Dataset Information

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 ?,?-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 ?g 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.

SUBMITTER: Kozekov ID

PROVIDER: S-EPMC2990652 | biostudies-literature | 2010 Nov

REPOSITORIES: biostudies-literature

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

Kozekov Ivan D ID Turesky Robert J RJ Alas Guillermo R GR Harris Constance M CM Harris Thomas M TM Rizzo Carmelo J CJ

Chemical research in toxicology 20101022 11

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 ...[more]

PMID: 20964440

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Project description:DNA interstrand cross-links (ICLs) are cytotoxic DNA lesions derived from reactions of DNA with a number of anti-cancer reagents as well as endogenous bifunctional electrophiles. Deciphering the DNA repair mechanisms of ICLs is important for understanding the toxicity of DNA cross-linking agents and for developing effective chemotherapies. Previous research has focused on ICLs cross-linked with the N7 and N2 atoms of guanine as well as those formed at the N6 atom of adenine; however, little is known about the mutagenicity of O6-dG-derived ICLs. Although less abundant, O6-alkylated guanine DNA lesions are chemically stable and highly mutagenic. Here, O6-2'-deoxyguanosine-butylene-O6-2'-deoxyguanosine (O6-dG-C4-O6-dG) is designed as a chemically stable ICL, which can be induced by the action of bifunctional alkylating agents. We investigate the DNA replication-blocking and mutagenic properties of O6-dG-C4-O6-dG ICLs during an important step in ICL repair, translesion DNA synthesis (TLS). The model replicative DNA polymerase (pol) Sulfolobus solfataricus P2 DNA polymerase B1 (Dpo1) is able to incorporate a correct nucleotide opposite the cross-linked template guanine of ICLs with low efficiency and fidelity but cannot extend beyond the ICLs. Translesion synthesis by human pol ? is completely inhibited by O6-dG-C4-O6-dG ICLs. Moderate bypass activities are observed for human pol ? and S. solfataricus P2 DNA polymerase IV (Dpo4). Among the pols tested, pol ? exhibits the highest bypass activity; however, 70% of the bypass products are mutagenic containing substitutions or deletions. The increase in the size of unhooked repair intermediates elevates the frequency of deletion mutation. Lastly, the importance of pol ? in O6-dG-derived ICL bypass is demonstrated using whole cell extracts of Xeroderma pigmentosum variant patient cells and those complemented with pol ?. Together, this study provides the first set of biochemical evidence for the mutagenicity of O6-dG-derived ICLs.

Project description:4-Hydroxy-2-nonenal (HNE), a major racemic product of lipid peroxidation, preferentially reacts with cysteine residues to form a stable HNE-cysteine Michael addition adduct possessing three chiral centers. Here, to gain more insight into sulfhydryl modification by HNE, we characterized the stereochemical configuration of the HNE-cysteine adducts and investigated their stereoselective formation in redox-regulated proteins. To characterize the HNE-cysteine adducts by NMR, the authentic (R)-HNE- and (S)-HNE-cysteine adducts were prepared by incubating N-acetylcysteine with each HNE enantiomer, both of which provided two peaks in reversed-phase high performance liquid chromatography (HPLC). The NMR analysis revealed that each peak was a mixture of anomeric isomers. In addition, mutarotation at the anomeric center was also observed in the analysis of the nuclear Overhauser effect. To analyze these adducts in proteins, we adapted a pyridylamination-based approach, using 2-aminopyridine in the presence of sodium cyanoborohydride, which enabled analyzing the individual (R)-HNE- and (S)-HNE-cysteine adducts by reversed-phase HPLC following acid hydrolysis. Using the pyridylamination method along with mass spectrometry, we characterized the stereoselective formation of the HNE-cysteine adducts in human thioredoxin and found that HNE preferentially modifies Cys(73) and, to the lesser extent, the active site Cys(32). More interestingly, the (R)-HNE- and (S)-HNE-cysteine adducts were almost equally formed at Cys(73), whereas Cys(32) exhibited a remarkable preference for the adduct formation with (R)-HNE. Finally, the utility of the method for the determination of the HNE-cysteine adducts was confirmed by an in vitro study using HeLa cells. The present results not only offer structural insight into sulfhydryl modification by lipid peroxidation products but also provide a platform for the chemical analysis of protein S-associated aldehydes in vitro and in vivo.

Dataset Information

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

Publications

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

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