Reaction of malondialdehyde-DNA adducts with hydrazines-development of a facile assay for quantification of malondialdehyde equivalents in DNA.
Malondialdehyde is a ubiquitous product of lipid peroxidation that reacts with DNA to form premutagenic lesions. Principal among them is pyrimido-[1,2-alpha]purin-10(3H)-one (M(1)G). M(1)G has recently been found to be a reactive electrophile in DNA that couples with amines at basic pH or hydroxylamines at neutral pH. We explored the reaction of M(1)G with hydrazines because of the possibility that the latter could act as bifunctional nucleophiles to strip the malondialdehyde equivalent from DNA. Pentafluorophenylhydrazine reacted rapidly with M(1)G to form a hydrazone conjugate. This hydrazone was stable at room temperature and did not cyclize to form the corresponding pyrazole. In contrast, phenylhydrazine and benzylhydrazine reacted with M(1)G to form phenylpyrazole and benzylpyrazole, respectively. Pentafluorobenzylhydrazine reacted rapidly with M(1)G to form pentafluorobenzylpyrazole and dG in near quantitative yield. This reaction formed the basis for a quantitative assay for the presence of M(1)G or M(1)G equivalents in DNA or protein that utilized gas chromatography/negative chemical ionization mass spectrometry. The assay was extended to the oxopropenyl donors, M(1)A, base propenal, and N(epsilon)-3-oxopropenyl-lysine. Analysis of DNA treated with bleomycin demonstrated a linear increase in the level of oxopropenyl groups that plateaued at approximately 1 oxopropenyl group/100 bases at a bleomycin concentration of 200 microM. Parallel analysis of M(1)G in the samples revealed that this adduct represents a small fraction of the total oxopropenyl units generated in DNA by treatment with bleomycin.
Otteneder
Michael
M
The A.B. Hancock, Jr. Memorial Laboratory for Cancer Research, Department of Biochemistry, Center in Molecular Toxicology, The Vanderbilt-Ingram Comprehensive Cancer Center, Vanderbilt University School of Medicine, Nashville Tennessee 37232-0146, USA.
Plastaras
John P
JP
Marnett
Lawrence J
LJ
D000225:Adenine / Q000031:analogs & derivatives* / Q000737:chemistry; D001761:Bleomycin; D018736:DNA Adducts / Q000737:chemistry*; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000737:chemistry; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods; D006834:Hydrazines / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008315:Malondialdehyde / Q000032:analysis / Q000737:chemistry*; D011687:Purines / Q000737:chemistry; D011743:Pyrimidines / Q000737:chemistry; D005464:Fluorobenzenes; D005466:Fluorocarbons
D000225:Adenine; D001761:Bleomycin; D018736:DNA Adducts; D003849:Deoxyguanosine; D006834:Hydrazines; D008315:Malondialdehyde; D011687:Purines; D011743:Pyrimidines; C107643:pyrimido(1,2-a)purin-10(3H)-one; C031120:(pentafluorophenyl)hydrazine; D005464:Fluorobenzenes; D005466:Fluorocarbons; C089188:3-(2'-deoxyribofuranosyl)pyrimido(1,2-a)purin-10(3H)-one; C085912:9-(3-oxoprop-1-enyl)adenine