P-32 POSTLABELING ANALYSIS OF DNA-ADDUCTS IN HUMAN SPERM CELLS FROM SMOKERS AND NONSMOKERS

To determine the feasibility of using human sperm cells for DNA P-32-p ostlabeling analyses, and to evaluate the baseline level and the possi ble presence of smoking-related DNA adducts in these cells, sperm DNA was isolated from specimens obtained from 12 heavy smokers, 12 light s mokers, and 12 nonsmokers. Background levels of radioactivity were min imized by using magnet transfer of P-32-labeled mononucleotides to new polyethyleneimine cellulose plates. Compared with placental tissues, few adducts were observed. Diffuse radioactivity observed in some of t he autoradiograms was minimally above background but the level of radi oactivity expressed as putative adducts/nucleotide was not related to smoking status. It was not clear, in some cases, whether this radioact ivity was associated with chemically bound adducts or was from nonspec ifically bound chemicals, radiolabeled enzymes, or other proteins. One major discrete DNA adduct of unknown chemical structure was detected in three of the 36 samples analyzed (one nonsmoker and two smokers). B ased on the level of radioactivity associated with various dilutions o f a benzo(a)pyrene-derived adduct, our limit of sensitivity was at lea st 1.2 adducts/10(9) nucleotides. Our study emphasizes the need to mor e clearly define the significance of background radioactivity associat ed with DNA adduct maps where the measured adduct levels approximate d etection limits defined by visual observance of adduct spots. This poi nt is particularly relevant given that the P-32-postlabeling procedure s rely, in part, on visual verification of the presence of DNA adducts .