Analysis of acrolein-derived 1, N ²-propanodeoxyguanosine adducts in human leukocyte DNA from smokers and nonsmokers

Cigarette smoking is a major source of human exposure to acrolein, a widespread environmental pollutant and toxicant that is also formed endogenously through metabolism of amino acids and polyamines and lipid peroxidation. Acrolein reacts with DNA, producing two pairs of regioisomeric 1,N ²-propanodeoxyguanosine adducts: (6R/S)-3-(2′-deoxyribos- 1′-yl)-5,6,7,8-tetrahydro-6-hydroxypyrimido[1,2-a]purine-10(3H)one (α-OH-Acr-dGuo) and (8R/S)-3-(2′-deoxyribos-1′-yl)-5,6,7,8- tetrahydro-8-hydroxypyrimido[1,2-a]purine-10(3H)one (γ-OH-Acr-dGuo). Previous studies indicate that these adducts might be involved in producing mutations in the p53 tumor suppressor gene, as observed in lung tumors in smokers, but there are only limited published data comparing acrolein-DNA adducts in smokers and nonsmokers. In this study, we developed a liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method to analyze Acr-dGuo adducts in human leukocyte DNA. The potential for artifactual formation was found in two steps of the assay: DNA isolation and DNA hydrolysis. This was eliminated by employing a Ficoll-Hypaque double density gradient to obtain leukocytes free of erythrocyte contamination and by adding glutathione to scavenge acrolein present in H₂O. The accuracy and precision of the method were confirmed. Acr-dGuo adducts were analyzed in leukocyte DNA from 25 smokers and 25 nonsmokers. γ-OH-Acr-dGuo was the predominant isomer in all samples, while α-OH-Acr-dGuo was detected in only three subjects. There was no significant difference between the total Acr-dGuo levels in smokers (7.4 ± 3.4 adducts/10⁹ nucleotides) and nonsmokers (9.8 ± 5.5 adducts/10⁹ nucleotides). Although our study is limited in size, these results, together with the results of previous analyses of acrolein-derived mercapturic acids in the urine of smokers and nonsmokers, suggest that glutathione conjugation effectively removes acrolein from external exposures such as cigarette smoking, protecting leukocyte DNA from damage.