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  • 1
    Electronic Resource
    Electronic Resource
    Determination of DNA single-strand breaks in lymphocytes of smokers and nonsmokers exposed to environmental tobacco smoke using the nick translation assay (1994)
    Springer
    Journal of molecular medicine 72 (1994), S. 930-936 
    Details
    ISSN: 1432-1440
    Keywords: DNA single-strand breaks ; DNA repair ; Nick translation ; Smoking ; Environmental tobacco smoke ; Biological monitoring
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The detection of DNA single-strand breaks (SSB) in human mononucleated white blood cells (MWBC) using a modified version of the nick translation assay is presented. This assay allows rapid and sensitive examination of SSB using only 5 ml heparinized blood for an eightfold determination. The assay was standardized by incubation of MBWC in vitro with N-methyl-N′-nitro-N-nitro-soguanidine (MNNG), a known genotoxic agent. In vitro incubation of MWBC with MNNG induced a dose-dependent increase in DNA-SSB at doses between 5 and 500 μM MNNG. The detection limit for the assay was 5 μM MNNG. To assess the suitability of this assay to detect SSB in vivo a controlled study was performed in which volunteer smokers (n = 5), nonsmokers (n = 5) exposed to environmental tobacco smoke (ETS), and nonsmoker controls (n = 5) were compared. The study lasted 4 experimental days, 2 control and 2 exposure days. On control days (days 1 and 3) smokers and nonsmokers sat in an unventilated 45 m3 room for 8 h. On the exposure days (days 2 and 4) each of the five smokers smoked 24 cigarettes in 8 h, while the five nonsmokers were exposed to the ETS generated by the smoking volunteers. High exposure to tobacco smoke was confirmed by dosimetry of carboxyhemoglobin (COHb), plasma nicotine and cotinine levels. Blood was drawn before and after each exposure on all 4 experimental days for determination of DNA-SSB in lymphocytes immediately after isolation of blood cells. COHb, plasma nicotine, and cotinine levels were considerably increased in both smokers and nonsmokers exposed to ETS on days 2 and 4. DNA-SSB were detected in all volunteers with intra-and interindividual day to day and morning to evening variations. After smoking, SSB increased on day 2 and on day 4 in smokers. In nonsmokers exposed to ETS no exposure-related variation in SSB levels was found. We conclude that the modified nick translation assay is sensitive enough to detect SSB induced in vivo by exposure to a genotoxic agent and could therefore be used in biological monitoring at the workplace.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00190755
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  • 2
    Electronic Resource
    Electronic Resource
    A novel technique for the detection of DNA single-strand breaks in human white blood cells and its combination with the unscheduled DNA synthesis assay (1993)
    Springer
    International archives of occupational and environmental health 65 (1993), S. 77-82 
    Details
    ISSN: 1432-1246
    Keywords: DNA single-strand breaks ; Biological monitoring ; Nick translation ; Unscheduled DNA synthesis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary A modified assay for the detection of DNA single-strand breaks (SSBs) in human mononucleated white blood cells (MWBCs) based on the nick translation (NT) reaction was developed and combined with the test for unscheduled DNA synthesis (UDS). Both assays were performed on disposable 96-well filtration plates and therefore allowed rapid and sensitive examination of SSBs and UDS. Only 5–8 ml of heparinized blood is required for an eightfold determination in both assays. The uptake of radioactive nucleotide precursors was demonstrated to depend linearly upon the NT reaction time and in both assay systems on the number of investigated cells. The best results and the lowest signal to noise ratio were obtained when the NT assay was performed at 25°C for 20 min. The test was standardized for 150000MWBCs/well and a polymerase I concentration of 20 U/ml. The same number of cells were used to measure UDS during a 4-h incubation at 37°C. We observed a dose-dependent increase in SSBs after in vitro incubation with N-methyl-N-nitrosoguanidine (MNNG), with a detection limit of 50 μM when MNNG was present for 1 h and of 5μM after 20-h incubation period. UDS in MWBCs was increased after treatment for 1 h with MNNG (200 μM) only if poly(ADP)ribose synthesis was inhibited by 3-aminobenzamide. UDS was induced by 320 μM methyl methanesulfonate, but SSBs could only be detected after inhibition of UDS by 100 μM hydroxyurea. The described modification of the NT procedure for the detection of SSBs in DNA of human MWBCs and its combination with the detection of UDS could serve as a useful tool for biological monitoring in occupational or environmental medicine.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00405723
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  • 3
    Electronic Resource
    Electronic Resource
    Detection of DNA single-strand breaks in lymphocytes of smokers (1993)
    Springer
    International archives of occupational and environmental health 65 (1993), S. 83-88 
    Details
    ISSN: 1432-1246
    Keywords: DNA single strand breaks ; Nick translation ; Unscheduled DNA synthesis ; Smoking ; DNA repair
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary In a controlled study, ten male volunteers (five smokers and five nonsmokers) were subjected to different smoking conditions and compared to five non-smokers, not exposed to cigarette smoke. During the 4 days of the study, nonsmoking periods were strictly controlled. On the first day the ten subjects were sham exposed. On the second day the five smokers smoked 24 cigarettes in 8 h, while the five nonsmokers were exposed to the environmental tobacco smoke. After another day of sham exposure the smoke exposure was repeated under the same conditions. Blood was drawn before and after exposure and DNA single-strand breaks (SSBs) were analyzed in lymphocytes immediately (1 h) after isolation of cells and after 4 h incubation at 37°C, using a modified assay based on the nick translation reaction. Base levels of unscheduled DNA synthesis (UDS) and UDS levels were determined after 1 h incubation with methyl methanesulfonate. Duplicate analysis using the same method was performed in a second laboratory after transportation of blood samples at 0°C on a train from Munich to Hamburg. Tobacco smoke exposure of the subjects increased COHb and plasma cotinine levels. SSBs could be detected in all probands with some inter-individual day-to-day and morning-to-evening variations. In four of five active smokers, SSB increases were found after smoking. In nonsmokers exposed to tobacco smoke no exposure-related variation in SSB levels could be detected. In lymphocytes which were incubated in culture medium (DME/H) for 4 h at 37°C, SSBs correlated significantly with the SSBs of fresh (NT1) samples but the SSB level was lower in almost all cases and the effect of smoking was not as pronounced as in the NT1 samples. Larger interindividual variations and higher values in general were detected after 8–9h of transportation. Therefore, we recommend immediate determination of SSBs as soon as possible after blood sampling. We conclude that the modified nick translation assay is sensitive enough to detect SSBs caused by an in vivo genotoxic exposure when possible interindividual differences are considered in the study design and could therefore be used in biological monitoring of exposures at the workplace.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00405724
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