Formation of deoxyguanosine cross-links from calf thymus DNA treated with acrolein and 4-hydroxy-2-nonenal

Abstract

Acrolein (AC) and 4-hydroxy-2-nonenal (HNE) are endogenous bis-electrophiles that arise from the oxidation of polyunsaturated fatty acids. AC is also found in high concentrations in cigarette smoke and automobile exhaust. These reactive α,β-unsaturated aldehyde (enal) covalently modify nucleic acids, to form exocyclic adducts, where the three-carbon hydroxypropano unit bridges the N1 and N(2) positions of deoxyguanosine (dG). The bifunctional nature of these enals allows them to undergo reaction with a second nucleophilic group and form DNA cross-links. These cross-linked enal adducts are likely to contribute to the genotoxic effects of both AC and HNE. We have developed a sensitive mass spectrometric method to detect cross-linked adducts of these enals in calf thymus DNA (CT DNA) treated with AC or HNE. The AC and HNE cross-linked adducts were measured by the stable isotope dilution method, employing a linear quadrupole ion trap mass spectrometer and consecutive reaction monitoring at the MS(3) or MS(4) scan stage. The lower limit of quantification of the cross-linked adducts is ∼1 adduct per 10(8) DNA bases, when 50 μg of DNA is assayed. The cross-linked adducts occur at levels that are ∼1-2% of the levels of the monomeric 1,N(2)-dG adducts in CT DNA treated with either enal.

Figure 1

Structures of AC, crotonaldehyde, 4-HNE, and their dG adducts

Figure 2

Structures of dG cross-linked adducts of AC, crotonaldehyde and HNE (9), and their chemically reduced analogs (10).

Figure 3

Proposed structures of principal product ions arising at the MS/MSⁿ scan stages. Mass is parentheses are the ¹⁵N or ²H labeled internal standards. The sites of isotopic incorporation are noted on Scheme 1.

Figure 4

Time course of 6-HO-PdG, 8-HO-PdG, 8-dG-AC-dG (measured as the reduced, 8-dG-(CH₂)₃-dG cross-link), HNE-dG and 8-dG-HNE-dG cross-link formation in CT DNA. Panel A: 1 mol AC reacted per mol base in CT DNA over a time period of 0, 1, 2, 3, 6 and 10 days; Panel B: Levels of 6-HO-PdG, 8-HO-PdG and 8-dG-AC-dG (measured as the reduced, 8-dG-(CH₂)₃-dG cross-link) formed in CT DNA treated with 0.1, 0.5, 1 or 5 mol AC per mol base in CT DNA for 10 days; and Panel C: Time course of HNE-dG and 8-dG-HNE-dG cross-link formation in CT DNA treated with 2 mol HNE per mol base in CT DNA over a time period up to 21 days.The upper panels depict the monomeric DNA adducts and lower panels depict the cross-linked DNA adducts.

Figure 5

A reconstructed ion chromatogram of the LC-ESI/MS/MS³ traces of CT-DNA treated with AC. Panel A depicts untreated CT DNA; Panel B depicts untreated CT DNA chemically reduced with Na(CN)BH₃ and NaBH₄; Panel C depicts CT DNA treated with 1 mol AC per mol base in CT DNA for 10 days, followed by chemical reduction. For untreated DNA, the level of spiking with non-reduced monomer internal standards was 2.0 adducts per 10⁶ bases, reduced monomer internal standards were added at a level of 1.0 adducts per 10⁶ bases, and the spiking level with [²H₆]-dG-(CH₂)₃-dG was 1.2 adducts per 10⁶ bases. The level of spiking with monomeric internal standards was 2.2 adducts per 10⁵ bases and the spiking level with [²H₆]-dG-(CH₂)₃-dG was 1.2 adducts per 10⁶ bases for the AC-treated CT DNA.

Figure 6

Reconstructed ion chromatograms of the LC-ESI/MS/MSⁿ traces of CT-DNA treated with HNE. A reconstructed ion chromatogram of the LC-ESI/MS/MS³ trace for HNE-dG and the reconstructed ion chromatogram of the LC-ESI/MS/MS⁴ trace for the 8-dG-HNE-dG cross-link. Both [²H₁₁]-HNE-dG and [²H₁₁]-8-dG-HNE-dG cross-link were added to CT DNA at a level of 1 adduct per 10⁷ bases. Panel A depicts untreated CT DNA and Panel B depicts CT DNA treated with 2 mol of HNE per mol base in CT DNA.

Scheme 1

Synthesis of the isotopically labeled internal standards

Scheme 2

Chemical reduction of AC and HNE-DNA adducts by Na(CN)BH₃/NaBH₄

Scheme 3

Cross-linking chemistry of enal adducts of dG