Analysis and biological relevance of advanced glycation end‐products of DNA in eukaryotic cells

Abstract

Advanced glycation end‐products (AGEs) of DNA are formed spontaneously by the reaction of carbonyl compounds such as sugars, methylglyoxal or dihydroxyacetone in vitro and in vivo. Little is known, however, about the biological consequences of DNA AGEs. In this study, a method was developed to determine the parameters that promote DNA glycation in cultured cells. For this purpose, the formation rate of N2‐carboxyethyl‐2′‐deoxyguanosine (CEdG), a major DNA AGE, was measured in cultured hepatic stellate cells by liquid chromatography (LC)‐MS/MS. In resting cells, a 1.7‐fold increase of CEdG formation rate was observed during 14 days of incubation. To obtain insights into the functional consequences of DNA glycation, CEdG was introduced into a luciferase reporter gene vector and transfected into human embryonic kidney (HEK 293 T) cells. Gene activity was determined by chemiluminescence of the luciferase. Thus, CEdG adducts led to a dose‐dependent and highly significant decrease in protein activity, which is caused by loss of functionality of the luciferase in addition to reduced transcription of the gene. When the CEdG‐modified vector was transformed into Escherichia coli, a loss of ampicillin resistance was observed in comparison to transformation with the unmodified plasmid. These results indicate that CEdG accumulates in the genomic DNA of resting cells, which could lead to diminished protein activity.

Associated links among mtDNA glycation, oxidative stress and colony sectorization in Metarhizium anisopliae

“…Prima facie therefore being AT rich would seem non-adaptive, but the consequent reduction in DNA glycation could provide a counteracting benefit. In contrast to hepatic stellate cells (Breyer et al, 2008), we did not observe glycation of genomic DNA in M. anisopliae. Presumably, the antioxidant defenses were sufficient in the fungus to protect against this, while mtDNA may be more accessible to AGEs.…”

“…BY4742) CFU per lg DNA in comparison to 20, 27 and 10 CFU per lg DNA, respectively, for the glycated plasmid. A similar reduction in efficiency was obtained when we transformed a glycated plasmid into E. coli (Mironova et al, 2005;Breyer et al, 2008).…”

“…DNA glycation may also be linked with aging or diseases (Baynes, 2002). Direct evidence for this has been lacking but recent studies have shown glycation of the chromosomal DNA of stationary phase Escherichia coli (Mironova et al, 2005) and the genomic DNA of resting hepatic stellate cells (Breyer et al, 2008).…”

Physicochemical analysis of structural changes in DNA modified with glucose

“…When native DNA and one-week-old glycated-DNA were analyzed under identical conditions they did not show mass value matching with CEdG (figure not shown). Furthermore, analysis of two, three and four weeks old glycated-DNA suggests CEdG formation and m/z value of 338 [24] was seen in all three cases (Fig. 10b-d).…”

“…In vivo, it has been shown that 3-deoxyglucosone (a glucose degradation product) induces embryonic malformation and teratogenicity, an effect that may be related to DNA AGEs [23]. The CEdG in a gene may lead to a reduced transcription and to a loss of gene function due to mis-sense or nonsense mutations [24].…”