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.
To investigate the accumulation of intracellular advanced glycation end products (AGEs), a method was established for the simultaneous analysis of glycation products of cytosolic proteins, nuclear DNA, and mitochondrial DNA (mtDNA). Nuclear DNA, mtDNA, and cytosolic proteins were simultaneously isolated from one cell lysate by differential centrifugation and combined mechanical and chemical cell disruption methods. The major DNA-AGE N(2)-carboxyethyl-2'-deoxyguanosine (CEdG) was quantified in nuclear DNA and mtDNA by ELISA, whereas the protein-AGEs N(ɛ)-(carboxymethyl)lysine (CML) and N(ɛ)-(carboxyethyl)lysine (CEL) were determined by western blot. The method was used to analyze NIH3T3 fibroblasts. In untreated cells, CEdG levels of mtDNA (14.84 ± 3.07 pg CEdG/μg mtDNA) were significantly higher compared with nuclear DNA (4.40 ± 0.64 pg CEdG/μg DNA; p < 0.001). Then, fibroblasts were analyzed after 7 days of senescence-like growth arrest. In senescent fibroblasts, the CEdG content of nuclear DNA significantly increased by 25%. However, the CEdG level of mtDNA significantly decreased to 52%; in parallel, an increase in mitochondrial mass and mtDNA was observed. Senescence did not lead to general accumulation of protein-AGEs, but two protein bands at 32 and 34 kDa showed a significant increase in the CML/CEL modification rate (208%, p < 0.001; 196%, p = 0.0016) in senescent fibroblasts compared with control cells.
The accumulation of somatic mutations in mitochondrial DNA (mtDNA) induced by reactive oxidative species (ROS) is regarded as a major contributor of aging and age-related degenerative diseases. ROS has also been shown to facilitate the formation of certain advanced glycation end-products in proteins and DNAs, and N2-carboxyethyl-2′-deoxyguanosine (CEdG) has been identified as a major DNA-bound AGE. Therefore, the influence of mitochondrial ROS on the glycation of mtDNA was investigated in primary embryonic fibroblasts derived from mutant mice (Sod2−/+) deficient in the mitochondrial antioxidant enzyme, manganese superoxide dismutase (MnSOD). In Sod2−/+ fibroblasts vs. wildtype fibroblasts, the CEdG content of mtDNA was increased from 1.90±1.39 pg/μg DNA to 17.14±6.60 pg/μg DNA (p<0.001). On the other hand, the CEdG content of nuclear DNA did not differ between Sod2+/+ and −/+ cells. Similarly, cytosolic proteins did not show any difference in the advanced glycation end-products or protein carbonyl contents between Sod2+/+ and −/+. Taken together, the data suggest that mitochondrial oxidative stress specifically promotes glycation of mtDNA and does not affect nuclear DNA or cytosolic proteins. Because DNA glycation can change DNA integrity and gene functions, glycation of mtDNA may play an important role in the decline of mitochondrial functions.
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