DETERMINATION OF DNA SINGLE-STRAND BREAKS IN LYMPHOCYTES OF SMOKERS AND NONSMOKERS EXPOSED TO ENVIRONMENTAL TOBACCO-SMOKE USING THE NICK TRANSLATION ASSAY

The detection of DNA single-strand breaks (SSB) in human mononucleated white blood cells (MWBC) using a modified version of the nick transla tion assay is presented. This assay allows rapid and sensitive examina tion of SSB using only 5 ml heparinized blood for an eightfold determi nation. 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 incre ase in DNA-SSB at doses between 5 and 500 mu M MNNG. The detection lim it for the assay was 5 mu M MNNG. To assess the suitability of this as say to detect SSB in vivo a controlled study was performed in which vo lunteer smokers (n = 5), nonsmokers (n = 5) exposed to environmental t obacco smoke (ETS), and nonsmoker controls (n = 5) were compared. The study lasted 4 experimental days, 2 control and 2 exposure days. On co ntrol days (days 1 and 3) smokers and nonsmokers sat in an unventilate d 45 m(3) room for 8 h. On the exposure days (days 2 and 4) each of th e five smokers smoked 24 cigarettes in 8 h, while the five nonsmokers were exposed to the ETS generated by the smoking volunteers. High expo sure to tobacco smoke was confirmed by dosimetry of carboxyhemoglobin (COHb), plasma nicotine and cotinine levels. Blood was drawn before an d after each exposure on all 4 experimental days for determination of DNA-SSB in lymphocytes immediately after isolation of blood cells. COH b, plasma nicotine, and cotinine levels were considerably increased in both smokers and nonsmokers exposed to ETS on days 2 and 4. DNA-SSB w ere 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 exposu re-related variation in SSB levels was found. We conclude that the mod ified 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.