Novel monoclonal antibody recognition of oxidative DNA damage adduct, deoxycytidine-glyoxal

Glyoxal, a reactive aldehyde, is a decomposition product of lipid hydroperoxides, oxidative deoxyribose breakdown, or autoxidation of sugars, such as glucose. It readily forms DNA adducts, generating potential carcinogens such as glyoxalated deoxycytidine (gdC). A major drawback in assessing gdC formation in cellular DNA has been methodologic sensitivity. We have developed an mAb that specifically recognizes gdC. Balb/c mice were immunized with DNA, oxidatively modified by UVC/hydrogen peroxide in the presence of endogenous metal ions. Although UVC is not normally considered an oxidizing agent, a UVC/hydrogen peroxide combination may lead to glyoxalated bases arising from hydroxyl radical damage to deoxyribose. This damaging system was used to induce numerous oxidative lesions including glyoxal DNA modifications, from which resulted a number of clones. Clone F3/9/H2/G5 showed increased reactivity toward glyoxal-modified DNA greater than that of the immunizing antigen. ELISA unequivocally showed Ab recognition toward gdC, which was confirmed by gas chromatography-mass spectrometry of the derivatized adduct after formic acid hydrolysis to the modified base. Binding of Ab F3/9 with glyoxalated and untreated oligomers containing deoxycytidine, deoxyguanosine, thymidine, and deoxyadenosine assessed by ELISA produced significant recognition (p 0.0001) of glyoxal-modified deoxycytidine greater than that of untreated oligomer. Additionally, inhibition ELISA studies using the glyoxalated and native deoxycytidine oligomer showed increased recognition for gdC with more than a 5-fold difference in IC50 values. DNA modified with increasing levels of iron (II)/EDTA produced a dose-dependent increase in Ab F3/9 binding. This was reduced in the presence of catalase or aminoguanidine. We have validated the potential of gdC as a marker of oxidative DNA damage and showed negligible cross-reactivity with 8-oxo-2'-deoxyguanosine or malondialdehyde-modified DNA as well as its utility in immunocytochemistry. Formation of the gdC adduct may involve intermediate structures; however, our results strongly suggest Ab F3/9 has major specificity for the predominant product, 5-hydroxyacetyl-dC.

Mass spectrometric analysis of HOCl- and free-radical-induced damage to lipids and proteins

In inflammatory diseases, release of oxidants leads to oxidative damage to biomolecules. HOCl (hypochlorous acid), released by the myeloperoxidase/H2O2/Cl- system, can cause formation of phospholipid chlorohydrins, or alpha-chloro-fatty aldehydes from plasmalogens. It can attack several amino acid residues in proteins, causing post-translational oxidative modifications of proteins, but the formation of 3-chlorotyrosine is one of the most stable markers of HOCl-induced damage. Soft-ionization MS has proved invaluable for detecting the occurrence of oxidative modifications to both phospholipids and proteins, and characterizing the products generated by HOCl-induced attack. For both phospholipids and proteins, the application of advanced mass spectrometric methods such as product or precursor ion scanning and neutral loss analysis can yield information both about the specific nature of the oxidative modification and the biomolecule modified. The ideal is to be able to apply these methods to complex biological or clinical samples, to determine the site-specific modifications of particular cellular components. This is important for understanding disease mechanisms and offers potential for development of novel biomarkers of inflammatory diseases. In the present paper, we review some of the progress that has been made towards this goal.

Micromechanics of agglomerate damage processes

This thesis reports a detailed investigation of the micromechanics of agglomerate behaviour under free-fall impact, double (punch) impact and diametrical compression tests using the simulation software TRUBAL. The software is based on the discrete element method (DEM) which incorporates the Newtonian equations of motion and contact mechanics theory to model the interparticle interactions. Four agglomerates have been used: three dense (differing in interface energy and contact density) and one loose. Although the simulated agglomerates are relatively coarse-grained, the results obtained are in good agreement with laboratory test results reported in the literature. The computer simulation results show that, in all three types of test, the loose agglomerate cannot fracture as it is unable to store sufficient elastic energy. Instead, it becomes flattened for low loading-rates and shattered or crushed at higher loading-rates. In impact tests, the dense agglomerates experience only local damage at low impact velocities. Semi-brittle fracture and fragmentation are produced over a range of higher impact velocities and at very high impact velocities shattering occurs. The dense agglomerates fracture in two or three large fragments in the diametrical compression tests. Local damage at the agglomerate-platen interface always occurs prior to fracture and consists of local bond breakage (microcrack formation) and local dislocations (compaction). The fracture process is dynamic and much more complex than that suggested by continuum fracture mechanics theory. Cracks are always initiated from the contact zones and propagate towards the agglomerate centre. Fracture occurs a short time after the start of unloading when a fracture crack "selection" process takes place. The detailed investigation of the agglomerate damage processes includes an examination of the evolution of the fracture surface. Detailed comparisons of the behaviour of the same agglomerate in all three types of test are presented. The particle size distribution curves of the debris are also examined, for both free-fall and double impact tests.

Phagocyte oxidation of phospholipids: comparison of hydroperoxide, chlorohydrin and epoxyisoprostane formation by LC-MS

Phagocytic cells produce a variety of oxidants as part of the immune defence, which react readily both with proteins and lipids, and could contribute to the oxidation of low density lipoprotein in atherosclerosis. We have investigated the oxidation of phospholipid vesicles by isolated human polymorphonuclear and mononuclear leukocytes, to provide a model of lipid oxidation in the absence of competing protein. PMA-stimulated cells were incubated with phospholipid vesicles contammg dipalmitoyl phosphatidylcholine (DPPC), palmitoyl-arachidonoyl phosphatidylcholine (PAPC), and stearoyl-oleoyl phosphatidylcholine (SOPC), before extraction of the lipids for analysis by HPLC coupled to electrospray mass spectrometry. In this system, oxidized phosphatidylcholines elute earlier than the native lipids owing to their decreased hydrophobicity, and can be identified according to their molecular mass. The formation of monohydroperoxides of P APC was observed routinely, together with low levels of hydroxides, but no chlorohydrin derivatives of P APC or SOPC were detected. However, the major oxidized product occurred at 828 m/z, and was identified as I-palmitoyl-2-(5,6-epoxyisoprostane E2)-sn-glycero-3-phosphocholine. These results show that phagocytes triggered by PMA cause oxidative damage to lipids predominantly by free radical mechanisms, and that electrophilic addition involving HOCl is not a major mechanism of attack. The contribution of myeloperoxidase and metal ions to the oxidation process is currently being investigated, and preliminary data suggest that myeloperoxidase-derived oxidants are responsible for the epoxyisoprostane phospholipid formation. The identification of an epoxyisoprostane phospholipid as the major product following phagocyte-induced phospholipid oxidation is novel and has implications for phagocyte involvement in atherogenesis.

Energy-based damage and fracture framework for viscoelastic asphalt concrete

A framework based on the continuum damage mechanics and thermodynamics of irreversible processes using internal state variables is used to characterize the distributed damage in viscoelastic asphalt materials in the form of micro-crack initiation and accumulation. At low temperatures and high deformation rates, micro-cracking is considered as the source of nonlinearity and thus the cause of deviation from linear viscoelastic response. Using a non-associated damage evolution law, the proposed model shows the ability to describe the temperature-dependent processes of micro-crack initiation, evolution and macro-crack formation with good comparison to the material response in the Superpave indirect tensile (IDT) strength test.