DETECTION OF DNA SINGLE-STRAND BREAKS IN LYMPHOCYTES OF SMOKERS

In a controlled study, ten male volunteers (five smokers and five nons mokers) 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 smo kers smoked 24 cigarettes in 8 h, while the five nonsmokers were expos ed to the environmental tobacco smoke. After another day of sham expos ure the smoke exposure was repeated under the same conditions. Blood w as drawn before and after exposure and DNA single-strand breaks (SSBs) were analyzed in lymphocytes immediately (1 h) after isolation of cel ls and after 4 h incubation at 37-degrees-C, using a modified assay ba sed on the nick translation reaction. Base levels of unscheduled DNA s ynthesis (UDS) and UDS levels were determined after 1 h incubation wit h methyl methanesulfonate. Duplicate analysis using the same method wa s performed in a second laboratory after transportation of blood sampl es at 0-degrees-C on a train from Munich to Hamburg. Tobacco smoke exp osure 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, SS B 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 determ ination 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 interindiv idual differences are considered in the study design and could therefo re be used in biological monitoring of exposures at the work-place.