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.
DNA single-strand breaks DNA repair Nick translation Smoking Environmental tobacco smoke Biological monitoring
Dulbecco's modification of Eagle's minimal essential medium