Analysis of Acrolein-Derived 1,N²-Propanodeoxyguanosine Adducts in Human Leukocyte DNA from Smokers and Nonsmokers

Abstract

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-AcrdGuo). 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 non-smokers. 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. 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 H 2 O. The accuracy and precision of the method were confirmed. Acr-dGuo adducts were analyzed in leukocyte DNA from 25 smokers and 25 non-smokers. γ-OH-Acr-dGuo was the predominant isomer in all samples, while α-OH-Acr-dGuo was detected in only 3 subjects. There was no significant difference between the total Acr-dGuo levels in smokers (7.4 ± 3.4 adducts/10 9 nucleotides) and non-smokers (9.8 ± 5.5 adducts/10 9 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 non-smokers, suggest that glutathione conjugation effectively removes acrolein from external exposures such as cigarette smoking, protecting leukocyte DNA from damage.

Toxicology and risk assessment of acrolein in food

“…Significantly lower levels of the a-OH-Acr-dGuo adduct were reported compared to g-OH-Acr-dGuo [57]. Zhang et al [58] also detected primarily g-OH-Acr-dGuo and hardly any a-OH-AcrdGuo adducts in leucocytes of smokers and non-smokers but found no differences in the adduct levels of g-OH-Acr-dGuo between smokers and non-smokers. Furthermore, human lung tissue of smokers or former smokers (1 wk to 26 years since last smoking) was examined for a-and g-OH-Acr-dGuo adducts whereby both isomers were detected.…”

“…Although a-OH-Acr-dGuo is formed after acrolein exposure in different organs in smaller amounts compared to g-OH-AcrdGuo [57,58], there could be an accumulation of a-OH-AcrdGuo in certain tissues such as the lung, so that similar levels of the two acrolein-guanosine adduct isomers develop [59].…”

Damage of DNA and proteins by major lipid peroxidation products in genome stability

“…These isomers were shown to have different mutagenic potency [42]. Other cyclic DNA adducts deriving from acrolein and crotonaldehyde occur in stereomeric forms as well [43,44].…”

High-throughput, Quantitative Analysis of Acrolein-derived DNA Adducts in Human Oral Cells by Immunohistochemistry

“…Acr reacts with dGuo in DNA to form two pairs of regioisomeric 1,N 2 -propanodeoxyguanosine adducts: α-OH-Acr-dGuo and γ-OH-Acr-dGuo ( Fig. 1) (Chung et al 1984;Zhang et al 2007Zhang et al , 2011. α-OH-Acr-dG in double-stranded DNA was found to be more mutagenic than γ-OH-Acr-dG in human cells and induced predominantly G→T transversions (Minko et al 2009;Yang et al 2002).…”