Identification of DNA Adducts of Methylglyoxal

Abstract

Methylglyoxal (MG) is a sugar degradation product, which is endogenously formed by fragmentation of triose phosphates during glycolysis, ketone body metabolism of acetone, and catabolism of threonine. Food, beverages, and medical products are important exogenous sources with concentrations of up to 100 microM MG. MG is a reactive dicarbonyl compound, which easily modifies amino groups of proteins (glycation reaction) and thereby induces proinflammatory responses. Moreover, increased mutation frequencies in mammalian cells after treatment with MG have been reported, which are caused by stable modifications of DNA bases. Thus far, two types of adducts have been identified, which are formed during the reaction of free guanine or 2'-deoxyguanosine with high MG concentrations. In this study, we investigated the prolonged exposure of DNA to physiological MG concentrations. DNA was incubated with MG, enzymatically hydrolyzed to release the free nucleosides, and then analyzed by LC-MS/MS. We detected four products, which were derived from the reaction of 2'-deoxyguanosine and 2'-deoxyadenosine with 1 and 2 equiv of MG each. The adducts with 1 equiv of MG were identified as N2-(1-carboxyethyl)-2'-deoxyguanosine (CEdG) and N6-(1-carboxyethyl)-2'-deoxyadenosine. LC-MS/MS was optimized for these compounds, and incubation of DNA was repeated using physiological concentrations of 10 microM MG. Thereby, CEdG proved to be the most sensitive and suitable marker for the reaction of DNA with MG (negative MRM mode, three mass transitions [M - 1](-) 338-->178, 338-->106, and 338-->149).

The DNA Polymerase Activity ofSaccharomyces cerevisiaeRev1 is Biologically Significant

“…caused by endogenous agents that similarly require Rev1's polymerase activity for bypass. An example of such an endogenous agent produced by normal metabolism is methylglyoxal, a reduced form of pyruvic acid that can react with DNA to produce N 2 -(1-carboxyethyl)-29-deoxyguanosine (N 2 -CEdG) as the major lesion (Frischmann et al 2005;Yuan et al 2008), which is also detectable in human samples (Schneider et al 2004;Li et al 2006). We assessed the percentage of survival of our strains in the presence of chronic exposure to methylglyoxal, choosing the rev1Dmms2D strain background for our experiments to maximize any observable phenotypes for the catalytic dead rev1 mutant.…”

Efficient and accurate bypass of N 2 -(1-carboxyethyl)-2′-deoxyguanosine by DinB DNA polymerase in vitro and in vivo

“…The concentration of MG in human cells can be elevated under various pathological conditions (e.g., diabetes) (19)(20)(21). It was found recently that MG induces modifications in calf thymus DNA mainly on dG to give N 2 -CEdG (22). N 2 -CEdG could also be detected in urine samples of healthy human subjects (23) and in kidney and aorta cells of diabetic and uremic patients (24).…”

“…1) (22). A competitive enzyme-linked immunosorbant assay showed that the adduct can be detected in urine samples of 121 healthy human subjects at levels ranging from 1.2-to 117-ng N 2 -CEdG equivalent per milligram of creatinine (23).…”

Identification of Adducts Formed in the Reactions of Malonaldehyde–glyoxal and Malonaldehyde–methylglyoxal with Adenosine and Calf Thymus DNA

“…Methylglyoxal forms adducts mainly with the guanine moiety: N 2 ,1-(1,2-dihydroxy-2-methyl)ethano-2'-deoxyguanosine (cMG-dGuo) and N 2 -(1-carboxyethyl)-2'-deoxyguanosine (CEdG) but also with the adenine [16] [17]. Moreover, a bis-adduct of 2'-deoxyguanosine and methylglyoxal, N 2 ,7-bis(1-hydroxy-2-oxopropenyl)-dGuo has been identified [18].…”

“…Replacement of glyoxal with methylglyoxal resulted in the formation of one new product marked M 1 MGx-A (Fig. 1, b), besides diastereoisomers of N 2 -(1-carboxyethyl)adenosine (CEA), the ribosyl analogue of the known methylglyoxal and 2'-deoxyadenosine adduct CEdA [16]. Large-scale reactions were performed to isolate sufficient amounts of the products for structural determination by spectrometric and spectroscopic methods.…”