Adjacent guanines as preferred sites for strand breaks in plasmid DNA irradiated with 193 nm and 248 nm UV laser light

Kainate-preferring glutamate receptors may contribute to the glutamatergic responses to seizures. The cloning of their encoding genes overcomes limitations of the receptor ligands available for their investigation. We have examined the expression of the high affinity kainate receptor subunits KA1 and KA2 mRNAs in the rat hippocampus, using electroconvulsive shock (ECS) as a seizure paradigm not confounded by neurotoxicity. A single shock reduced the levels of KA1 mRNA in the CA3c region, while increasing the expression of KA2 mRNA in the dentate gyrus. Following repeated ECS (5 shocks over 10 days), KA1 mRNA was reduced in CA3c and in CA3a-b but was unchanged in dentate gyrus. KA2 mRNA, on the other hand, significantly increased in dentate gyrus, and to a lesser extent in CA3c and CA1. All changes in KA1 and KA2 mRNAs had returned to baseline 3 weeks after the last shock. We also measured the expression of cyclophilin mRNA, and found it to be reduced in all hippocampal subfields, and in the parietal cortex, after a single ECS. It returned to control levels after repeated ECS but was again reduced following 3 weeks recovery from repeated ECS. These results indicate that the expression of KA1 and KA2 not only change in opposite directions in the rat hippocampus after ECS, but that the alterations are anatomically and temporally regulated. In the respect that cyclophilin is regarded as a housekeeping gene, the reduction in its mRNA suggests that ECS may have more persistent and widespread effects on brain gene expression than previously suspected.     

Non-random distribution of DNA double-strand breaks induced by particle irradiation

Induction of DNA double-strand breaks (dsbs) in mammalian cells is dependent on the spatial distribution of energy deposition from the ionizing radiation. For high LET particle radiations the primary ionization sites occur in a correlated manner along the track of the particles, while for X-rays these sites are much more randomly distributed throughout the volume of the cell. It can therefore be expected that the distribution of dsbs linearly along the DNA molecule also varies with the type of radiation and the ionization density. Using pulsed-field gel and conventional gel techniques, we measured the size distribution of DNA molecules from irradiated human fibroblasts in the total range of 0.1 kbp-10 Mbp for X-rays and high LET particles (N ions, 97 keV/microns and Fe ions, 150 keV/microns). On a mega base pair scale we applied conventional pulsed-field gel electrophoresis techniques such as measurement of the fraction of DNA released from the well (FAR) and measurement of breakage within a specific NotI restriction fragment (hybridization assay). The induction rate for widely spaced breaks was found to decrease with LET. However, when the entire distribution of radiation-induced fragments was analysed, we detected an excess of fragments with sizes below about 200 kbp for the particles compared with X-irradiation. X-rays are thus more effective than high LET radiations in producing large DNA fragments but less effective in the production of smaller fragments. We determined the total induction rate of dsbs for the three radiations based on a quantitative analysis of all the measured radiation-induced fragments and found that the high LET particles were more efficient than X-rays at inducing dsbs, indicating an increasing total efficiency with LET. Conventional assays that are based only on the measurement of large fragments are therefore misleading when determining total dsb induction rates of high LET particles. The possible biological significance of this non-randomness for dsb induction is discussed.     

Asbestos and DNA double strand breaks

A radiosensitive DNA repair-deficient xrs-5 cell line was used to study asbestos cytotoxicity and DNA double strand breaks (DSBs). Although xrs-5 cells did not show any increase in sensitivity to chrysotile fibers in short-term (4-h) treatment when compared with wild-type CHO cells, longer-term exposure (24 h) gave significantly lower cell survival accompanied by a cell growth delay as well as a higher DNA DSB induction in this mutant cell line. These results suggest an important role played by DNA DSBs at the initial stage of asbestos injury.     

Differential effects of DNA supercoiling on radical-mediated DNA strand breaks

Supercoiling is an important feature of DNA physiology in vivo. Given the possibility that the reaction of genotoxic molecules with DNA is affected by the alterations in DNA structure and dynamics that accompany superhelical tension, we have investigated the effect of torsional tension on DNA damage produced by five oxidizing agents: gamma-radiation, peroxynitrite, Fe2+/ EDTA/H2O2, Fe2+/H2O2, and Cu2+/H2O2. With positively supercoiled plasmid DNA prepared by a recently developed technique, we compared the quantity of strand breaks produced by the five agents in negatively and positively supercoiled pUC19. It was observed that strand breaks produced by gamma-radiation, peroxynitrite, and Fe2+/EDTA/H2O2 were insensitive to DNA superhelical tension. These results are consistent with a model in which chemicals that generate highly reactive intermediates (e.g., hydroxyl radical), but do not interact directly with DNA, will be relatively insensitive to the changes in DNA structure and dynamics caused by superhelical tension. In the case of Fe2+ and Cu2+, metals that bind to DNA, only Cu2+/H2O2 proved to be sensitive to DNA superhelical tension. Strand breaks produced by Cu2+/H2O2 in the positively supercoiled substrate occurred at lower Cu concentrations than in negatively supercoiled DNA. Furthermore, a sigmoidal Cu2+/H2O2 damage response was observed in the negatively supercoiled substrate but not in positively supercoiled DNA. The results with Cu2+ suggest that the redox activity, DNA binding orientation, or DNA binding affinity of Cu1+ or Cu2+ is sensitive to superhelical tension, while the results with the other oxidizing agents warrant further investigation into the role of supercoiling in base damage.     

DNA strand breaks in human nasal respiratory epithelium are induced upon exposure to urban pollution

All organisms have the ability to respond and adapt to a myriad of environmental insults. The human respiratory epithelium, when exposed to oxidant gases in photochemical smog, is at risk of DNA damage and requires efficient cellular adaptative responses to resist the environmentally induced cell damage. Ozone and its reaction products induce in vitro and in vivo DNA single strand breaks (SSBs) in respiratory epithelial cells and alveolar macrophages. To determine if exposure to a polluted atmosphere with ozone as the main criteria pollutant induces SSBs in nasal epithelium, we studied 139 volunteers, including a control population of 19 children and 13 adult males who lived in a low-polluted Pacific port, 69 males and 16 children who were permanent residents of Southwest Metropolitan Mexico City (SWMMC), and 22 young males newly arrived to SWMMC and followed for 12 weeks. Respiratory symptoms, nasal cytology and histopathology, cell viabilities, and single-cell gel electrophoresis were investigated. Atmospheric pollutant data were obtained from a fixed-site monitoring station. SWMMC volunteers spent >7 hr/day outdoors and all had upper respiratory symptoms. A significant difference in the numbers of DNA-damaged nasal cells was observed between control and chronically exposed subjects, both in children (p<0.00001) and in adults (p<0.01). SSBs in newly arrived subjects quickly increased upon arrival to the city, from 39.8 +/- 8.34% in the first week to 67.29 +/- 2.35 by week 2. Thereafter, the number of cells with SSBs remained stable in spite of the continuous increase in cumulative ozone, suggesting a threshold for cumulative DNA nasal damage. Exposure to a polluted urban atmosphere induces SSBs in human nasal respiratory epithelium, and nasal SSBs could serve as a biomarker of ozone exposure. Further, because DNA strand breaks are a threat to cell viability and genome integrity and appear to be a critical lesion responsible for p53 induction, nasal SSBs should be evaluated in ozone-exposed individuals.     

Atomic force microscopy investigation of radiation-induced DNA double strand breaks

We have used atomic force microscopy (AFM) to study radiation-induced DNA double strand breaks. Double-stranded plasmid DNA was irradiated with 18-MeV electrons in aqueous buffer, using a medical linear accelerator. Doses of 50, 100, 150, and 200 Gy were delivered to DNA samples, and atomic force microscopy was used to measure the length of each DNA fragment. From these measurements, we obtained the average length of the irradiated DNA for each sample and found a linear-quadratic relationship between the average length and radiation dose.     

Single strand breaks and mutagenesis in yeast induced by photodynamic treatment with chloroaluminum phthalocyanine

The CD53 antigen is a member of the tetraspan family of proteins with unknown function. Stimulation of rat IR938F B-cell lymphoma cells with monoclonal antibody MRC OX44 (anti-rat CD53) triggered a homotypic adhesion reaction which reached a maximum effect at 24 hr. This effect occurred at 37 degrees C but not at 4 degrees C. Adhesion was prevented by removal of divalent cations, Ca2+ and Mg2+, with EGTA and EDTA as chelating agents. The adhesion induced by MRC OX44 was inhibited by cycloheximide and actinomycin D, suggesting that de novo protein synthesis was required for this effect. The addition of mAb WT1 against rat LFA-1 (CD11a) antigen had no effect on adhesion, suggesting that the cell-cell interaction is not mediated by the expression of LFA-1 antigen. The intracellular signals required to induce adhesion were inhibited by two tyrosine kinase inhibitors, genistein and piceatannol. Wortmannin, a selective inhibitor of phosphoinositide 3-kinase activity, completely blocked adhesion. Two protein kinase C inhibitors, H7 and bisindolylmaleimide, inhibited the adhesion, suggesting that part of the signal is mediated by PKC. Electron microscopy of aggregated cells showed that the interaction is localized to short membrane regions, where contact areas of higher density in opposing zones from both cells were detected. We postulate that there is a common adhesion mechanism that is modulated by several tetraspan family members and associated proteins. This adhesion structure might represent a novel form of cell communication among lymphoid cells.     

Detection of apoptosis at the single-cell level by direct incorporation of fluorescein-dUTP in DNA strand breaks

Apoptosis induced in different cell lines can be detected and quantified in one step. Direct labeling of apoptotic cells (DLAC) was performed by incorporation of fluorescein-dUTP (F-dUTP) in DNA strand breaks by terminal deoxynucleotidyl transferase (TdT). Nick-end-labeling using F-dUTP obviates the need for second-step revelation reagents without reducing the specificity of detection. Cells were analyzed by microscopy or flow cytometry for objective quantification of apoptotic cells in controlled dilution samples. Furthermore, we demonstrate that DLAC is compatible with surface labeling by monoclonal antibodies, allowing dual-color analysis. The data presented here illustrate the simplicity and potential of this method, which allows the preservation of fragile cells and the possibility of combining apoptosis detection with immunostaining.     

A study of apoptosis in human glomerulonephritis as determined by in situ non-radioactive labelling of DNA strand breaks

With a morbidity of 0.8%, epilepsy in childhood is one of the most frequent chronic diseases of the CNS. Improved diagnostic and therapeutical options of the last two decades made it possible that today 60-70% of the patients are without attacks for a long period and are fully socially integrated. Epilepsy is clinically classified into generalized epilepsies; which are of symptomatic origin in 50% of the cases, and focal epilepsies, which are even more frequent caused by CNS-injuries. The diagnosis of epilepsy results from the synopsis of the clinical picture of the attack and the electro-encephalogram (EEG), in case of doubts from the analysis of the video-EEG. The anti-convulsive drug therapy is undertaken as long-term treatment when free of attacks for 3-5 years and has to be monitored clinically as well as biochemically. The option of epilepsy-surgical procedures in a center of epilepsy should be considered in cases of a resistance to drug therapy. The tight cooperation with the family physician is of great importance for the ambulant epileptic care, others like social service, careers guidance and psychologic service have to be integrated.     

Myocardial DNA strand breaks are detected in biopsy tissues from patients with dilated cardiomyopathy

A 68-year-old woman was admitted to Kinki University Hospital because of progressive renal failure. She had been well until two months before admission. Laboratory data were as follows: serum creatinine 4.1 mg/dl, BUN 69 mg/dl, MPO-ANCA 33 EU, anti-glomerular basement membrane antibodies (AGBMA) 118 U. Histological findings showed cellular and fibrocellular crescents in many glomeruli. Therefore, we diagnosed rapidly progressive glomerulonephritis (RPGN) due to MPO-ANCA and anti-GBM associated renal disease. The patient was started on prednisolone and double filtration plasmapheresis (DFPP) therapy. Subsequently, the values of MPO-ANCA and AGBMA decreased. However, the patient's condition suddenly worsened and she died of interstitial pneumonia. Autopsy examination revealed crescentic glomerulonephritis and alveolar hemorrhage with linear deposition of IgG along the glomerular and alveolar capillary walls by immunofluorescence studies. We considered this to be a rare case of Goodpasture's syndrome associated with not only anti-GBM antibodies, but also MPO-ANCA.     

Comparison of strand breaks in plasmid DNA after positional changes of Auger electron-emitting iodine-125

To elucidate the kinetics of the induction of DNA strand breaks by low-energy Auger electron emitters, we compared the yields of DNA breaks in supercoiled pUC19 DNA after the decay of 125I (1) in proximity to DNA after minor-groove binding (125I-iodoHoechst 33342, 125IH) and (2) at a distance from DNA (125I-iodoantipyrine, 125IAP). Iodine-125 bound to the minor groove in DNA or free in solution is equally effective per decay in producing single-strand breaks (SSBs), while 125I bound to the minor groove is 6.7-fold more efficient than 125I free in solution in producing double-strand breaks (DSBs) (1.08 +/- 0.13 compared to 0.16 +/- 0.01 DSB/decay). Consequently, SSB to DSB ratios for 125IAP and gamma radiation (20.7 +/- 2.9 and 43.8 +/- 1.5, respectively) are greater than that for 125IH (2.9 +/- 0.4). Finally, the decay of 125IH leads to fragmentation of plasmid DNA beyond SSBs and DSBs.     

Prolongation of the p53 response to DNA strand breaks in cells depleted of PARP by antisense RNA expression

The observation that 3-aminobenzamide, which inhibits a variety of ADP-ribose transferases, prolongs the gamma-irradiation-induced increase in intracellular p53 concentration suggested that one or more of such enzymes may determine the duration of the p53 response during G1 arrest. The role of poly(ADP-ribose) polymerase (PARP), an abundant nuclear enzyme activated by DNA strand breaks, in the p53 response to y-irradiation was investigated in Burkitt's lymphoma AG876 cells stably transfected with an inducible PARP antisense construct. Immunoblot analysis revealed that the cellular content of PARP was reduced to virtually undetectable levels after incubation of transfected cells for 72 h with the inducer dexamethasone. In noninduced antisense cells, the p53 concentration reached a maximum 2 h after exposure to 6.3 Gy of gamma-radiation and returned to control values by 4 h. In contrast, the p53 response in PARP-depleted antisense cells peaked at 4 h, with the levels of p53 remaining elevated for up to 12 h after y-irradiation. The maximal increase in p53 concentration was similar in both induced and noninduced cells. These results thus indicate that PARP activity, in part, determines the duration, but not the magnitude, of the p53 response to DNA damage.     

Homologous recombination in plant cells is enhanced by in vivo induction of double strand breaks into DNA by a site-specific endonuclease

Induction of double strand breaks (DSBs) is coupled to meiotic and mitotic recombination in yeast. We show that also in a higher eukaryote induction of DSBs is directly correlated with a strong enhancement of recombination frequencies. We cotransfected Nicotiana plumbaginifolia protoplasts with a plasmid carrying a synthetic I-SceI gene, coding for a highly sequence specific endonuclease, together with recombination substrates carrying an I-SceI-site adjacent to their homologous sequences. We measured efficiencies of extrachromosomal recombination, using a well established transient beta-glucuronidase (GUS) assay. GUS enzyme activities were strongly increased when a plasmid carrying the I-SceI gene in sense but not in antisense orientation with respect to the promoter was included in the transfections. The in vivo induced DSBs were detected in the recombination substrates by Southern blotting, demonstrating that the yeast enzyme is functional in plant cells. At high ratios of transfected I-SceI-genes to I-SceI-sites the majority of the I-SceI-sites in the recombination substrates are cleaved, indicating that the induction of the DSBs is the rate limiting step in the described recombination reaction. These results imply that in vivo induction of transient breaks at specific sites in the plant genome could allow foreign DNA to be targeted to these sites via homologous recombination.     

In vitro induction of primary DNA double-strand breaks in leukemic and normal blood cells by ultraviolet irradiation

The yield of UV-induced DNA double-strand breaks was studied for white blood cells ("light" fraction) derived from peripheral blood, and from patients with lymphomas, chronic lymphoid leukemia (CLL), and chronic myeloid leukemia (CML). The method employed was constant-field electrophoresis of plug-embedded DNA in agarose gel. Characteristic dose-response curves were obtained for various cell populations. Lymphoid cells, both from healthy subjects and CLL patients, revealed less damage to DNA under UV-irradiation, whereas CML cells were much more affected. Possible interpretation of these results includes species-specific differences in UV-induced DNA damage, as well as sufficient DNA crosslinking, thus interfering with DNA dsbs detection in irradiated cells.     

Nitric oxide synergistically enhances DNA strand breakage induced by polyhydroxyaromatic compounds, but inhibits that induced by the Fenton reaction

This paper deals with the muscle force of the cervical spine in patients with cervical syndrome compared to the muscle activity in non-patients. It has thus been observed that both the flexors' and the extensors' force of the cervical spine is much greater in non-patients (the flexors' P < 0.01 and the extensors' P < 0.01). The precise measurements having been carried out by a dynamometer. The groups being tested comprised 50 persons each, 25 men and 25 women. The average age of the patients group was 43.1 years, namely between 21 and 57 years, while that of the non-patients group was 42.3 years, between 24 and 56 years. As regards the professional engagement of both groups and, accordingly, the strain on the cervical spine and the corresponding musculature, they were approximately the same.     

Developing a model of DNA replication to be used for Monte Carlo calculations that predict the sizes and shapes of molecules resulting from DNA double-strand breaks induced by X irradiation during DNA synthesis

A Monte Carlo computer program was written to introduce double-strand breaks (DSBs) randomly into cellular DNA that is configured according to different models of DNA replication. Then, from a review of the literature using DNA fiber autoradiography and other studies relating to rates of replication of DNA that is organized in approximately 3-Mbp regions or bands, a particular model for DNA replication was developed. Using this model, Monte Carlo calculations were made to predict the types and sizes of molecules that would result from introducing DSBs into DNA when synchronous cells are irradiated in the middle of S phase. Then results of the Monte Carlo calculations were compared with migration profiles obtained by pulsed-field gel electrophoresis (PFGE) for molecular size distributions of linear DNA molecules. For these comparisons, CHO cells irradiated in S phase also were pulse-labeled at the time of irradiation with [3H]dThd for 15 min to compare the migration patterns of 3H-labeled replicating DNA with those of the mass of S-phase DNA, measured by imaging with a CCD camera. For the Monte Carlo calculations, we assumed from the reports in the literature that molecules containing replication bubbles with and without forks would be trapped in the PFGE plug. We also assumed that those molecules that are < or = 8 Mbp, both linear and with replication forks, would be released into the lane. However, approximately 75% of the 3H-labeled DNA that is released from the plug migrated much more slowly than linear molecules, which we attributed to the slow migration of 3H-labeled molecules having replication forks not attached to bubbles. The percentages of both mass of S-phase DNA and 3H-labeled replicating DNA released from the plug, as determined by PFGE, were compared with comparable values determined from Monte Carlo calculations. A DNA replication model that provides good agreement between the PFGE results and Monte Carlo calculations is described. Furthermore, Monte Carlo methodology is presented that can be used for comparing data obtained with PFGE with results of Monte Carlo calculations that are based on different models of DNA replication and different assumptions for the migration of various types of replicating molecules.     

Comparative study of the repair kinetics of chromosomal aberrations and DNA strand breaks in proliferating and quiescent CHO cells

Repair kinetics observable at the level of exchange-type chromosomal aberrations (dicentric chromosomes), using fractionation and delayed-plating techniques, have been compared with repair kinetics of radiation-induced DNA double-strand breaks, measured with PFGE, and with repair kinetics of all strand breaks, measured with the alkali-unwinding technique. Only data from quiescent or proliferating CHO K1 cells obtained in the same laboratory were used. We determined repair kinetics in terms of the time constant tau (equal to half-time/log(e)2). The repair kinetics (tau approximately 11-14 min) observed in the split-dose formation of dicentric chromosomes agrees with fast repair kinetics of double-strand breaks (tau approximately 11-13 min), thus permitting us to identify the latter as the 'primary lesions' whose pairwise interaction leads to the beta D2 yield term of the aberrations. The repair kinetics observed for dicentric chromosomes formed under delayed-plating conditions (tau approximately 75 min), which mainly affects the alpha D yield term, is attributed to an intermediate interchromosomal product temporarily existing in the course of aberration formation; it is suggested that this product is mechanistically correlated with the slow repair kinetics of 'clustered damage' to DNA seen with the applied molecular methods (tau approximately 90 min).     

Stimulation of mitochondrial respiration, induced by laser irradiation in the presence of rhodamine dyes

The effect of micromolar concentration of rhodamine 123 (methylrhodamine) and ethyl and amyl esters of unsubstituted rhodamine on oxygen consumption by rat liver mitochondria was studied under irradiation by an argon laser (488 and 514 nm). Irradiation of mitochondria in the presence of rhodamine stimulates their respiration. Light-induced stimulation of respiration is not inhibited by free radical scavenger ionol and by inhibitor of the permeability transition pore cyclosporine A. Stimulation of respiration by moderate doses of radiation is reversed in the dark. Increase in radiation dose resulted in only partial reversal of stimulated respiration in the dark. Rhodamine efficacy in stimulation of mitochondrial respiration depends on its structure (amyl > ethyl > methylrhodamine).     

Graft decentration in penetrating keratoplasty: nonmechanical trephination with the excimer laser (193 nm) versus the motor trephine

Seven right-handed participants performed bimanual circling movements in either a symmetrical or an asymmetrical coordination mode. Movements were paced with an auditory metronome at predetermined frequencies corresponding to transition frequency, where asymmetrical patterns became unstable, or at two-thirds transition frequency where both symmetrical and asymmetrical patterns were stable. The pacing tones were presented in either a high (1000 Hz) or low (500 Hz) pitch, and the percentage of high-pitched tones during a 20 s trial varied between 0% and 70%. Participants were instructed to count the number of high-pitched pacing tones that occurred during a trial of bimanual circling. Overall, the symmetrical pattern was more stable than the asymmetrical pattern at both frequencies. Errors on the tone-counting task were significantly higher during asymmetrical circling than symmetrical circling but only at the transition movement frequency. The results suggest that cognitive processes play a role in maintaining coordination patterns within regions of instability.