary results showed that the level of DNA tail moments was relatively high (average: 2.77; range: 1.56-5.38) but without any significant difference between the four sample sites. One of the main conclusions is that more knowledge is needed of the level of DNA damage in the wild living mussels before they can be used for monitoring of genotoxic effects in the marine environment.
Key words: comet assay, waste water, Blue Mussel
NEOPLASMA, 46, Supplement, 1999, pp. 9-10
T. Ollikainen, K. Linnainmaa, V.L. Kinnula
Finnish Institute of Occupational Health, Helsinki, Finland
Department of Internal Medicine, University of Oulu, Finland
Transformed human pleural mesothelial cells (MeT-5A) were exposed to 1-4 microg/cm2 crocidolite asbestos fibers, to 10-100 ng/ml tumor necrosis factor alpha (TNF-alpha) for up to 48 h, and studied for the induction of DNA damage using the comet assay. Significant but reversible increases in the mean tail moments were observed, but not in the distribution of comet tails in the histograms in the cells exposed to 1 microg/cm2 crocidolite for 6 h. At higher concentrations of asbestos fibers all the indices in the Comet assay showed significant and irreversible change. All the doses of TNF-alpha caused marginal increase in the mean tail moments. The mean tail moments were highest in the cells with concurrent treatment with TNF-alpha and crocidolite. In the cells pretreated with inhibitors of antioxidant enzymes (aminotriazole for catalase and buthionine sulfoximine for gamma-glutamylcysteine synthetase) asbestos fibers slightly increased oxidant-related fluorescence of dichlorofluorescein (DCFH) but did not cause any further increases in the mean tail moments. This study shows that asbestos fibers cause DNA single strand breaks in human mesothelial cells. Since the inhibition of antioxidant enzymes did not have an effect on the DNA damage caused by fibers, other mechanisms than reactive oxygen metabolites may be involved in the induction of DNA damage by mineral fibers.
Key words: Asbestos fibers, single strand breaks, human mesothelial cells.
NEOPLASMA, 46, Supplement, 1999, pp. 11-12
A. Trzeciak, J. Kowalik, M. Wojewódzka, J. Błasiak
Department of Molecular Genetics, University of Łódź, Banacha 12/16, 90-237 Łódź,
Department of Radiobiology and Health Protection, Institute of Nuclear Chemistry and Technology, Dorodna 16, 03-195 Warsaw, Poland
Hexavalent chromium compounds are well-recognized carcinogens. Chromium is present in some workplaces as well as in water resources and the food chain, so it can interact with DNA of the mucosa of the gastrointestinal tract. In order to elucidate the genotoxic potency of chromium in human gastric mucosa (GM) cells, the DNA-damaging effect of potassium dichromate (K2Cr2O7) was investigated using alkaline single cell gel electrophoresis. A parallel test with human peripheral blood lymphocytes was also performed. Both types of cells were incubated at 37°C with 500 microM of K2Cr2O7 for 1 h and, after washing, were placed in a chromium-free medium to examine DNA repair. Chromium introduced damage to DNA in both GM cells and lymphocytes. The effect induced by K2Cr2O7 in GM cells was comparable with that in lymphocytes. Treated cells were able to recover within a 60-min incubation in chromium-free medium at 37°C. These results indicate that hexavalent chromium compounds, which may be found in the diet, can interact directly with DNA of the mucosa of the stomach, so the single cell electrophoresis is a useful technique to assess chemicals for genotoxicity activities in tumor target tissues.
Key words: Chromium genotoxicity, comet assay, DNA damage, DNA repair, human lymphocytes, human gastric mucosa cells
NEOPLASMA, 46, Supplement, 1999, pp. 13-15
H. Petrovská, M. Somorovská, B. Vallová, M. Dušinská
Department of Molecular and Cellular Toxicology, Institute of Preventive and Clinical Medicine, Limbová 14, 833 01 Bratislava, Slovak Republic
The alkaline comet assay modified with endonuclease III was used to investigate the potential mutagenic effects of air samples from 12 localities in Slovakia during the course of a year. As target cells, freshly isolated human peripheral lymphocytes and transformed human liver HepG2 cells were used. DNA strand breaks and oxidised pyrimidines with AP sites were measured after the treatment with different concentrations of extracts from the 48 air samples. 32 samples were used to test organic compounds present in air samples (dichloromethane extracts), and 16 samples for evaluation of aqueous extracts from filters. Preliminary analyses show that 2/3 of samples extracted by dichloromethane induced higher DNA damage in HepG2 cells and almost 1/3 in lymphocytes. 3/4 of aqueous extracts induced a strong positive effect in HepG2 cells, none of them in lymphocytes. Comparing preliminary results from all localities and from different samplings during the course of the year, we found winter samples to be more mutagenic than summer samples.
Key words: Comet assay, air pollution, primary human lymphocytes, transformed human liver HepG2 cells, oxidative DNA damage, genotoxicity testing
NEOPLASMA, 46, Supplement, 1999, pp. 16-17
Purkynje Military Medical Academy, P.O. BOX 35/T, 500 01 Hradec Králové, Czech Republic
A special pure diet containing a low amount of vitamins and 2% of cholesterol increased the level of DNA damage in male rat lymphocytes and aortal endothelium cells two times in comparison with controls, as measured by the modified comet assay using endonuclease III for the detection of oxidised bases. The vitamin mixture added to the cholesterol diet reduced this increase of DNA damage in a dose-dependent manner. Oxidative damage to the endothelial cell DNA induced probably by the increased plasma cholesterol level might play an important role in the initiation of atherogenesis and can be reduced by food antioxidants.
Key words: rat lymphocytes, aortal endothelium cells, vitamin mixture, cholesterol diet
NEOPLASMA, 46, Supplement, 1999, pp. 18-19
P. Jałoszyński, M. Tądrowska, M. Kujawski, M. Wąsowicz, R. Szulc, K. Szyfter
Institute of Human Genetics, Polish Academy of Sciences, Strzeszyńska 32, 60-479
1st Department of Anesthesiology and Intensive Therapy, K. Marcinkowski University of Medical Sciences, Długa 1/2, 61-848 Poznań, Poland
In previous studies, we described genotoxic properties of two inhalation anesthetics -
halothane and isoflurane. Genotoxicity was tested in vitro in human peripheral
blood lymphocytes by alkaline comet assay to assess anesthetic-induced DNA damage. We
found that halothane was genotoxic at a concentration as low as 0.1 mM and cytotoxic at 1
mM whereas the genotoxic effect of isoflurane was detectable at 1 mM without apparent
cytotoxicity. In the present study the above drugs were used as a control to estimate the
genotoxicity of sevoflurane, a recently introduced modern anesthetic. Sevoflurane was
tested using a similar in vitro protocol at concentrations 1 and 10 mM. Under
applied experimental conditions no significant effect of sevoflurane was detected whereas
halothane and isoflurane increased DNA migration in a way consistent with our former
results. We conclude that sevoflurane does not induce comet assay-detectable DNA damage,
at least at studied concentrations and under applied conditions.
Further, in order to study genotoxic effects of anesthetics in vivo, blood samples of operating room personnel (29 persons) were analysed. The base level of DNA damage measured with the alkaline comet assay in peripheral blood lymphocytes did not differ from controls (20 persons without contact with anesthetic drugs). The lack of statistically significant differences in base level of DNA damage between the exposed and control group suggests that a chronic exposure to anesthetic gases is not followed by genotoxicity, assuming that the comet assay is a proper technique to monitor such long-term, low-level exposures.
Key words: Comet assay, genotoxicity in vitro and in vivo, inhalation anesthetics, sevoflurane
NEOPLASMA, 46, Supplement, 1999, pp. 20-22
M. Somorovská, J.Tulinská, M. Barančoková, M. Zámečníková, A. Collins, A. Líšková, B. Vallová, H. Petrovská, E. Jáhnová , P. Vodička, L. Fuortes, M. Dušinská
Institute of Preventive and Clinical Medicine, Limbová 14, 833 01 Bratislava, Slovak
National Institute of Public Health, Bratislava, Slovakia
Rowett Research Institute, Aberdeen AB21 9SB, UK
Institute of Experimental Medicine, Czech Academy of Sciences, Vídeňská 1083, 14200 Prague 4, Czech Republic
University of Iowa, Department of Preventive Medicine and Environmental Health, Iowa City, USA
The alkaline comet assay was used in parallel with chromosome aberrations as well as
immunological endpoints to assess the effect of exposure in two factories in Slovakia - a
rubber factory and a plastic lamination plant. We examined 29 exposed workers from
the rubber factory exposed to a complex mixture of contaminants belonging mostly to
polycyclic aromatic hydrocarbons, and two control groups (22 subjects each). In the
lamination plant, styrene was the only contaminant at the workplace. Monitoring in this
plant consisted of 17 hand-laminators directly exposed to styrene, 10 medium exposed
sprayers and 18 controls.
In both factories exposed workers had significantly elevated levels of DNA breaks compared with controls (p<0.001). In both studies we found a correlation between strand breaks in lymphocytes and years of exposure (p<0.001, r=0.57, p<0.001, r=0.545 resp.). Cytogenetic results also showed significantly higher numbers of chromosome aberrations in both exposed populations. We observed a clear correlation of chromosome aberrations with DNA strand breaks.
Immune parameters were also changed in workers exposed to both the single contaminant (styrene) and the complex mixture.
Key words: Biomonitoring, comet assay, chromosomal aberrations, micronuclei, immune parameters
NEOPLASMA, 46, Supplement, 1999, pp. 23-25
G. Russev, I. Mikhailov, B. Anachkova
Institute of Molecular Biology, Bulgarian Academy of Sciences Acad. G. Bonchev Street, Bl. 21, 1113 Sofia, Bulgaria
Comet assay was used to monitor the appearance and accumulation of DNA breaks in mammalian cells treated with mimosine. The results showed that the rate of break accumulation was the same in exponentially growing mouse erythroleukemia cells and in quiescent human peripheral blood lymphocytes. It did not depend on the stage of the cell cycle and was not connected with the mechanism of DNA replication. Mimosine caused a slow-down of DNA replication, changes in the progression of the cells through the cell cycle and apoptosis. The conclusion was drawn that these effects of mimosine on DNA synthesis and the cell cycle were a result of the introduction of breaks into DNA.
Key words: Mimosine, DNA synthesis, DNA breaks, mouse erythroleukemia cells
NEOPLASMA, 46, Supplement, 1999, pp. 26-27
M. De Boeck, A. Hartwig, D. Lison, M. Kirsch-Volders
Free University of Brussels (VUB), Laboratory of Cell Genetics, Pleinlaan 2, 1050
University of Karlsruhe, Department of Food Chemistry, 76128 Karlsruhe, Germany
Université catholique de Louvain, Unité de Toxicologie Industrielle et Medécine du Travail, Clos Chapelle-aux-Champs, 1200 Brussels, Belgium
NEOPLASMA, 46, Supplement, 1999, pp. 28
UMR IC/CNRS 218, Institut Curie, Section de Recherche, 26 rue d’Ulm, 75248 Paris Cedex 05, France
The kinetics of rejoining of DNA single- and double-strand breaks were measured by the comet assay and the graded-field gel electrophoresis technique in DNA ligase-deficient cells and in their normal counterparts after exposure to UV-C and gamma radiation. Human 46BR cells were deficient in DNA ligase I. Hamster EM9 and EM-C11 cells were deficient in DNA ligase III as a consequence of mutations in the XRCC1 gene. Hamster XR-1 cells had mutation in the XRCC4 gene, whose product stimulates DNA ligase IV activity. Results obtained on the DNA rejoining capabilities of these cell lines with respect to DNA breaks processed in different repair pathways support the conclusion that i) DNA ligase I is involved only in nucleotide excision repair; ii) DNA ligase IV plays an important role only in DNA double-strand break repair and iii) DNA ligase III is implicated in base excision repair and in DNA double-strand break repair, but probably not in nucleotide excision repair.
Key words: DNA strand break rejoining, graded-field electrophoresis, normal and DNA ligase-deficient cells
NEOPLASMA, 46, Supplement, 1999, pp. 29-30
D. Renault, D. Brault, Y. Lossouarn, F. Périn , V. Thybaud
Rhône-Poulenc Rorer, Nonclinical Safety Assessment, Vitry sur Seine, France
Institut Curie-Biologie, Centre Universitaire, Orsay, France
NEOPLASMA, 46, Supplement, 1999, pp. 31
M. Kruszewski, M. Wojewódzka, T. Iwaneńko, A. Okuyama, T. Żebrowska, N. Jarocewicz, I. Szumiel
Department of Radiobiology and Health Protection, Institute of Nuclear Chemistry and
Technology, 03-195 Warsaw, Poland
Department of Experimental Hematology and Cord Blood Bank, M.Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, W.K. Roentgena 5, 02-781 Warszawa, Poland
Banyu Tsukuba Research Institute, 3 Okubo, Tsukuba 300-26, Japan
DNA damage repair was evaluated in two mouse lymphoma L5178Y (LY) cell lines differing in sensitivity to ionizing radiation. The cells were treated with OK-1035, a potent DNA-dependent protein kinase (DNA-PK) inhibitor, and irradiated (8 Gy). The level of initial damage was similar in both LY cell lines. In L5178Y cells the repair of 8 Gy-induced DNA damage proceeded identically in the inhibitor treated and untreated cells. In contrast, the level of residual damage in OK-1035 treated LY-R cells was considerably higher than in untreated cells. The inhibitor affected LY-R cells preferentially in G1 and S phases, in agreement with cell-cycle specificity of DNA-PK. The results may indicate that DSB repair defect previously identified in LY-S cells is due to a lack of function of DNA-PK.
Key words: DNA repair, mouse lymphoma cell lines, DNA-dependent protein kinase, comet assay
NEOPLASMA, 46, Supplement, 1999, pp. 32-33
T.S. Kumaravel, A. Lohani, E. Mambo, M.K. Evans
Laboratory of Molecular Genetics, Gerontology Research Center, National Institute on Aging, National Institutes of Health, 5600 Nathan Shock Drive, Baltimore, MD 21224-6825, USA
We examined the role of DNA-PKcs in the adaptive response using the severe combined immunodeficient (SCID) mouse model that lacks functionally active DNA-PKcs molecules. We used DNA repair as measured by single cell gel electrophoresis (Comet Assay) and apoptosis as endpoints to evaluate the adaptive response in two SCID cell lines. Additionally, we have examined the expression of the base excision repair (BER) enzyme AP endonuclease (APE) and the expression of NER related proteins p21, GADD45 and ERCC3 in an attempt to identify the induction of BER or NER pathways in adapted and non-adapted cells. Our data suggests that the adaptive response is present in SCID cells, implying that DNA-PKcs is not required for the adaptive response. In SCID as well as in normal fibroblasts, the expression of APE was markedly increased and prolonged in the adapted cells. This suggests that low priming doses of ionizing radiation may accentuate the BER pathway induced by the subsequent high dose and confer a survival advantage measured by lower percentages of apoptosis and more efficient repair of DNA damage. The presence of the adaptive response in radiation-sensitive SCID cells known to be defective in double strand break repair and nucleotide excision repair may be related to an intact and inducible base excision repair mechanism.
Key words: Adaptive response, severe combined immunodeficient mouse, DNA-PKcs, comet assay, gamma irradiation, apoptosis
NEOPLASMA, 46, Supplement, 1999, pp. 34-37
C. Schindewolf, K. Lobenwein, G. Lüke, M. Gomolka, D. Soewarto, C. Fella, R. Balling, M. Hrabé De Angelis, T. Jung
Institut für Säugetiergenetik, GSF Research Center, Ingolstädter Landstr.1, 85764
Bundesamt für Strahlenschutz, Institut für Strahlenhygiene, Ingolstädter Landstr. 1, 85764 Oberschleissheim, Germany
Radiation sensitivity is a serious problem in cancer therapy which leads to an
early termination or interruption of therapy in about 15% of the cases due to
uncontrollable side effects. With the exception of rare genetic syndromes such as AT
(Ataxia Telangiectasia) or NBS (Nijmegen breakage syndrome), the background for the
inheritance of genetic susceptibility to radiation is unknown.
Recently, a large scale genetic screen of mouse mutants has been established within the German Human Project as a collaboration between the Institute of Mammalian Genetics, GSF Research Center and the Gene Center of the Ludwig Maximillian University in Munich. The goal of this ENU (ethyl-nitroso-urea) mutagenesis screen is the production of mutant mice by chemical mutagenesis which are characterized by specific phenotypic abnormalities and can serve as animal models for human diseases and genetic susceptibility. All mice are phenotypically screened for about 150 biochemical, immunological, and morphological parameters, respectively. The genetic screen is expected to generate between 1000 and 2000 new mouse mutants within the next 5 to 10 years.
In order to utilise the resources of a genetic screen of this magnitude for screening for radiation sensitivity, it is necessary to establish an in vitro assay system which is amenable to automatization. Hence, we are using the comet assay to detect mice mutants which display a genetic susceptibility to ionising radiation. Currently, we have established comet assay analysis parameters able to detect radiation sensitive mouse mutants and to control the variance within the mouse population in the ENU screen. The principle goal of our effort is to isolate genes which are responsible for DNA repair and radiation sensitivity in mouse and man.
Key words: Radiation sensitivity, mouse mutants, ethyl-nitroso-urea screen
NEOPLASMA, 46, Supplement, 1999, pp. 38-39
M. Wojewódzka, G.P. Van der Schans, I. Szumiel
Institute of Nuclear Chemistry and Technology, 03-195 Warszawa, Poland
TNO Medical Biological Laboratory, 2280 AA Rijswijk, The Netherlands
NEOPLASMA, 46, Supplement, 1999, pp. 40
F. Kassie, H-M. Qin, S. Rabot, S. Knasmüller
Institute of Cancer Research, University of Vienna, Vienna, Austria
National Institute for Agricultural Research, INRA, France
The single cell gel electrophoresis (SCGE) assay also known as comet assay is a relatively new genotoxicity test that detects, among other kinds of genotoxic effects, single and double strand breaks, alkali labile sites and DNA-cross linking. We employed the SCGE assay in vivo to assess genotoxic effects of IQ in organs of rats which are amenable to develop tumor upon treatment of the animals with the carcinogen. In subsequent experiments, we investigated the potential protective effects of benzyl isothiocyanate (BITC), glucotropaeolin (GT) and garden cress (GC) juice against the effects of IQ. The results showed a concordance between findings in SCGE assay and long term carcinogenicity tests in that pronounced genotoxic effects were induced in organs of rats where tumors were preferentially induced by IQ. These effects were drastically reduced upon pretreatment with BITC, GT and GC juice for three consecutive days.The antigenotoxic effect of BITC, GT, and GC juice might be attributed, at least partly, to induction of glutathione S- transferase (GST) and UDP-glucuronyl transferase (UDPGT) by these agents. In conclusion, SCGE assay was found to be sensitive enough to detect the genotoxicity of IQ and it could also be applied to identify dietary protective substances.
Key words: IQ, benzyl isothiocyanate, glucotropaeolin, garden cress juice, genotoxic effects, protective substances, protective effects
NEOPLASMA, 46, Supplement, 1999, pp. 41-43
E. Cordelli, P. Altavista, G. Leter, L. Castaldi, P. Villani
Section of Toxicology and Biomedical Sciences, ENEA CR Casaccia, Rome, Italy
In vivo and in vitro cell populations exhibit heterogeneous responses
to many genotoxic agents which can be partially explained by the relative position of the
cells in the mitotic cycle. In this study, the possibility to simultaneously detect
oxidative DNA damage and cell cycle position has been investigated in C3H10T1/2 cell line.
Exponential and plateau phase cells were treated with hydrogen peroxide and DNA damage and
repair were evaluated by the alkaline comet assay and the micronucleus test. Our results
show a dose-related increase of DNA damage after H2O2 treatment
in both culture conditions.
The percentage of cells in the various phases of cell cycle determined by comet assay was compared with the data obtained by DNA content flow cytometric analysis, and a good agreement between the results of the two techniques was found. Untreated exponentially growing cells in G1 phase showed a lower tail moment than S and G2/M cells. The same cell cycle dependence was evidenced in cells treated with low doses of H2O2, while, at higher doses, all cells showed a similar high level of damage.
DNA repair was assessed analyzing cells by comet assay 2 h after treatment. The results showed that DNA repair was almost complete for all H2O2 doses tested.
These results confirm the sensitivity of the Comet Assay in assessing DNA damage and repair, and support its usefulness in evaluating cell cycle-related differential sensitivity to genotoxic agents.
Key words: Alkaline comet assay, cell cycle, DNA damage, repair capability
NEOPLASMA, 46, Supplement, 1999, pp. 44-45
B. L. Pool-Zobel, S. Roser, G. Rechkemmer
Department of Nutritional Toxicology, Institute for Nutrition, Friedrich Schiller
University of Jena, 07743 Jena, Germany
Institute for Nutritional Physiology, Federal research Centre for Nutrition, Haidt-und-Neu Str. 9, 76131 Karlsruhe, Germany
NEOPLASMA, 46, Supplement, 1999, pp. 46
W. Dyga, A. Cebulska-Wasilewska
Institute of Nuclear Physics, Department of Environmental and Radiation Biology, ul. Radzikowskiego 152, 31-342 Kraków, Poland
The aim of this paper was to study variation in the individual susceptibility to the induction of the DNA damage by genotoxic agents. Human lymphocytes were isolated from whole blood samples collected from 48 male donors. Among the donors 24 males were treated as reference group (no occupational exposure); average age was 38.6, and among them 58% were recent or former smokers. The other 24 males were occupationally exposed to pesticides; average age was 38.9, and 64% of them were recent or former smokers. Differences in sensitivity of human lymphocytes to X-rays and variability of the DNA damage repair capacity were investigated by use of the single cell gel-electrophoresis method (SCGE), also known as the comet assay. Previously cryopreserved lymphocytes were defrosted and then irradiated with 2 Gy of X-rays. Viability of the cells and DNA damage in lymphocytes prior to irradiation were also investigated. In order to evaluate repair capacity of the cells, DNA damage was estimated immediately after irradiation and after two hours of incubation in presence or absence of phytohemagglutinin (PHA), a cell division-stimulating agent. The same procedures were performed on the samples from people exposed and unexposed to pesticides. Preliminary results revealed no statistically significant difference between exposed and unexposed groups, in the mean values of the DNA damage levels measured either prior to or immediately after irradiation, although some differences in repair capacities were observed. In the reference group, after two hours of incubation 85% and 86% of the radiation-induced damage was repaired (in absence or presence of PHA respectively). In the exposed group, after two hours of incubation 67% and 82% of the radiation induced damage was repaired (in absence or presence of PHA respectively). In absence of PHA, a lower repair capacity in lymphocytes of people exposed to pesticides (expressed as a higher average level of residual DNA damage) was statistically significant.
Key words: Single cell gel electrophoresis, DNA damage, X-ray, lymphocytes, repair
NEOPLASMA, 46, Supplement, 1999, pp. 47-49
M. Gomolka, G. Lüke, E. Konhäuser, K. Hetzl, C. Schindewolf, K. Lobenwein, S. Hornhardt, R. Balling, M. Hrabé De Angelis, T. Jung
Bundesamt für Strahlenschutz, Institut für Strahlenhygiene, Ingolstädter Landstr. 1,
85764 Oberschleissheim, Germany
Institut für Säugetiergenetik, GSF Research Center, Ingolstädter Landstr.1, 85764 Neuherberg, Germany
In the framework of a large genetic mouse screen, we are searching for mice bearing deficiencies in their DNA repair system for radiation-induced strand breaks. The alkaline version of the comet assay was chosen as a phenotypic detection system. In order to establish positive and negative controls in the genetic screen, we analysed two DBA/2 mouse cell lines with known different radiation sensitivities. According to cell survival analysis, the L5178YS (LYS) cell line is twice as sensitive to X-ray irradiation as the resistant cell line (LYR). In addition, the two cell lines display differences in their DNA repair efficiency. Cells were g-irradiated with doses ranging form 1 to 4 Gy to establish a dose-effect relationship. Repair efficiency was tested after irradiation with 2 Gy on ice and repair times ranging from 30 min to 120 min at 37°C. Slides were evaluated by a new software developed in collaboration with Till Photonics (Munich, Germany) and Narendra Singh (Seattle, USA). The comet assay demonstrated significant differences between the two cell lines in initial radiation damage and repair in various parameters. The most informative parameters were Olive tail moment, tail extended moment (Kent) and tail integrated intensities. Damage after different doses of irradiation, expressed in relative tail moments, was always higher in LYR cells than in LYS cells. Repair efficiencies for the two cell lines showed a dependence on the growth time of the cell culture; this was probably due to the cell cycle phase of the cell culture. After 72 h of growth, followed by irradiation, repair in LYS cells was more efficient during the first 30 minutes than repair in LYR cells. In contrast, 24 h growth of the cell cultures resulted in a more efficient repair in LYR cells. In addition, relative tail moments decreased also for control cells after incubation on ice or at 37°C. After 60 minutes incubation, necrotic and apoptotic cells appeared in controls as well as in irradiated cell samples. In general, we conclude that the experimental system and the software we utilised are capable of detecting DNA damage in correlation to the irradiation doses and differences in repair can be demonstrated between the cultured cell lines. Since the cell lines differ in background DNA damage, owing to culture conditions and cell isolation, and in cell cycle distribution compared to freshly isolated mouse lymphocytes they are unsuitable positive and negative controls for our screening assay.
Key words: DBA/2 mouse cell lines, alkaline comet assay, DNA repair system
NEOPLASMA, 46, Supplement, 1999, pp. 50-52
B. Novotná, D. Öoms, M. Vilhelmová, J. Vaňková, R. Hooghe
Institute of Experimental Medicine, Czech Academy of Sciences, Praha, Czech Republic
Flemish Institute for Technological Research, Mol, Belgium
Institute of Molecular Biology, Czech Academy of Sciences, Praha, Czech Republic
Treatment of 3-day-old chick embryos with 5-bromo-2-deoxyuridine (BrdU) significantly modified membrane glycosylation of blood cells. After the induction of DNA lesions and during the DNA repair process, membrane carbohydrates were more sialylated than in controls whereas the loss of sialic acid, indicated by increased binding of sialic acid-specific lectins to blood cells, accompanied apoptotic cleavage of DNA. Flow-cytometry analysis after the labeling of cells with lectins thus may contribute to the interpretation of comet assay results. On the other hand, annexin V - generally recommended for identification of dying cells - gave no clue regarding the choice between diverse apoptotic pathways made in BrdU-modified cells.
Key words: DNA fragmentation, membran glycosylation, chick embryo.
NEOPLASMA, 46, Supplement, 1999, pp. 53-54
E. Horváthová, D. Slameňová, B. Košíková, Ľ. Ružeková, J. Lábaj
Cancer Research Institute, Slovak Academy of Sciences, 833 91 Bratislava, Slovak
Institute of Chemistry, Slovak Academy of Sciences, 842 38 Bratislava, Slovak Republic
The possible protective effects of natural and synthetic antioxidants on DNA in hamster V79 cells exposed to hydrogen peroxide (H2O2) and N-methyl-N´-nitro-N-nitrosoguanidine (MNNG) were investigated. The levels of DNA damage were measured using single cell gel electrophoresis. It was assessed that natural antioxidants lignin biopolymer and vitamin E (alpha-tocoferol) and a synthetic antioxidant stobadine (STB) exhibited a protective effect against the overall DNA damage induced after H2O2-treatment. In addition, it was observed that lignin NG. These findings suggest that the antioxidant nature of lignin biopolymer enables a rbiopolymer and stobadine (but not vitamin E) also protected against DNA damaged by MNeduction of the levels of frank breaks and of oxidized DNA bases in H2O2-treated cells and its adsorptive capacity enables binding of N-nitroso compounds and reduction of alkylation in MNNG-treated cells. The reduced level of DNA damage in STB-pre-treated samples could be due to its antioxidant properties in the case of H2O2-treatment and to a direct reduction of MNNG activity in the case of the alkylating agent.
Key words: Hamster cells V79, hydrogen peroxide, N-methyl-N´-nitro-N-nitrosoguanidine, vitamin E, stobadine, lignin
NEOPLASMA, 46, Supplement, 1999, pp. 55-57
A.R.Collins, C.M.Gedik, S.G.Wood
Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen AB21 9SB, UK
NEOPLASMA, 46, Supplement, 1999, pp. 58
J. Błasiak, E.Gloc-Fudała, A.Trzeciak
University of Łódź, Department of Molecular Genetics, Banacha 12/16, 90-237 Łódź, Poland
Ethyl alcohol can be mutagenic, carcinogenic and teratogenic in man and its mutagenicity can be attributed to its first and major metabolite, acetaldehyde, which was reported to form adducts with DNA and proteins and DNA strand breaks. It was suggested that DNA crosslinks may be the predominant DNA adducts of acetaldehyde but these results were obtained with the alkaline elution technique which provides no information on the extent of DNA damage at the individual cell level. The comet assay is a technique, which allows detection of double- and single-strand DNA breaks caused by a broad spectrum of mutagens. It is also a suitable tool for the detection of crosslinking agents. Human peripheral blood lymphocytes were incubated for 1 h at 37°C with 3, 10, 30 and 100 mM of acetaldehyde. The alkaline comet assay was used to assess DNA damage. A dose-dependent decrease in the migration of DNA of acetaldehyde-treated cells was observed. The results obtained suggest that acetaldehyde may form crosslinks with DNA in human lymphocytes. The nature of the crosslinks remains unknown and needs further investigation. Our results contradict those obtained by Singh and Khan who reported DNA strand breaking activity of acetaldehyde.
Key words: Acetaldehyde genotoxicity, ethanol metabolism, comet assay, DNA damage, DNA crosslinks, formaldehyde; human lymphocytes
NEOPLASMA, 46, Supplement, 1999, pp. 59-60
J. Błasiak, J. Kowalik, A. Trzeciak, M. Wojewódzka
Department of Molecular Genetics, University of Łodź Banacha 12/16, 90-237 Łodź,
Department of Radiobiology and Health Protection, Institute of Nuclear Chemistry and Technology, Dorodna 16, 03-159 Warsaw, Poland
Cisplatin is a widely used anticancer drug, but its application is limited by severe side effects. To reduce these effects many other platinum drugs have been synthesised. In the present work the toxicities of cisplatin, oxoplatin and a conjugate (NH3)2Pt(SO3) were compared in terms of cell viability and DNA damage and repair in human lymphocytes, using the trypan blue exclusion test and the comet assay, respectively. Cisplatin and oxoplatin did not cause a significant change in the viability of the lymphocytes even at the highest concentration used (750 microM), but the conjugate dramatically diminished viability - at 100 microM only about 60% of the lymphocytes were viable (p < 0.05), at 750 microM - less than 20% (p < 0.001). Cisplatin and oxoplatin formed crosslinks with DNA in lymphocytes, whereas the conjugate induced DNA strand-breaks. The lesions induced by cisplatin and oxoplatin were slowly removed, but damage induced by (NH3)2Pt(SO3) was not repaired after 5 h even at a drug concentration of 10 microM. Severe cytotoxic and genotoxic effects exerted by (NH3)2Pt(SO3) in normal human lymphocytes preclude its intravenous application in cancer therapy.
Key words: Cisplatin, oxoplatin, Se-Pt conjugate [(NH3)2Pt(SeO3)], genotoxic effects of anticancer drugs, DNA damage, DNA repair, comet assay
NEOPLASMA, 46, Supplement, 1999, pp. 61-63
J. Kowalik, A. Trzeciak, M. Wojewódzka, J. Błasiak
Department of Molecular Genetics, University of Łodź, Banacha 12/16, 90-237 Lodz,
Poland, Tel: (+48) 42 635 44 89; Fax: (+48) 42 635 44 84
Department of Radiobiology and Health Protection, Institute of Nuclear Chemistry and Technology, Dorodna 16, 03-159 Warsaw, Poland
Curcumin is a yellow pigment extracted from the rhizomes of Curcuma longa (Zingiberaceae) a herb widely cultivated in Asia. It has been used for centuries as a coloring agent in food, drugs and cosmetics. Because of its intensive colour and original taste curcumin is readily used as a spice, for example in curry or many kinds of mustard. It is a component of the diet in many areas of the world. Curcumin is reported to have a broad spectrum of biological effects including antiinflammatory and antineoplastic properties. It is considered as a natural antioxidant. However, there are some reports demonstrating prooxidant properties of curcumin. Since curcumin may have the opposite effects in a biological environment, we took an interest in curcumin as a potential genotoxic compound. The sensitive method of alkaline single cell electrophoresis was applied in this study to detect possible DNA damage. Human peripheral blood lymphocytes were exposed to curcumin for 1 h at concentrations of 15, 25 and 50 microM. Curcumin at 25 and 50 microM caused a significant dose-dependent increase in DNA migration measured as tail moment. After repair time of 1 h the extent of DNA damage returned to the control values. These results indicate that curcumin has a genotoxic potency in human lymphocytes. In spite of antioxidative properties of curcumin, it is possible in some conditions that curcumin may participate in generating reactive oxygen species able to damage DNA.
Key words: Curcumin, turmeric, DNA damage, DNA repair, comet assay
NEOPLASMA, 46, Supplement, 1999, pp. 64-65
T. Godard, V. Fessard, S. Huet, A. Mourot, E. Deslandes, D. Pottier, O. Hyrien, F. Sichel, P. Gauduchon, J.m. Poul
AFSSA (Agence Française de Sécurité Sanitaire des Aliments), Laboratoire des
Médicaments Vétérinaires, Unité de Toxicologie, Javené F-35133 Fougeres, France
CJF-INSERM 96-03 & Grecan, Université de Caen, Laboratoire de Cancérologie Expérimental, Centre Régional François Baclesse, Route de Lion-sur-Mer, 14076 Caen cedex 05 France
The comet assay was used to evaluate in vitro and in vivo genotoxicity of etoposide, a topoisomerase II inhibitor known to induce DNA strand breaks, and chlorothalonil, a fungicide widely used in agriculture. For in vivo studies, rats were sacrificed at different times after treatment and the clastogenicity of substances was assessed in whole blood, bone marrow, thymus, liver, kidney cortex and in the distal part of the intestine. One hour after injection, etoposide induced DNA damage in cells from all organs studied except in kidney cells, and especially in bone marrow cells, thymocytes and leukocytes. As observed after in vitro comet assay on Chinese Hamster Ovary (CHO) cells, dose- and time-dependent DNA effects were observed with a total disappearance of damage 24 hours after treatment. Apoptotic cells detected in vitro, 48 hours after 1 hour exposure to etoposide, were not found in vivo, but a decrease in cell numbers was observed for whole blood, bone marrow and thymus. After treatment with chlorothalonil, no DNA strand breaks were observed on rat organs using the comet assay whereas a clear dose-related DNA damage was observed in vitro. The discrepancy between in vivo and in vitro models could be explain by metabolic and mechanistic reasons. Our results suggest that the in vivo comet assay is able to detect the target organs of etoposide and that chlorothalonil is devoid of appreciable in vivo clastogenic activity.
Key words: Comet assay, rat organs, CHO cells, etoposide, chlorothalonil
NEOPLASMA, 46, Supplement, 1999, pp. 66-68
M. Dušinská, A. Mateášik, D. Jr. Chorvát, I. Lajdová, V. Spustová, A. Chorvátová
Department of Molecular and Genetic Toxicology, Institute of Preventive and Clinical
Medicine, Bratislava, Slovak Republic
Department of Clinical Pharmacology, Institute of Preventive and Clinical Medicine, Bratislava, Slovak Republic
Department of Biomedicine, Institute of Preventive and Clinical Medicine, Bratislava, Slovak Republic
International Laser Centre, Bratislava, Slovak Republic
We studied the mechanism of action of the photosensitising fluorescent dye Merocyanine (MC 540) at membrane and DNA levels. The spectrofluorimetric method was used to characterise MC 540 binding to the cell membrane. Human lymphocytes from 6 healthy subjects were exposed to MC 540 in PBS and excitation spectra from 400 to 575 nm (emission wavelength 585 nm) were measured in the dark and after 1 min and 5 min of exposure to white light. At the same time strand breaks and oxidised bases were measured in nuclei by the comet assay modified with lesion-specific enzymes. We found that irradiation in combination with MC 540 caused oxidation of purines and to a lesser extent pyrimidines in lymphocyte DNA, with only limited DNA strand breakage; the dye was able to enter the cell plasma membrane. These results suggest that MC 540 could be a promising dye for the study of mechanisms of oxidative damage in model conditions.
Key words: Comet assay, merocyanine, photosensitising fluorescent dye, oxidative DNA damage
NEOPLASMA, 46, Supplement, 1999, pp. 69-70
T. Gichner, O. Ptacek, D.A. Stavreva, M.J. Plewa
Institute of Experimental Botany, Na Karlovce 1a, 160 00 Prague 6, Czech Republic
University of Illinois at Urbana-Champaign, USA
NEOPLASMA, 46, Supplement, 1999, pp. 71
K.J. Angelis, M. McGuffy, M. Menke, I. Schubert
Institute of Experimental Botany, CAS, Na Karlovce 1, 160 00 Praha 6, Czech Republic,
Fax: +420 2 243 10 113, e-mail: email@example.com
Institute fur Pflanzengenetik und Kulturpflanzenforschung, D-06466 Gatersleben, Germany
By using isolated nuclei instead of whole cells, the comet assay can be adopted for
virtually any plant. In this report we compareVicia faba, a "standard"
plant for cytogenetic studies, and Arabidopsis thaliana, at present the most widely
used model plant.
X-rays, N-methyl-N-nitrosourea (MNU) and methylmethanesulfonate (MMS) were studied for their potential to induce comets. We show a substantial dependence of comet formation on electrophoretic conditions after X-ray irradiation rather than on conditions of lysis and unwinding.
With the A/N assay (alkaline unwinding and neutral electrophoresis), less DNA damage - both DNA breaks and AP sites (detected as sites sensitive to exonuclease III) - is observed when pretreatment with a low dose of alkylating agents MNU or MMS is followed by a high (challenge) dose, compared with application of only the high dose.
Surprisingly, such adaptation followed by comet assay is not induced in Arabidopsis.
Key words: Arabidopsis, Vicia, Ap sites
NEOPLASMA, 46, Supplement, 1999, pp. 72-73
P. Poli, A. Buschini, A. Ficarelli, F.M. Restivo, T.M.A.D. Zucchi, C. Rossi
Instituto di Genetica, Universita di Parma, Parma, Italy
Departamento de Parasitologia, Instituto de Cięncias Biomédicas, Universidade de Săo Paulo, Brazil. e-mail: firstname.lastname@example.org
The best experimental conditions for Aspergillus nidulans were when treatment with Novozim 234 was performed after the slide preparation. The comet images appeared in treated cells. A modified and improved SCGE on different tissues (root, stem and leaf) of Impatiens balsamina was performed, using genotoxic compounds or environmental mixtures. Our modification resulted in an increased yield of nuclei and a more uniform distribution of nuclei in the agarose layer. The system could be also useful in the estimation of genotoxicity in situ.
Key words: Comet assay, Aspergillus nidulans, Impatiens balsamina.
NEOPLASMA, 46, Supplement, 1999, pp. 74-75
G. Speit, O. Merk
Universitätsklinik Ulm, Abteilung Medizinische Genetik, D - 89070 Ulm, Germany
The comet assay detects a broad spectrum of DNA lesions but the standard protocol seems to be unsuited for the detection of crosslinking agents. Crosslinking (DNA-DNA or DNA-protein) can lead to an decrease in DNA migration and modifications of the comet assay have been established to study the effect of crosslinkers by measuring the inhibition of spontaneous or mutagen-induced DNA migration. We comparatively studied the sensitivity of the comet assay for the detection of various crosslinkers and tried to relate the effects in the comet assay to other genotoxic and cytotoxic effects to better understand the biological significance of comet assay results with crosslinking agents.
Key words: Alkaline comet assay, crosslinking agents, V79
NEOPLASMA, 46, Supplement, 1999, pp. 76-78
D. Slameňová, E. Horváthová, Ľ. Ružeková, A.R. Collins
Cancer Research Institute of Slovak Academy of Sciences, Department of Mutagenesis and
Carcinogenesis, Vlárska 7, 833 91 Bratislava, Slovak Republic
Rowett Research Institute, Greenburn Road Bucksburn, Aberdeen AB21 9SB, U.K.
We characterized MNNG- and H2O2- induced DNA strand breaks in hamster V79 cells and UV- induced DNA strand breaks in human VH10 cells by two modifications of the comet assay (SCGE) using (a) lesion-specific enzymes endonuclease III (endo III) and formamidopyrimidine-DNA-glycosylase (FPG) as well as enzyme exonuclease III (exo III) recognizing alkali-labile sites and (b) pH-variations of the comet assay. SCGE at pH=13 showed that DNA of H2O2-treated V79 cells incubated for 90 min contains both endo III- and FPG-sensitive sites but that the occurrence of endo III- and FPG-sensitive sites is uncertain in MNNG-treated V79 cells. SCGE processed in parallel at pH 13.0 and pH 12.1 showed that: (1) strand breaks detected in DNA of MNNG- and MMS- treated V79 cells originated from alkali-labile sites; (2) strand breaks detected immediately after the treatment of cells with H2O2 represent mainly breaks stable at both pH values; (3) the same level of DNA strand breaks accumulated in UV-irradiated human VH10 cells during incubation with DNA repair inhibitors can be revealed at both pH values. SCGE at pH 12.1 showed that the level of DNA strand breaks in MNNG- treated V79 cells was increased mainly after digestion with exoIII and that the level of DNA strand breaks in H2O2-treated and 90 min-incubated V79 cells was increased by both endo III and FPG to the same extent as at pH 13.0. Digestion by both endo III and FPG together led to an additive effect.
Key words: Hamster cells V79, human cells VH10, the comet assay, DNA repair enzymes, DNA repair inhibitors, N-methyl-N’-nitro-N-nitrosoguanidine (MNNG), hydrogen peroxide (H2O2), ultraviolet (UV) light
NEOPLASMA, 46, Supplement, 1999, pp. 79-81
A.R.Collins, B.H.Collins, M. Dušinská , K. Angelis
Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen AB21 9SB, UK
Institute of Preventive and Clinical Medicine, Limbova 14, 901 33 Bratislava, Slovakia
Institute of Experimental Botany, CAS, Na Karlovce 1a, 160 00 Praha 6, Czech Republic
NEOPLASMA, 46, Supplement, 1999, pp. 82
B.vallová, Z. Kováčiková, M. Ďurčík, M. Vičanová, E. Tátrai , M. Dušinská
Institute of Preventive and Clinical Medicine, Limbová 14, 83301 Bratislava, Slovak
National Institute of Occupational Health, Nagyvárad tér 2, H-150 Budapest, Hungary
The comet assay was applied to measure DNA breaks and oxidised bases in freshly isolated alveolar macrophages and epithelial type II cells from the rat lung. The cells were exposed to radon for 60 min. Radon exposure was estimated at 1.25-2.45 MBq.h.m-3. Strand breaks were significantly elevated above the background level after irradiation of epithelial type II cells. In contrast, no strand breaks were induced in alveolar macrophages, but a high level of oxidised bases, mostly purines, was found. Alveolar macrophages and epithelial type II cells freshly isolated from the rat lung provide an exceptionally suitable cell model for investigation of potential hazards of air-borne environmental contaminants.
Key words: Comet assay, radon, radon progeny, oxidative DNA damage, alveolar macrophages, epithelial type II cells
NEOPLASMA, 46, Supplement, 1999, pp. 83-84
L. M. Pedersen, P. Mřller, B.Danesvar, H. Wallin, H. Autrup, S. Loft, L. O. Dragsted
Natl Inst Occupational Health, Copenhagen
Institute of Food Safety and Toxicology, Sřborg
Steno Institute of Public Health, Aarhus University, Aarhus
Institute of Public Health, Copenhagen University, Copenhagen, Denmark.
NEOPLASMA, 46, Supplement, 1999, pp. 85
Z. Krivošíková, M. Dušinská, K. Šebeková, V. Spustová, A. Heidland, R. Dzúrik
Department of Pharmacotherapy, Institute of Preventive and Clinical Medicine,
Department of Molecular and Genetic Toxicology Institute of Preventive and Clinical Medicine, Bratislava, Slovakia
Kuratorium fur Dialyse und Nierentransplantation,Würzburg, Germany
DNA damage was evaluated in isolated rat lymphocytes of sham operated rats (Sham), remnant kidney rats (Nx) and remnant kidney rats treated with losartan (NxL). The study duration was three months. Nx rats were slightly hypertensive while NxL rats revealed lower blood pressure levels. Mean values of DNA strand breaks were significantly higher in Nx if compared to Sham group, while losartan significantly prevented DNA damage expressed in % DNA in tail (Sham 15.92±1.14, Nx 54.49±1.74, NxL 23.29±2.61, p<0.001 vs. Sham and NxL). Serum creatinine levels correlated positively with DNA breaks if Sham and Nx groups were evaluated together (r=0.507, p<0.0004). In conclusion, rats in advanced renal insufficiency undergo expressive DNA damage and the administration of losartan effectively prevents it.
Key words: DNA damage, Comet assay, rat remnant kidney, renal failure, losartan.
NEOPLASMA, 46, Supplement, 1999, pp. 86-87
D.Florjan, W.Niedzwiedz, K.Schneider, A.Cebulska-Wasilewska
Institute of Nuclear Physics, Radiation and Environmental Biology Department, Radzikowskiego 152, 31-342 Kraków, Poland
The aim of this study was to evaluate the effectiveness of DNA and chromosomal damage induction in human lymphocytes exposed in vitro to neutrons from a Californium-252 isotopic source at very low dose rate, i.e. 0.001 Gy/min, at a dose range of 0 - 0.8 Gy (chronic radiation). SCGE assay, classical cytogenetics and FISH with whole painting probes for chromosomes 1 and 4, and pancentromeric probe were applied to investigate this problem. There is an increase in DNA damage and aberration frequency with dose of radiation. The analysis of the results revealed a linear dose-dependent response in the whole investigated dose range both for DNA damage and chromosomal aberrations. The level of translocations observed is comparable with the frequencies of dicentrics and rings.
Key words: Comet assay, FISH, Californium Neutron Irradiations.
NEOPLASMA, 46, Supplement, 1999, pp. 88-89
D. Vlček, K. Závacká, M. Dušinská
Department of Genetics, Faculty of Natural Science, Comenius University, Bratislava,
Department of Molecular and Genetic Toxicology, Institute of Preventive and Clinical Medicine, Limbová 14, 801 03, Bratislava, SK
We applied the modified comet assay to investigate different DNA lesions in the alga Chlamydomonas reinhardtii arising either spontaneously or after treatment with MMS and menandione. For this purpose the wild type (W1), and two repair-deficient strains of the C. reinhardtii were used. The mutant strain Uvs 12 is deficient in nuclear excision repair pathway, whereas mutant strain Uvs E1 is blocked in recombination repair. The sensitivity of strains to alkylation was studied with the alkylating agent MMS. Menadione was applied to induce oxidative DNA damage. To detect oxidative DNA lesions we used both endonuclease III (for oxidised pyrimidines and AP sites) and formamidopyrimidinglycosylase (for oxidised and ring-opened purines, and AP sites). DNA alkylation was detected with AlkA (3-methyladenine DNA glycosylase II). Both mutants were more sensitive than W1 for induction of strand breaks after MMS treatment. Using Alk A we detected a higher level of DNA alkylations in mutant Uvs E1. MMS induced a low proportion of AP sites after MMS treatment in all three strains. Compared with wild type both mutants were more sensitive to DNA oxidation.
Key words: Chlamydomonas reinhardtii, the comet assay, DNA damage and repair, MMS, menandione, DNA glycosylases
NEOPLASMA, 46, Supplement, 1999, pp. 90-91
D.A. Stavreva, M. Strnad, L. Havlíček, T. Gichner
Institute of Experimental Botany, Academy of Sciences of the Czech Republic, Na
Karlovce 1a, 160 00 Prague 6, Czech Republic
Laboratory of Growth Regulators, Institute of Experimental Botany & Palacký University, Šlechtitelů 11, 78371 Olomouc, Czech Republic
Three purine analogues (olomoucine, bohemine and roscovitine), known to have an essential role in the regulation of the cell division cycle, were assayed for their DNA-damaging activity in tobacco suspension cells with the comet assay. At concentrations not affecting cell viability, none of the tested purine analogues increased the DNA migration. After treatment with concentrations that markedly lowered cell viability, bohemine and roscovitine slightly but significantly increased the tail moment values. After treating tobacco cells with ethyl methanesulfonate and hydrogen peroxide we obtained a direct concentration-response for induced DNA damage with treatments that did not affect cell viability.
Key words: SCGE assay, purine analogues, tobacco cells.
NEOPLASMA, 46, Supplement, 1999, pp. 92-93
M. McGuffie, M. Menke, I. Schubert, K.J. Angelis
Institute of Experimental Botany, CAS, CZ-160 00 Praha 6, Czech Republik
Institute of Plant Genetics & Crop Plant Research (IPK), D-06466 Gatersleben, Germany
NEOPLASMA, 46, Supplement, 1999, pp. 94
G.Bačová, A.Gábelová, M. Bacigálová
Cancer Research Institute, 833 91 Bratislava, Slovakia
Several modifications of lysis and the same electrophoresis conditions have been applied on K562 cells in order to find optimal conditions of single cell gel electrophoresis for manifestation of double strand breaks. The most significant difference in DNA migration between untreated K562, and treated cells was observed when the lysis was performed at 50oC. Two lysis modifications have been choosen and applied on human lymphocytes, but neither of these conditions were suitable for human lymphocytes because of extensive DNA migration in untreated cells.
Key words: Single cell gel electrophoresis, lysis modifications, neutral comet assay, tail moment
NEOPLASMA, 46, Supplement, 1999, pp. 95-96
S. Sauvaigo, M-J. Richard, J. Cadet
DRFMC/SCIB/LAN, CEA Grenoble, 17 rue des Martyrs, F-38054 Grenoble Cedex 9
LBSO, Université Joseph Fourier, F-38043 Grenoble Cedex 3, France
We have used the Comet assay to assess the repair of UV-induced damage in human fibroblasts (normal and from a xeroderma pigmentosum patient) after UVB irradiation. The repair was monitored by strand breaks formation on one hand and by a specific anti-cyclobutane pyrimidine dimer (CPD) antibody signal, on the other hand. This latter antibody, that was fluorescently labeled, could be used directly on the cell DNA embeded in the agarose gel. Normal fibroblasts showed a bi-phasic repair kinetic with a first 50% signal disappearance within 15 h correlated with a return of the tail moment to its basal level. After a transient enhancement of the tail moment, the complete diseappearance of the antibody signal occurs within 48 h after irradiation. In the XP cell line, unwinding of the DNA occured but was not correlated with the antibody signal disappearance. The same approach was also used to assess the extend of formation of another UV-induced damage : pyrimidine[6-4]pyrimidone photoproducts after UVC irradiation.
Key words: UV radiation, Xeroderma pigmentosum, DNA repair
NEOPLASMA, 46, Supplement, 1999, pp. 97-98
A. Rapp, C. Bock, H. Dittmar, K.O. Greulich
Institut für Molekulare Biotechnologie, Postfach 100813, D-07708 Jena, Germany, Phone/Fax **49.3641.656405/10, e-mail: email@example.com
The conventional comet assay allows to study, at the single cell level, the overall
sensitivity of genomes towards noxes such as radiation, environmental toxins or
therapeutics. Comet-FISH, a combination of comet assay and FISH, goes a step
further and gives additional information on the region-specific sensitivity for DNA
breakage. This technique provides the tool to examine whether DNA strand breaks occur
randomly all over the genome or in preferred genomic regions.
In this work we realised the comet-FISH technique using a special three layer agarose gel with a low temperature chemically modified hybridisation similar to classical FISH techniques. The electrophoretically separated DNA fragments that occur after UV-A (365nm) irradiation were used as the target for the hybridisation instead of metaphase chromosomes. In this way comet-FISH was first time realised for whole chromosome probes. Additionally centromere-, telomere- and region-specific probes as well as unique gene specific probes were used.
Here we present the data obtained with four probes located on chromosome 8 and the use of this method in the measurement of breakage sensitivity of these genomic regions. The chromosome 8 was used because of its relative high overall breakage sensitivity compared to the other autosomes.
The results of these experiments suggest, that DNA damage of UV-A irradiation is preferentially induced in the peripheral region of 8q compared to the central regions of the chromosomal arm, though a more detailed study is required to confirm this hypothesis definitively.
Key words: Comet FISH, UV-A light, peripheral human blood lymphocyte
NEOPLASMA, 46, Supplement, 1999, pp. 99-101
A.P. McGlynn, G.R .Wasson, V.J. Mc Kelvey-Martin, G. Mc Kerr, C.S. Downes
Cancer and Ageing Research Group, School of Biomedical Sciences, University of Ulster at Coleraine
NEOPLASMA, 46, Supplement, 1999, pp. 102
M. Menke, K.J. Angelis, I. Schubert
Institute of Plant Genetics & Crop Plant Research (IPK) D-06466 Gatersleben,
Institute of Experimental Botany, CAS, CZ-16000 Prague 6, Czech Republic
NEOPLASMA, 46, Supplement, 1999, pp. 103
C. Alapetite, P.Thirion, A. de la Rochefordiere, J-M. Cosset, E. Moustacchi
UMR 218 CNRS, Institut Curie - Recherche
Département de radiotherapie Institut Curie - Section Médicale, 26 rue d’Ulm, 75005 Paris, France
NEOPLASMA, 46, Supplement, 1999, pp. 104
A. Cebulska-Wasilewska, W. Dyga, S. Krasnowolski, A. Wierzewska, E. Budzanowska
Department of Radiation and Environmental Biology, INP, 31-342 Kraków, Poland
L.Rydygier Hospital, Department of Dermatology, Oś. Złotej Jesieni 1, 31-826 Kraków, Poland
NEOPLASMA, 46, Supplement, 1999, pp. 105
G. Emri, E. Remenyik, Cs. Varga, J. Hunyadi, I. Horkay
Department of Dermatology, University Medical School of Debrecen, Hungary
Hygiene & Epidemiology, University Medical School of Debrecen, Hungary
UVB-phototherapy and photochemotherapy (PUVA, 8-methoxypsoralen plus UVA-irradiation) have been widely used for the treatment of various skin diseases for many years. The mechanism of the biological effects of UV-light, which contributes to the therapeutic efficiency, is not well understood, particularly for PUVA. The DNA is one of the essential UV-targets within the cell. The aim of the present study was to investigate DNA damage and repair following the treatment of HaCaT-cells by PUVA in comparison with the effects of UVB and UVA-irradiation. Single cell gel electrophoresis (the comet assay) was used to evaluate DNA strand breaks and alkali labile sites caused by UV or PUVA. Our results suggest that the mechanism of the effects of UVB and UVA for the formation and repair of DNA damage is quite different from that of PUVA. Immediately after irradiation, comet formation was seen only in the case of UVA (0-5 J/cm2). After UVB (0-60 mJ/cm2), comet formation was detected only 1 h after irradiation, when the length and intensity of the comet in UVA-irradiated cells were considerably diminished. Neither UVA nor UVB caused DNA-damage detectable by neutral comet-assay within the dose ranges studied. After PUVA-treatment (300 ng/ml 8-MOP and 2 J/cm2 UVA) we observed increasing comet-formation with a peak of 1.5 h after irradiation by neutral assay, then the comet-tails decreased slowly during the next few hours. These findings suggest that UVA and UVB induce DNA single strand breaks before and during DNA repair, respectively, whereas DNA double strand breaks are occurred following PUVA treatment. It is hypothesised that these latter DNA lesions are repair intermediary products of the DNA cross-links caused by PUVA.
Key words: Neutral comet assay, UV- and PUVA-induced DNA strand breaks
NEOPLASMA, 46, Supplement, 1999, pp. 106-107
A. Gábelová, G. Bacová
Cancer Research Institute, 833 91 Bratislava, Slovak Republic
NEOPLASMA, 46, Supplement, 1999, pp. 108
P. Moller, M. Dybdahl, H. Wallin, U. Vogel, G. Frentz, B.A. Nexo
National Institute of Occupational Health. Lerso Parkalle 105, DK-2100 Copenhagen
Institute of Preventive Medicine, Kommune Hospitalet
Psoriasis is a skin disease characterized by high proliferation rate of epidermal keratinocytes. The treatment of psoriasis includes several DNA damaging agents, and this has caused concern that psoriasis patients are at risk of developing basal cell carcinoma (BCC). We have investigated the DNA repair incision rate following UVC irradiation by the comet assay in isolated lymphocytes in a case-control study comprising psoriasis patients with and without BCC and non-cancer patients with and without psoriasis. Previously, we have found that lymphocytes from psoriasis patients with BCC had a high rate of incisions following UVC irradiation. Here we report that the lymphocytes from BCC patients also had the highest level of UVC-mediated DNA repair incisions in blood samples taken two years later. We have reported earlier that lymphocytes from psoriasis patients with BCC had the lowest level of DNA repair activity, determined by unscheduled DNA synthesis and host cell reactivation. This suggests that the DNA repair-defect in BCC patients can be distinguished by high UVC incision rate and low DNA repair activity.
Key words: Basal cell carcinoma, comet assay, DNA repair, psoriasis, UVC
NEOPLASMA, 46, Supplement, 1999, pp. 109-110
U.Öztok, M.Yýlmaz, A.Karakoç, N.Çakýr, A.E.Karakaya, S.Şardaş
Gazi University Faculty of Pharmacy Dep. of Toxicology, 06330, Ankara, Turkey
Gazi University Faculty of Medicine Dep. of Internal Medicine, Ankara, Turkey
Gazi University Faculty of Medicine Dep. of Endocrinology and Metabolism, Ankara, Turkey
Diabetic patients, both insulin-dependent (IDDM) and non-insulin dependent (NIDDM), exhibit abnormal antioxidant status, auto-oxidation of glucose and excess glycosylated proteins. Oxidative stress in diabetes leads to tissue damage, with lipid peroxidation and inactivation of proteins. Reduced antioxidant defenses in diabetic patients are associated with an increased risk of free radical mediated diseases. Dietary antioxidant compounds, including ascorbic acid, tocopherol offer some protection against these complications through their roles as inhibitors of glycation and as free radical scavengers. It has been reported that reactive oxygen generation in long standing diabetes may also result in oxidative damage to DNA. Also there is evidence to suggest that reactive oxygen is involved in the pathogenicity and complications arising from IDDM, but there is little to suggest a role of oxidative stress in the pathogenesis of NIDDM. In order to investigate this hypothesis further, peripheral blood samples were taken from 30 control individuals and 63 IDDM and NIDDM patients and examined by comet assay for DNA strand breakage. Statistically significant differences (p<0.05) were detected between control and diabetic patient groups. DNA damage in the comet assay was at higher level in NIDDM patients and slightly lower level in IDDM patients which may indicate that these cells are handling more oxidative damage on a regular basis. Also, a synergistic effect of smoking on DNA damage (with high levels of tailed nuclei) was observed in smoking diabetic patients in comparison with smoking non-diabetic controls.
Key words: Diabetic patient, comet assay, oxygen radical
NEOPLASMA, 46, Supplement, 1999, pp. 111-113
T.H.Ward , J. Richards , A.T. McGown , J.Butler
Department of Drug Development and Imaging, Paterson Laboratories, Christie Hospital,
Department of Biological Sciences, University of Salford, Salford ,UK
We have used a modified version of the comet assay to detect DNA cross-linking in primary ovarian cancer cell cultures. This assay combined with parallel measurements on cytotoxicity and gene expression has proved valuable in highlighting a possible novel mechanism of resistance. In addition an interesting correlation between the expression of two genes was obtained. The comet assay could become a valuable tool in patient profiling prior to the start of chemotherapy.
Key words: Ovarian cancer, chemosensitivity, DNA cross-links, enzyme profiling
NEOPLASMA, 46, Supplement, 1999, pp. 114-116
A. Kantar, L. Tiano, G. Falcioni, G. P. Littarru, R. Fiorini, G.V. Coppa, O. Gabrielli
Departments of Pediatrics, University of Ancona, Italy
Biochemistry, University of Ancona, Italy
Department of MCA Biology, University of Camerino, Italy
Data from various studies indicate that a delicate antioxidant enzyme balance exists in cells. The fine-tuning of antioxidant enzymes becomes imperative if the cell is to function successfully in an oxygen-rich environment. The human condition known as Down syndrome (DS) or trisomy 21 has provided some provocative clues regarding the balance between oxidants and antioxidants. DS is one situation where the antioxidant balance is affected due to gene dosage. The human cytosolic Cu/Zn - superoxide dismutase (SOD) gene is located on chromosome 21 and cells from subjects with DS contain 50% more than the normal amount of the antioxidant enzyme. A surprising variety of problems are caused by over-expression of the "housekeeping" enzyme SOD. Alterations in oxygen handling have been observed in DS. A causative role for reactive oxygen species (ROS) in the symptoms of DS has been hypothesised and a large body of supportive circumstantial evidence has been accumulated. In this study DNA damage was evaluated in DS using the comet assay. Our data demonstrate an increase in oxidative damage to DNA after an oxidative stress induced by superoxide anion and an increase in basal oxidised sites of DNA in DS. These data support the notion that ROS are involved in the development of some features of DS.
Key words: Down syndrome, reactive oxygen species, antioxidant enzymes, comet assay
NEOPLASMA, 46, Supplement, 1999, pp. 117-118
M. Dušinská, M. Somorovská, B. Vallová, H. Petrovská, A. Horská, A. Stupáková, K. Rašlová, M. Šustrová, J. Lietava, V. Mezencevová, V. Spustová, K. Štefíková, Z. Krivošíková, R. Dzúrik, K. Gazdíková, K. Rojková, A.R.Collins
Institute of Preventive and Clinical Medicine, Limbová 14, 833 01 Bratislava, Slovak
Institute of Medical Biology, Medical Faculty University of Pavel Josef Safarik, SNP 04060 Košice, Slovak Republic
2nd Department of Internal Medicine, Comenius University, Bratislava, SK
Rowett Research Institute, Aberdeen AB21 9SB, UK
NEOPLASMA, 46, Supplement, 1999, pp. 119
A.M. Eastham, B. Marples, A.E. Kiltie, C.J. Orton, C.M.L. West , T.H. Ward
Department of Genome Damage and Repair, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester. M20 4BX UK
NEOPLASMA, 46, Supplement, 1999, pp. 120
V. Mezencevová, K. Rojková, M. Dušinská
Institute of Medical Biology, Medical Faculty University of Pavel Josef Safarik, SNP
1 04060 Košice, SK
Department of Biochemistry and Microbiology, Faculty of Chemical Technology, Slovak Technical University, Bratislava, SK
Department of Molecular and Genetic Toxicology, Institute of Preventive and Clinical Medicine, Limbová 14, 801 03, Bratislava, SK
We applied the modified comet assay for detection of strand breaks and oxidised bases in lymphocytes of psoriatic patients before and after the treatment with therapeutic coal tar. Ten psoriatic patients were treated for 14-21 days. Lymphocytes of psoriatic patients taken before the treatment had significantly higher levels of DNA strand breaks compared with matched controls. Moreover, after the treatment, DNA breaks as well as oxidised bases in lymphocytes were significantly elevated compared with levels of DNA damage before the treatment. The frequencies of chromosomal aberrations in the same group of patients were also higher after the treatment compared with no treatment. Patients who smoke had more DNA damage than non-smokers; this effect seems stronger after the treatment with coal tar. Numbers of strand breaks in all patients correlated with range of disease (size of damaged skin), both before (r=0.677; p=0.035) and after the treatment (r=0.697; p=0.025).
Key words: Psoriasis, therapeutic coal tar, the comet assay, DNA damage
NEOPLASMA, 46, Supplement, 1999, pp. 121-122