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 IC(50) 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.
Mistry
Nalini
N
Oxidative Stress Group, Department of Clinical Biochemistry, University Hospitals of Leicester National Heath Service Trust, Leicester, United Kingdom.
Podmore
Ian
I
Cooke
Marcus
M
Butler
Paul
P
Griffiths
Helen
H
Herbert
Karl
K
Lunec
Joseph
J
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000737:chemistry; D001666:Binding Sites, Antibody; D002461:Cell Line, Transformed; D004247:DNA / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D018736:DNA Adducts / Q000032:analysis / Q000276:immunology / Q000378:metabolism*; D004249:DNA Damage; D003841:Deoxycytidine / Q000031:analogs & derivatives / Q000276:immunology / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D008401:Gas Chromatography-Mass Spectrometry; D006037:Glyoxal / Q000276:immunology / Q000378:metabolism* / Q000633:toxicity; D015603:Keratinocytes; D051379:Mice; D008807:Mice, Inbred BALB C; D018384:Oxidative Stress
D000911:Antibodies, Monoclonal; D018736:DNA Adducts; D003841:Deoxycytidine; D006037:Glyoxal; D004247:DNA