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 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Einhaus
M
M
Abteilung Arbeitsmedizin, Universität Wien, Austria.
Holz
O
O
Meissner
R
R
Krause
T
T
Warncke
K
K
Held
I
I
Scherer
G
G
Tricker
A R
AR
Adlkofer
F
F
Rüdiger
H W
HW
D000328:Adult; D004249:DNA Damage; D005821:Genetic Techniques; D006801:Humans; D008214:Lymphocytes / Q000378:metabolism*; D012680:Sensitivity and Specificity; D012907:Smoking / Q000009:adverse effects*; D014028:Tobacco Smoke Pollution / Q000009:adverse effects*
D014028:Tobacco Smoke Pollution