Determination of DNA single-strand breaks in lymphocytes of smokers and nonsmokers exposed to environmental tobacco smoke using the nick translation assay

• Published in: The Clinical investigator Vol. 72; no. 11; p. 930
• Main Authors: Einhaus, M, Holz, O, Meissner, R, Krause, T, Warncke, K, Held, I, Scherer, G, Tricker, A R, Adlkofer, F, Rüdiger, H W
• Format: Journal Article
• Language: English
• Published: Germany 01.11.1994
• Subjects: Adult; DNA Damage; Genetic Techniques; Humans; Lymphocytes - metabolism; Sensitivity and Specificity; Smoking - adverse effects; Tobacco Smoke Pollution - adverse effects
• ISSN: 0941-0198
• DOI: 10.1007/BF00190755

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-nitrosoguanidine (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 microM MNNG. The detection limit for the assay was 5 microM 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 nonsmokers 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 (CO-Hb), 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.