Mechanism of Repair of Acrolein- and Malondialdehyde-Derived Exocyclic Guanine Adducts by the α-Ketoglutarate/Fe(II) Dioxygenase AlkB

The structurally related exocyclic guanine adducts α-hydroxypropano-dG (α-OH-PdG), γ-hydroxypropano-dG (γ-OH-PdG), and M[subscript 1]dG are formed when DNA is exposed to the reactive aldehydes acrolein and malondialdehyde (MDA). These lesions are believed to form the basis for the observed cytotoxicity and mutagenicity of acrolein and MDA. In an effort to understand the enzymatic pathways and chemical mechanisms that are involved in the repair of acrolein- and MDA-induced DNA damage, we investigated the ability of the DNA repair enzyme AlkB, an α-ketoglutarate/Fe(II) dependent dioxygenase, to process α-OH-PdG, γ-OH-PdG, and M[subscript 1]dG in both single- and double-stranded DNA contexts. By monitoring the repair reactions using quadrupole time-of-flight (Q-TOF) mass spectrometry, it was established that AlkB can oxidatively dealkylate γ-OH-PdG most efficiently, followed by M[subscript 1]dG and α-OH-PdG. The AlkB repair mechanism involved multiple intermediates and complex, overlapping repair pathways. For example, the three exocyclic guanine adducts were shown to be in equilibrium with open-ring aldehydic forms, which were trapped using (pentafluorobenzyl)hydroxylamine (PFBHA) or NaBH[subscript 4]. AlkB repaired the trapped open-ring form of γ-OH-PdG but not the trapped open-ring of α-OH-PdG. Taken together, this study provides a detailed mechanism by which three-carbon bridge exocyclic guanine adducts can be processed by AlkB and suggests an important role for the AlkB family of dioxygenases in protecting against the deleterious biological consequences of acrolein and MDA.National Institutes of Health (U.S.) (Grant R01 CA080024)National Institutes of Health (U.S.) (Grant R01 CA26731)National Institutes of Health (U.S.) (Center Grant P30 ES02109)National Institutes of Health (U.S.) (Training Grant T32 ES007020

Differential Base Stacking Interactions Induced by Trimethylene Interstrand DNA Cross-Links in the 5′-CpG-3′ and 5′-GpC-3′ Sequence Contexts

Synthetically derived trimethylene interstrand DNA cross-links have been used as surrogates for the native cross-links that arise from the 1,N 2-deoxyguanosine adducts derived from R,β-unsaturated aldehydes. The native enal-mediated cross-linking occurs in the 5′-CpG-3 ′ sequence context but not in the 5′-GpC-3 ′ sequence context. The ability of the native enal-derived 1,N 2-dG adducts to induce interstrand DNA cross-links in the 5′-CpG-3 ′ sequence as opposed to the 5′-GpC-3 ′ sequence is attributed to the destabilization of the DNA duplex in the latter sequence context. Here, we report higher accuracy solution structures of the synthetically derived trimethylene cross-links, which are refined from NMR data with the AMBER force field. When the synthetic trimethylene cross-links are placed into either the 5′-CpG-3′ or the 5′-GpC-3 ′ sequence contexts, the DNA duplex maintains B-DNA geometry with structural perturbations confined to the cross-linked base pairs. Watson-Crick hydrogen bonding is conserved throughout the duplexes. Although different from canonical B-DNA stacking, the cross-linked and the neighbor base pairs stack in the 5′-CpG-3 ′ sequence. In contrast, the stacking at the cross-linked base pairs in the 5′-GpC-3 ′ sequence is greatly perturbed. The π-stacking interactions between the crosslinked and the neighbor base pairs are reduced. This is consistent with remarkable chemical shift perturbations of the C 5 H5 and H6 nucleobase protons that shifted downfield by 0.4-0.5 ppm. In contrast

DNA damage by lipid peroxidation products: implications in cancer, inflammation and autoimmunity

Oxidative stress and lipid peroxidation (LPO) induced by inflammation, excess metal storage and excess caloric intake cause generalized DNA damage, producing genotoxic and mutagenic effects. The consequent deregulation of cell homeostasis is implicated in the pathogenesis of a number of malignancies and degenerative diseases. Reactive aldehydes produced by LPO, such as malondialdehyde, acrolein, crotonaldehyde and 4-hydroxy-2-nonenal, react with DNA bases, generating promutagenic exocyclic DNA adducts, which likely contribute to the mutagenic and carcinogenic effects associated with oxidative stress-induced LPO. However, reactive aldehydes, when added to tumor cells, can exert an anticancerous effect. They act, analogously to other chemotherapeutic drugs, by forming DNA adducts and, in this way, they drive the tumor cells toward apoptosis. The aldehyde-DNA adducts, which can be observed during inflammation, play an important role by inducing epigenetic changes which, in turn, can modulate the inflammatory process. The pathogenic role of the adducts formed by the products of LPO with biological macromolecules in the breaking of immunological tolerance to self antigens and in the development of autoimmunity has been supported by a wealth of evidence. The instrumental role of the adducts of reactive LPO products with self protein antigens in the sensitization of autoreactive cells to the respective unmodified proteins and in the intermolecular spreading of the autoimmune responses to aldehyde-modified and native DNA is well documented. In contrast, further investigation is required in order to establish whether the formation of adducts of LPO products with DNA might incite substantial immune responsivity and might be instrumental for the spreading of the immunological responses from aldehyde-modified DNA to native DNA and similarly modified, unmodified and/or structurally analogous self protein antigens, thus leading to autoimmunity

Synthesis of New trans-2-Benzyl-3-(furan-2-yl)-4-substituted-1,2,3,4-tetrahydroisoquinolinones

The reaction of homophthalic anhydride and N-(furan-2-yl-methylidene)- benzylamine in different solvents and varying temperatures was studied in detail. Mixtures of the expected trans- and cis-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acids trans-5 and cis-5, alongwith by-products 6 and 7 were obtained in dichloroethane or benzene. In pyridine, used for the first time, the reaction became completely diastereoselective, giving only the trans isomer. The carboxylic acid group of trans-5 was transformed in four steps into cyclic aminomethyl groups which yielded various new tetrahydroisoquinolinones trans- 11a-g, incorporating both a known fragment of pharmacological interest and various pharmacophoric substituents