DETECTION OF DNA SINGLE-STRAND BREAKS IN LYMPHOCYTES OF SMOKERS

In a controlled study, ten male volunteers (five smokers and five nonsmokers) were subjected to different smoking conditions and compared to five nonsmokers, 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-degrees-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-degrees-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 interindividual 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-degrees-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 work-place.