Mutagenicity and Sequence Specificity of Acrolein-DNA Adducts

Abstract

Acrolein (Acr) is a major toxicant in cigarette smoke (CS); it can interact with DNA forming two major adduct isomers: alpha-OH-Acr-dG and gamma-OH-Acr-dG. Previously, we found that the Acr-DNA binding pattern in the human p53 gene coincides with the p53 mutational pattern in CS-related lung cancer; hence, we proposed that Acr is a major lung cancer etiological agent [ Feng , Z. , Hu , W. , Hu , Y. , and Tang , M.-s. ( 2006 ) Acrolein is a major cigarette-related lung cancer agent: Preferential binding at p53 mutational hotspots and inhibition of DNA repair . Proc. Natl. Acad. Sci. U.S.A. 103 , 15404 - 15409 ]. This hypothesis has been brought into question with recent work that failed to detect Acr-induced mutations in the pSP189 system [ Kim , S. I. , Pfeifer , G. P. , and Besaratinia , A. ( 2007 ) Lack of mutagenicity of acrolein-induced DNA adducts in mouse and human cells . Cancer Res. 67 , 11640 - 116472 ]. To resolve this controversy, we determined the level and the type of Acr-dG formation, and the mutagenicity of Acr-dG adducts in the same pSP189 system. We also mapped the Acr-dG adduct distribution at the nucleotide level and the Acr-dG-induced mutational spectrum in this system. We found that (1) gamma-OH-Acr-dG is the major adduct formed in Acr-modified DNA based on the LC-ESI-MS/MS analysis; (2) the mutation frequency is proportional to the extent of Acr modifications, the majority of which are G:C to T:A and G:C to A:T mutations; and (3) sequences with a run of Gs are the mutational hotspots. Using the UvrABC nuclease incision method to map the Acr-dG distribution in the supF gene sequence, we confirmed that Acr-DNA adducts preferentially form in guanine-rich sequences that are also mutational hotspots. These results reaffirm that Acr-dG adducts are mutagenic and support our hypothesis that Acr is a major etiological agent for CS and cooking fume-related lung cancer.