Detection and Quantitation of Acrolein-Derived 1,N²-Propanodeoxyguanosine Adducts in Human Lung by Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry

Abstract

Acrolein, a widely distributed environmental pollutant, reacts with dGuo in DNA to form two pairs of 1,N2-propano-dGuo adducts: (6R/S)-3-(2'-deoxyribos-1'-yl)-5,6,7,8-tetrahydro-6-hydroxypyrimido[1,2-a]purine-10(3H)one (alpha-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 (gamma-OH-Acr-dGuo). alpha-OH-Acr-dGuo is more mutagenic and mainly induces G --> T transversions. A recent study demonstrated that acrolein-DNA adducts are preferentially formed in p53 mutational hotspots in human lung cancer, but there are no reports on the presence of these adducts in the human lung. To directly investigate this question, we have developed a sensitive and specific liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method for the quantitative analysis of Acr-dGuo adducts in DNA. Our method is based on the enzymatic hydrolysis of DNA isolated from the human lung in the presence of [13C10,15N5]Acr-dGuo as internal standards. Acr-dGuo adducts are enriched from the hydrolysates by solid-phase extraction and analyzed by LC-ESI-MS/MS using selected reaction monitoring. The method is accurate and precise, and the identity of the adducts was confirmed by monitoring different transitions from the same parent ion and by carrying out reactions with NaOH and NaBH4, which produced N2-(3-hydroxypropyl)-dGuo or 1,N2-(1,3-propano)-dGuo from gamma-OH-Acr-dGuo and alpha-OH-Acr-dGuo, respectively. Thirty DNA samples from lung tissue were analyzed, and Acr-dGuo adducts were detected in all samples. Both alpha-OH- and gamma-OH-Acr-dGuo were observed in most of the samples; total adduct concentrations ranged from 16-209 adducts/109 nucleotides. These results demonstrate for the first time that both types of Acr-dGuo adducts are present in human lung DNA. There was no difference in adduct levels between current and ex-smokers. Collectively, the results support a plausible role for acrolein as one cause of p53 mutations in the human lung.

Interstrand DNA Cross-Links Induced by α,β-Unsaturated Aldehydes Derived from Lipid Peroxidation and Environmental Sources

“…[36][37][38] Enal adducts are lower oxidation state homologues of 21 and the notion that acrolein, crotonaldehyde, and 4-HNE undergo similar chemistry was confirmed by the observation that γ-OH-PdG (9) ring-opens to the N 2 -(3-oxopropyl)-dG aldehyde (1) when placed opposite dC. 39 We further hypothesized that these aldehydes react with other nucleobases in the complementary DNA strand, forming interstrand cross-links, which exist as equilibrium mixtures of carbinolamine (17), imine (18), or pyrimidopurinone (19) species. The aldehyde in structure 1 also yields peptide-and protein-DNA conjugates (20); 40,41 however, analysis of this literature is beyond the scope of this Account.…”

“…17,[66][67][68] Since they equilibrate with non-cross-linked species, and require the presence of the 5′-CpG-3′ sequence, they may be present at low levels in tissue samples. Nevertheless, it has been reported that acrolein preferentially binds at 5′-CpG-3′ sites, a consequence of cytosine methylation at these sequences.…”

“…57 The identification of the cross-link in duplex DNA as the carbinolamine (17) and not the pyrimidopurinone (19) 56 was accomplished by isotope-edited NMR experiments in which oligodeoxynucleotide 43 containingγ-OH-15 N 2 -PdG was annealed with its complementary strand. A 15 N-HSQC filtered spectrum revealed the NOE between X 7 15 N 2 H and the imino proton X 7 N1H (Figure 8), precluding the pyrimidopurinone structure (19).…”

“…15,16 The principal acrolein adduct is γ-OH-PdG (9), 10,12 although the regioisomeric 6-hydroxypyrimido[1,2-a]purin-10(3H)-one (α-OH-PdG, 10) has also been observed. 12,17 The γ-OH-PdG adduct (9) exists as a mixture of C8-OH epimers. With crotonaldehyde, addition at N 2 -dG creates a stereocenter at C6.…”

“…[2][3][4]17,21 The binding pattern of acrolein-DNA adducts is similar to the p53 mutational pattern in human lung cancer, implicating acrolein as a major cigarette-related lung cancer agent. 22 Acrolein is mutagenic in bacterial and mammalian cells, 23,24 including human, 25,26 and is carcinogenic in rats.…”