Analysis and biological relevance of advanced glycation end-products of DNA in eukaryotic cells Viola Breyer1,*, Matthias Frischmann1,*, Clemens Bidmon1, Annelen Schemm2, Katrin Schiebel2 and Monika Pischetsrieder1 Department of Chemistry and Pharmacy, University of Erlangen-Nuremberg, Germany Institute for Biochemistry, University of Erlangen-Nuremberg, Germany Keywords advanced glycation end-products; DNA; eukaryotic cells; Maillard reaction; N 2-carboxyethyl-2¢-deoxyguanosine Correspondence M Pischetsrieder, Department of Chemistry and Pharmacy, Henriette Schmidt-Burkhardt Chair of Food Chemistry, Schuhstr 19, 91052 Erlangen, Germany Fax: +49 9131 8522587 Tel: +49 9131 8524102 E-mail: pischetsrieder@lmchemie uni-erlangen.de Website: http://www.lebensmittelchemie pharmazie.uni-erlangen.de *These authors contributed equally to this work (Received 23 October 2007, revised 17 December 2007, accepted 19 December 2007) 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 doi:10.1111/j.1742-4658.2008.06255.x Sugars and other reactive carbonyl compounds bind spontaneously to nucleophilic amino groups of amino acids and proteins in a nonenzymatic process (glycation) [1] It is well established that proteins are readily glycated in vivo The first glycation product to be detected in vivo was hemoglobin (Hb) A1c, the Amadori product of Hb A [2] Hb A1c is now an established clinical marker for medium-term hyperglycemia in diabetic patients In vivo, early glycation products, such as the Amadori product, are further converted into the heterogeneous group of advanced glycation end-products (AGEs) AGEs accumulate on serum proteins and in various tissues, particularly during aging, diabetes, and renal failure [3] Elevated AGE levels contribute to the development of diabetic and uremic complications, such as atherosclerosis [4], nephropathy, and retinopathy [5] In analogous reactions, glycation may also affect DNA In vitro, nucleobases and dsDNA react with Abbreviations AGE, advanced glycation end-product; CEdG, N2-carboxyethyl-2¢-deoxyguanosine; DAD, diode array detector; dG, 2¢-deoxyguanosine monohydrate; DHA, dihydroxyacetone; Hb, hemoglobin; LC, liquid chromatography; TMB, 3,3¢,5,5¢-tetramethylbenzidine dihydrochloride; TY, tryptan yeast medium 914 FEBS Journal 275 (2008) 914–925 ª 2008 The Authors Journal compilation ª 2008 FEBS V Breyer et al sugars in a similar way as proteins [5–7] The exocyclic amino group of 2¢-deoxyguanosine is particularly prone to glycation reactions, leading to the formation of N2-carboxyethyl, N2-carboxymethyl, N2-(1-carboxy3-hydroxypropyl), and N2-(1-carboxy-3,4,5-trihydroxypentyl) modifications, as well as cyclic dicarbonyl adducts [6,8–10] The two diastereomers of N2-carboxyethyl-2¢-deoxyguanosine (CEdGA,B; Scheme 1) are stable reaction products that are formed from a variety of glycating agents, such as glucose, ascorbic acid, glyceraldehyde, dihydroxyacetone (DHA), or methylglyoxal [10–12] Recently, carboxyethylated nucleobases were detected in human urine [13], indicating the formation of DNA AGEs in the healthy human organism A significantly increased number of CEdGpositive cells were immunostained in glomeruli of patients with diabetic nephropathy as compared to healthy controls [14], as well as in glomeruli of diabetic rats [15] DNA AGEs are potentially genotoxic compounds because they induce depurination [9] as well as singlestrand breaks and lead to mutations [16] in vitro In vivo, it was shown, for example, that 3-deoxyglucosone, a glucose degradation product, induces embryonic malformation and teratogenicity, effects that may be related to DNA AGEs [17] DNA glycation in cultured cells was observed using radioactively labeled glucose [18] or a 32P-postlabeling technique [19] Furthermore, the presence of CEdGA,B was detected in cultured cells by HPLC–diode array detector (DAD) after immunoaffinity chromatography [20] In order to investigate factors that influence cellular DNA glycation, reliable analytical methods are required to measure the DNA glycation rate in cell models In this study, we developed a liquid chromatography (LC)-MS ⁄ MS method for the analysis of the CEdG formation rate in genomic DNA of cultured cells Furthermore, the influence of CEdG DNA advanced glycation end-products in cells formation on cellular protein expression was investigated Results LC-MS ⁄ MS analysis of CEdGA,B In this study, an LC-MS ⁄ MS method was developed to determine the formation rate of CEdGA,B in genomic DNA of cultured HSC-T6 hepatic stellate cells In the first step, chromatographic conditions were optimized with a CEdGA,B standard to minimize the detection limit For sufficient sensitivity of the mass analysis, the analytes should elute with a maximal proportion of organic solvent into the ion source On the other hand, the LC should lead to the separation of CEdGA,B from major interfering compounds Thus, optimal chromatography conditions were achieved using an ammonium formate ⁄ acetonitrile buffer that eluted CEdGA,B as a peak pair at 8.3 and 9.5 (Fig 1A) The sensitivity was greatly increased by the use of ammonium formate to support ESI and by mass analysis in the negative mode [21] As guanosine, which coelutes with CEdGB under these chromatographic conditions, leads to quenching of the analyte signal, it was necessary to remove RNA thoroughly during sample work-up by isolation of the nuclei and adequate RNase treatment Deoxycytidine, which coelutes with CEdGA, showed no interference with the analysis After the extraction step, the DNA was hydrolyzed enzymatically and subjected to LCMS ⁄ MS Thus, a detection limit of about 0.5 ngỈmL)1 CEdGA,B was achieved Identification of CEdGA,B and assessment of the glycation rate For unequivocal identification of CEdGA,B in the genomic DNA of HSC-T6 cells, several parameters Scheme Formation of DNA-bound CEdGA,B from different carbonyl sources FEBS Journal 275 (2008) 914–925 ª 2008 The Authors Journal compilation ª 2008 FEBS 915 DNA advanced glycation end-products in cells V Breyer et al 1800 A Mass transition (m/z) 1600 338 – 178 338 – 106 338 – 149 Intensity (cps) 1400 1200 CEd GA 1000 800 CE dGB 600 400 200 B 8.0E+05 1.2E+06 Intensity 10 11 Time (min) 12 13 14 178 [M- CO2-dRib - H] 15 338 [M-CO2-H]- Fig LC-MS ⁄ MS analysis of CEdGA,B (A) LC-MS ⁄ MS chromatogram of a synthesized CEdGA,B standard showing the three main mass transitions (338 fi 178, 338 fi 106, 338 fi 149) (B) Product ion scan of CEdG The three main mass transitions 338 fi 178, 338 fi 106 and 338 fi 149 were used as qualifiers; the mass transition 338 fi 178 was used as quantifier 106 4.0E+05 0.0E+00 294 [M- CO2- H] 149 25 50 75 100 125 150 175 200 225 250 275 300 325 350 m/z Accumulation of CEdGA,B in hepatic stellate cells in vitro In order to investigate factors that influence DNA glycation, a cell culture model was established that allowed the analysis of cellular CEdGA,B formation HSC-T6 hepatic stellate cells were chosen as an adequate model, because of the high yield of extractable 916 Intensity (cps) A 4.0E+06 3.0E+06 2.0E+06 1.0E+06 0.0E+00 10 15 20 Time (min) B Intensity (cps) were applied: (a) the appearance of a peak pair with retention times of 8.3 and 9.5 min, and (b) the presence and (c) the correct proportion of the three major mass transitions of CEdGA,B that were used for MS ⁄ MS detection The main transition is 338 fi 178 m ⁄ z, which arises from the loss of deoxyribose and CO2, followed by the transitions 338 fi 106 m ⁄ z, and 338 fi 149 m ⁄ z (Fig 1B) Furthermore, some of the biological samples were spiked with synthesized CEdGA,B standard to verify peak assignment CEdGA,B was quantified by MS ⁄ MS using the transition 338 fi 178 m ⁄ z as quantifier and then normalized by the concentration of 2¢-deoxyguanosine The nucleotide 2¢-deoxyguanosine monohydrate (dG) eluted with a retention time of 10.6 min, and the three major mass transitions 266 fi 150 m ⁄ z, 266 fi 133 m ⁄ z, and 266 fi 107 m ⁄ z in the MS ⁄ MS identified the correct peak (Fig 2A) For quantification, the main transition at 266 fi 150 m ⁄ z was used 500 450 400 350 300 250 200 150 100 50 CEdGB CEdGA 10 11 12 13 14 15 Time (min) Fig LC-MS ⁄ MS analysis of CEdGA,B in the DNA of HSC-T6 cells (A) Analysis of dG in genomic DNA The LC-MS ⁄ MS chromatogram shows the main transitions for dG (266 fi 150, 266 fi 133, 266 fi 108) (B) Detection of CEdGA,B in the genomic DNA of cultured HSC-T6 cells after enzymatic hydrolysis The LC-MS ⁄ MS chromatogram shows the main mass transition for CEdG (338 fi 178) DNA from these cells and their relatively high glycation rate as compared to several other tested cell lines Although CEdGA,B concentrations were rather low FEBS Journal 275 (2008) 914–925 ª 2008 The Authors Journal compilation ª 2008 FEBS V Breyer et al DNA advanced glycation end-products in cells 300 Glycation rate (%) 250 200 150 100 50 0 days of incubation 14 days of incubation Fig Increase in the glycation rate of genomic DNA from HSC-T6 cells, which were incubated for 14 days in reduced medium The DNA was extracted, enzymatically hydrolyzed, and analyzed by LCMS ⁄ MS For each data point, cells from 10 culture flasks were combined The results from two experiments are shown under normal growing conditions, DNA glycation could be unequivocally detected when a sufficient number of cells was analyzed (Fig 2B) In order to investigate whether CEdG has the potential to accumulate in genomic DNA of nongrowing cells, HSC-T6 cells were kept in minimal medium containing 1.5% fetal bovine serum for weeks The composition of the medium was adjusted to minimize growth and cell death during 14 days of incubation The cell number was determined by the total amount of 2¢-deoxyguanosine One half of the confluent HSC-T6 cells was harvested immediately and the other half after 14 days of incubation in minimal medium Then, genomic DNA was extracted from the cells, digested, and analyzed by LCMS ⁄ MS For each data point, the DNA of 10 culture flasks was pooled, and a 1.7-fold increase of CEdG formation rate was measured (Fig 3) Influence of CEdG adducts on luciferase activity in HEK 293 T cells in vitro As these data indicated that CEdG accumulates in resting cells, we investigated whether CEdG adducts of DNA have an impact on protein expression For this purpose, CEdG was introduced into the luciferase reporter gene vector pGL3 Control CEdG-modified and unmodified vectors were transfected in parallel into HEK 293 T cells, and gene expression was investigated by measurement of luciferase activity CEdG was specifically introduced into the vector pGL3 Control by mixtures containing 0.1 lgỈlL)1 plasmid and 100 lm, mm or mm DHA, respectively CEdG concentrations were determined by an anti-CEdG ELISA Samples were taken every h over a period of h The results are shown in Table The amount of CEdG formed during the DHA incubation of the vector pGL3 Control increased with both incubation time and quantity of glycating reagent The reaction mixture containing the highest concentration of DHA generated the highest CEdG levels CEdG-modified and unmodified vectors (as control) were transfected into HEK 293 T cells via calcium phosphate precipitation A b-galactosidase vector was cotransfected in order to determine the transfection efficiency The relative luciferase activities were determined using a luciferase reporter gene assay Relative luciferase activities were defined as the ratio of the firefly luciferase to b-galactosidase value of each sample to the mean ratio of the unmodified pGL3 Control vector The results of the luciferase reporter gene assay with the CEdG-modified vector showed that the plasmid with the highest CEdG level yielded the lowest protein activity Incubation of the vector pGL3 Control with 100 lm DHA resulted in a significant reduction of about 50% (P < 0.01) of the relative luciferase activity (Fig 4A) The modified plasmids derived by the incubation with mm DHA led to a highly significant reduction in the protein activity of about 70% (P < 0.001) (Fig 4B) A significant decrease of 90% (P < 0.01) was observed for plasmids incubated with mm DHA (Fig 4C) These data indicate that CEdG modification led to reduced protein activity, increasing with higher modification rates of the encoding vector Table Concentrations of CEdG (nmolỈlg)1 plasmid) measured by ELISA CEdG adducts were introduced by incubation of 0.1 lgỈlL)1 plasmid and 100 lM, mM and mM DHA for 0, 3, and h at 70 °C, respectively Each data point represents the mean value of three ELISA measurements DHA concentration 0h 3h 6h 9h 100 lM mM mM Not detectable Not detectable Not detectable 1.58 · 10)4 2.00 · 10)3 6.42 · 10)3 6.37 · 10)4 8.36 · 10)3 1.55 · 10)2 1.54 · 10)3 9.95 · 10)3 1.94 · 10)2 FEBS Journal 275 (2008) 914–925 ª 2008 The Authors Journal compilation ª 2008 FEBS 917 DNA advanced glycation end-products in cells V Breyer et al 100 100 100 80 80 * ** 60 40 60 RLU (%) C 120 RLU (%) B 120 RLU (%) A 120 ** 40 *** *** 80 60 40 20 20 0 ** 20 Control 0h 3h 6h 9h Control 0h 3h 6h 9h ** Control 0h 3h ** 6h 9h Fig Chemiluminescence measurement of luciferase activity after transformation of a CEdG-modified vector pGL3 Control into HEK 293 T cells For CEdG formation, 0.1 lgỈlL)1 plasmid was incubated with 100 lM (A), mM (B) and mM (C) DHA for 0, 3, and h at 70 °C Relative luciferase units (RLU) were defined as the ratio of the firefly luciferase to a cotransfected b-galactosidase mean value relative to the unmodified pGL3 Control vector Each assay was performed in triplicate, and mean and standard deviations are shown *P < 0.05, **P < 0.01, ***P < 0.001 Cytotoxic effect on transfected HEK 293 T cells To ensure that the observed reduction of the luciferase activity was not a result of cytotoxic activity of the CEdG-modified plasmid DNA, the viability of transfected HEK 293 T cells was determined The data shown in Fig indicate that transfection in general had a cytotoxic effect Transfection with the unmodified vector reduced the cell viability from 92.7% to 64.8%, whereas CEdG-modified plasmid DNA led to a decrease to 49.8% The difference between transfection with CEdG-modified and unmodified plasmid DNA is around the significance level of 0.05 Survival of transfected HEK 293 T cells (%) 100 90 80 70 60 50 40 In addition to the luciferase gene, the vector pGL3 Control contains the Amp+ gene, which confers resistance to ampicillin as a selection marker for propagation in bacterial cells This gene was used to determine the potential of CEdG–DNA adducts to induce mutations by the parallel transformation of equal amounts of CEdG-modified and unmodified DNA into the electrocompetent Escherichia coli strain JM 109 An additional mock transformation and plating without ampicillin resistance selection resulted in a strong cytotoxic effect of the transformation procedure itself and so in far fewer colonies on culture plates The addition of DNA further increased the cytotoxic effect, but the effect was not significantly different between unmodified and CEdG-modified DNA (data not shown) In contrast, a 10 000-fold reduction of viable cells was observed when bacteria transformed with modified DNA (5 mm DHA, h) were compared with those transformed with unmodified DNA after plating on ampicillin-supplemented plates Furthermore, the reduction of the number of ampicillin-resistant colonies correlated with increasing CEdG concentration (Fig 6) 30 20 Restriction enzyme digestion of CEdG-modified plasmid DNA 10 Mock transfection Control plasmid CEdG modified plasmid Fig Cytotoxic effect of unmodified and CEdG-modified DNA on transfected HEK 293 T cells Equal amounts of the unmodified control plasmid DNA and the CEdG-modified plasmid DNA were transfected into HEK 293 T cells via calcium phosphate precipitation For the introduction of CEdG adducts, 0.1 lgỈlL)1 plasmid DNA and mM DHA were incubated for h at 70 °C The cell viability was determined 24 h after transfection Mean and standard deviation are shown 918 Influence of CEdG adducts on the functionality of the ampicillin resistance gene Restriction enzymes recognize a palindromic DNA sequence and cut the DNA endonucleolytically within this sequence To determine whether CEdG-modified plasmid DNA is differentially recognized and digested by restriction enzymes, CEdG-modified and unmodified plasmid DNA was digested in parallel by six restriction endonucleases, which were known to cut the plasmid DNA at a single position, at two positions, or at three positions, respectively Enzymes were chosen FEBS Journal 275 (2008) 914–925 ª 2008 The Authors Journal compilation ª 2008 FEBS V Breyer et al DNA advanced glycation end-products in cells enzymes, the CEdG-modified plasmid DNA often yielded higher amounts of the open circular undigested and of only partially digested plasmid as compared to the unmodified plasmid DNA (Fig 7) (cfu·mL–1) A 1.E+09 1.E+08 1.E+07 1.E+06 1.E+05 1.E+04 1.E+03 1.E+02 1.E+01 1.E+00 Quantification of plasmid transcription Control In order to determine whether reduced luciferase activity after transfection with CEdG-modified plasmid DNA is due to reduced expression of the gene, mRNA of transfected cells was isolated, reverse transcribed, and amplified with luciferase-specific primers For normalization, unmodified b-galactosidase plasmid was cotransfected and amplified in parallel Two independent transfection experiments resulted in a 60% reduced expression level of the luciferase gene (Fig 8) 9h B 7.E+04 (cfu·mL–1) 6.E+04 5.E+04 4.E+04 3.E+04 2.E+04 1.E+04 0.E+00 3h 6h 9h Fig Colony forming units (cfmL)1) after electroporation of an unmodified and a CEdG-modified vector in competent E coli JM 109 cells and plating on ampicillin-supplemented TY plates (A) Comparison of unmodified (treated similarly but in the absence of DHA) and CEdG-modified DNA (9 h, mM DHA) (B) Incubation time-dependent reduction of colony forming units (cfmL)1) of CEdG-modified plasmid DNA (0.1 lgỈlL)1 DNA, mM DHA for 3, and h at 70 °C) so that they had at least two GC pairs in the recognition site When the agarose gel electrophoresis patterns of undigested samples of CEdG-modified and unmodified plasmids were compared, differences in the amounts of supercoiled, open circular and linear DNA were observed After hydrolysis by the restriction Discussion In this study, we developed a highly sensitive method to measure cellular DNA glycation rate in vitro by LC-MS ⁄ MS, with the goal of studying parameters that promote cellular DNA glycation The two diastereomers of CEdG were chosen as important DNA glycation products, because they are formed from a large variety of glycating agents and represent stable adducts that can accumulate during a lifetime [12] Furthermore, CEdG was detected in vivo, where its formation was related to diseases, such as diabetic nephropathy [13–15] In order to detect low amounts of DNA AGEs present in cultured cells, maximum analytical sensitivity of the LC-MS ⁄ MS method had to be achieved by several measures In particular, the solvent composition of the HPLC gradient, the thorough removal of RNA from the samples and the use of negative ionization for Fig Restriction digests of CEdG-modified and unmodified pGL3 Control vector For the introduction of CEdG adducts, 0.1 lgỈlL)1 plasmid was incubated with mM DHA for h at 70 °C The digestions were performed at 37 °C overnight using NcoI, AfeI, MfuI, BspMI, BspHI, and PvuI, respectively For control, the vector was treated in parallel, but in the absence of DHA (U, unmodified; M, CEdG modified; k, marker k EcoRI ⁄ HindIII; SS, single stranded; SC, supercoiled; L, linear; OC, open circular) FEBS Journal 275 (2008) 914–925 ª 2008 The Authors Journal compilation ª 2008 FEBS 919 Relative luciferase expression (%) DNA advanced glycation end-products in cells V Breyer et al 100 90 80 70 60 50 40 30 20 10 Control mM DHA, h Fig Quantification of mRNA expression CEdG-modified (5 mM DHA for h at 70 °C) pGL3 Control vector plasmid DNA (0.1 lgỈlL)1) and the unmodified vector (control) were transfected in parallel into HEK 293 T cells The isolated RNAs were reverse transcribed and amplified with luciferase- and b-galactosidase-specific primers Amplification products were separated on agarose gels, and the amount was determined semiquantitatively Relative luciferase activity was defined as the ratio of luciferase to a cotransfected b-galactosidase mean value relative to the unmodified pGL3 Control vector The experiment was performed in duplicate MS analysis were critical parameters This novel method was then sensitive enough to detect CEdGA,B in cultured cells, when a sufficient number of cells was combined Thus, the influence of culture conditions on cellular CEdG formation and the relative glycation rate could be determined Among several tested cell lines, hepatic stellate cells (HSC-T6) showed the highest glycation rate Even after only h of incubation of the confluent cells, CEdGA,B could be measured by LC-MS ⁄ MS This system proved to be suitable for the investigation of factors that influence cellular DNA glycation First, we investigated whether CEdG accumulates in resting HSC-T6 cells After 14 days of incubation, a 1.7-fold increase of CEdG content in the genomic DNA of the cells was measured These results indicated that CEdG can accumulate at least in vitro under conditions of limited cell proliferation In vivo, nonproliferating cells are found in postmitotic tissue, such as the brain DNA repair activity in adult brain is only present at a very low level [22], leading to an accumulation of DNA damage and DNA adducts during aging [23,24] CEdG formation is caused by the reaction of reactive carbonyl compounds with DNA As reactive carbonyl compounds are ubiquitously present in cells, the current results suggest that DNA glycation contributes to the observed accumulation of DNA damage during aging DNA glycation could be further promoted by conditions of increased carbonyl stress, such as diabetes or uremia [25] It was hypothesized before that DNA glycation is a critical mechanism in aging: 920 DNA–carbonyl adducts may be readily removed by the cellular repair systems As these processes are error-prone, they may gradually lead to DNA mutations and – as a consequence – to a gradual loss of genomic integrity [26] On the basis of the present study, a second mechanism can be added in which DNA glycation leads to an accumulation of CEdG adducts in postmitotic tissues with decreased repair efficiency Unrepaired CEdG adducts may influence both replication and transcription by steric hindrance and ⁄ or mispairing Less efficient replication may be caused by modification of the origin of replication or by inhibition of the replication machinery It has been shown that glycation decreases the stability of the N-glycosidic bond in DNA This leads to an increased hydrolysis rate and to depurination in vitro [9] The resulting destabilization of the DNA may also incapacitate the DNA polymerase As a consequence, generalized DNA degradation may occur, which could lead to apoptosis, indicating a cytotoxic effect of CEdG-modified DNA A cytotoxic effect around the significance level was observed in HEK 293 T cells after transfection, whereas transformation of CEdG-modified plasmids into E coli cells showed only a slight cytotoxic effect Hence, eukaryotic cells may have developed a mechanism to eliminate cells with an increased level of glycated DNA To analyze the consequences of DNA glycation for protein expression, different levels of CEdG adducts were introduced into the luciferase-containing reporter gene vector pGL3 Control by a preparative method using DHA The advantage of this method is that defined concentrations of CEdG can be introduced into the DNA by varying DHA concentration and ⁄ or incubation time Under these conditions, formation of byproducts was not observed [9] As a consequence, any observed biological effects can clearly be related to the presence of CEdG In contrast, incubation of DNA with other glycation precursors may lead to ambiguous results, due to the presence of modifications different from CEdG There may be different reasons for the CEdGdependent loss of luciferase activity observed after transfection of DHA-treated DNA into HEK 293 T cells: DNA modification could lead to a generally reduced transcription level, or may inactivate the promoter by preventing transcription factors from binding to the template Furthermore, it is possible that CEdG-induced mutations result in a nonfunctional protein Gene mutations were observed before, when E coli cells were transfected with carboxyethylated DNA [16] Mutation events, mainly transposition, were also detected when a plasmid, preincubated with FEBS Journal 275 (2008) 914–925 ª 2008 The Authors Journal compilation ª 2008 FEBS V Breyer et al a reaction mixture of glucose 6-phosphate and lysine, was transfected into murine lymphoid cells [27] These changes may be caused, for example, by glycation or oxidation reactions that have taken place during the pretreatment In addition, transfection of a glyoxaltreated plasmid into COS-7 cells led to mutations, which were mainly caused by G:C fi T:A transversions [28,29] Glyoxal, however, is not a precursor for CEdG [30] Treatment with mm DHA for h leads to 67 modified bases per plasmid Therefore, theoretically, two or three CEdG adducts were created in the promoter region and a further 21 in the luciferaseencoding gene Agarose gel electrophoresis of the CEdG-modified and unmodified plasmid showed, for the latter, a higher prevalence of the intact supercoiled structure Both heat treatment and CEdG modification have been shown previously to diminish the amount of supercoiled DNA [11,31] As CEdG-modified and unmodified plasmids have been heated in parallel, the observed changes in the DNA structure are most likely caused by the CEdG adducts Comparative restriction digests of CEdG-modified and unmodified plasmid DNA clearly showed that enzymatic interaction with CEdG-modified DNA is disturbed, resulting in a reduced completeness of the digest The inability to digest the CEdG-modified DNA by restriction enzymes can be caused by a steric hindrance of DNA adducts and by the loss of the symmetry of the recognition site Steric hindrance and error-prone repair mechanisms might also be one cause for reduced transcription factor binding decreasing the amount of transcripts Analysis of the amount of luciferase mRNA of transfected cells by semiquantitative RT-PCR resulted in an approximately 60% reduction of transcripts Therefore, reduced expression of functional luciferase is caused, at least in part, by a reduced amount of mRNA Experiments in bacteria demonstrated that, apart from a cytotoxic effect of the transformation procedure itself, transfection with CEdG-modified plasmid DNA had only a minor influence on cell viability as compared to transfection with the unmodified vector Therefore, the 10 000-fold reduction in the formation of ampicillin-resistant colonies is due to the loss of the expression of a functional Amp+ gene, which most likely is caused by mutations altering the correct translation of the protein Both the influence of CEdG modifications on the relative luciferase activity and the influence on the ampicillin resistance resulted in an inhibition of functional gene expression As different transfection ⁄ trans- DNA advanced glycation end-products in cells formation methods were used (calcium phosphate precipitation for HEK 293 T cells and electroporation for E coli), it is unlikely that the observed effects are caused only by decreased transfection ⁄ transformation efficiency It can be assumed that the CEdG adducts in a gene lead to a reduced transcription rate, due to reduced priming or inefficient transcription, and to a loss of gene function, due to missense or nonsense mutations Experimental procedures Synthesis of CEdGA,B dG (Fluka, Buchs, Switzerland; 1.71 g, 0.2 m) and 2.70 g of DHA (1 m) were suspended in 30 mL of m sodium phosphate buffer (pH 7.4) and incubated at 70 °C in a shaking water bath dG dissolved at 70 °C during the course of the reaction After 24 h, the reaction mixture was pipetted slowly into 130 mL of cold ethanol to precipitate sodium phosphate After filtration, ethanol was removed by vacuum distillation with a rotary evaporator The mixture was separated by preparative HPLC in order to obtain both diastereomers of CEdG A Jasco HPLC system (Gross-Umstadt, Germany) with a PU-1580 intelligent pump, an LG-1580-02 ternary gradient unit, a DG-1580-53 three-line degasser and an MD-1510 multiwavelength detector was used Chromatographic conditions were as follows: Macherey-Nagel Nucleosil 100 column (Dueren, Germany), 21 · 250 mm with lm particle size; eluent A, 50 mm aqueous ammonium formate buffer (pH 4.5); eluent B, methanol HPLC grade (Fisher Scientific, Loughborough, UK); eluent C, water; gradient elution (time ⁄ %B; eluent A was constantly 10%), ⁄ 20, 30 ⁄ 50, 35 ⁄ 90, 45 ⁄ 90, 50 ⁄ 20, 70 ⁄ 20, flow rate mLỈmin)1 Two main fractions, which eluted at about 15 and 19 min, were collected, lyophilized, and purified a second time with 100% water as eluent MS and NMR data identified the two products as the diastereomers of CEdG CEdGA and CEdGB were assigned randomly according to the appearance in the HPLC chromatograms LC-MS ⁄ MS instrument setup An Agilent 1100 series HPLC system (Palo Alto, USA) with degasser, binary pump, column compartment, and DAD, a Perkin-Elmer PE200 autosampler (Boston, USA) and an Applied Biosystems API 2000 ESI-MS ⁄ MS instrument (Foster City, CA, USA) were used Chromatographic conditions were as follows: Agilent Zorbax Eclipse XDBC8 column, 4.6 · 150 mm, with lm particle size; FEBS Journal 275 (2008) 914–925 ª 2008 The Authors Journal compilation ª 2008 FEBS 921 DNA advanced glycation end-products in cells V Breyer et al eluent A, mm aqueous ammonium formate buffer (freshly prepared) (pH 6.2); eluent B, acetonitrile HPLC grade (Fisher Scientific); gradient elution (time ⁄ %A), ⁄ 95, ⁄ 40, ⁄ 10, 17 ⁄ 10, 20 ⁄ 95, 25 ⁄ 95, flow rate 300 lLỈmin)1 Unmodified DNA and RNA bases were detected by DAD at their absorption maximum of 254 nm MS parameters were as follows: negative ionization; ion spray voltage )4500 V; nebulizer gas, 30 lb in)2, heater gas, 75 lb in)2; heater gas temperature, 420 °C; declustering potential, )21 V; focusing potential, )340 V; entrance potential, )10.5 V LC-MS ⁄ MS (negative MRM mode): collision gas N2, collision gas setting 9, transitions 338 ⁄ 178 (quantifier), 338 ⁄ 106, 338 ⁄ 149 (CEdG), 266 ⁄ 150 (quantifier), 266 ⁄ 133, 266 ⁄ 108 (dG), scan time 150 ms per mass transition For the MRM method, six different collision energies between –20 V and )50 V were used, so that maximum intensity was achieved for each fragment ion DNA extraction from HSC-T6 cells After incubation, the confluent cells of 10 tissue culture flasks were scraped and combined in one 15 mL reaction tube DNA was extracted by a modified chaotropic method [32] After centrifugation (1700 g, °C, min), 1.5 mL of lysis buffer A [320 mm saccharose, mm MgCl2Ỉ6H2O, 10 mm Tris, 1% Triton X-100 (Fluka), pH adjusted to 7.5 with m HCl] was added to the pellet The mixture was shaken vigorously and centrifuged (10 min, 1500 g, °C) This step was repeated once Next, 35 lL of 10% SDS (Fluka) and 600 lL of buffer B (10 mm Tris, mm Na2-EDTA, pH adjusted to 8.0 with m NaOH) were added to the pellet and shaken vigorously After centrifugation (5 min, 1500 g, °C), the supernatant was incubated with 15 lL of RNase A ⁄ T1-Mix (Fermentas, St Leon-Rot, Germany; 75 U, 15 min, 50 °C) and 30 lL of proteinase K (Fermentas; 600 lg, 60 min, 37 °C) In the next step, 1.2 mL of NaI (7.6 m) and mL of isopropanol were added, and the DNA was precipitated by shaking carefully After centrifugation (15 min, 5000 g, °C), the pellet was washed with mL of isopropanol (40%), centrifuged (15 min, 5000 g, °C), washed again with mL of ethanol (70%), centrifuged again (15 min, 5000 g, °C), and air dried The DNA was then dissolved in 100 lL of water and subjected to enzymatic hydrolysis Enzymatic DNA hydrolysis For enzymatic DNA hydrolysis, a modified protocol according to Crain [33] was used After addition of 10 lL of ammonium acetate buffer (0.1 m, pH 5.3) and 10 lL of S1 nuclease EC 3.1.30.1 (Fermentas) (10 U), the samples were incubated for h at 45 °C Then, 10 lL of ammonium bicarbonate buffer (1 m, pH 8.0) and 10 lL of 922 phosphodiesterase EC 3.1.4.1 (Sigma-Aldrich, Munich, Germany; 0.008 U) were added and incubated for h at 37 °C Finally, the samples were incubated with alkaline phosphatase EC 3.1.3.1 (1 U; Fluka) for h at 37 °C and centrifuged through a 10 kDa cut-off filter (Nanosep spin columns; Pall Life Science, Dreieich, Germany) for 10 at 14 000 g to remove the enzymes The combined filtrates were frozen immediately, stored at )21 °C, and lyophilized directly before LC-MS ⁄ MS analysis Storage of the hydrolyzed samples for more than 24 h at room temperature or for several days at °C can lead to overestimation of the CEdG concentration LC-MS ⁄ MS measurement The dry samples were resolved in 70 lL of water, 50 lL of which was injected into the LC-MS ⁄ MS instrument CEdGA,B and dG were identified by their retention times as well as by their specific mass transitions The detection limit for CEdGA,B was about 0.5 ngỈmL)1 in the solution that was injected into the HPLC The relative glycation rate was determined by the ratio of the peak area of CEdGA,B and dG The glycation rate of the lowest control was set to 100% Cell culture Hepatic stellate cells (HSC-T6) were incubated in growth media (MEM; Biochrom, Berlin, Germany) with Earle’s salts containing 20% fetal bovine serum (Biochrom) and 1% penicillin ⁄ streptomycin solution (10 000 ImL)1; Biochrom) For each experiment, 10 tissue culture flasks (75 cm2) were used The growth medium was removed from the confluent cells The cells were washed twice with NaCl ⁄ Pi for cell culture (Biochrom) Half of the cells were harvested immediately, combined, washed three times with NaCl ⁄ Pi, and stored frozen for further analysis The other half of the cells were incubated with 10 mL of reduced medium [MEM with Earle’s salts containing 1% fetal bovine serum and 1% penicillin ⁄ streptomycin (10 000 ImL)1)] per flask for 14 days After the incubation time, cells were harvested by scraping, and the medium was removed by centrifugation The cell pellets of the samples were combined and washed three times with NaCl ⁄ Pi The experiment was performed in duplicate HEK 293 T cells were cultured in MEM with Earle’s salts supplemented with 10% fetal bovine serum, 1% l-glutamine (200 mm; PAA Coelbe, Germany) and 1% penicillin ⁄ streptomycin (10 000 E, 10 000 lgỈlL)1) at 37 °C in a humidified atmosphere containing 5% CO2 Plasmid DNA preparation The plasmid pGL3 Control (Promega, Mannheim, Germany) was amplified in the competent E coli strain FEBS Journal 275 (2008) 914–925 ª 2008 The Authors Journal compilation ª 2008 FEBS V Breyer et al JM 109, and DNA was extracted using a commercial purification kit (Jetstar Plasmid Kit; Genomed, Loehne, Germany) according to the manufacturer’s instructions Purified plasmid DNA was checked by gel electrophoresis after restriction endonuclease digestion and quantified by absorbance measurement (260 and 280 nm) Treatment of plasmid DNA with DHA For the introduction of CEdG, 0.1 lgỈlL)1 of the reporter vector pGL3 Control plasmid was incubated with 100 lm, mm or mm DHA (VWR International, Darmstadt, Germany), respectively, in NaCl ⁄ Pi (pH 7.4) at 70 °C for h Samples were taken every h Modified vectors derived by incubation with 100 lm DHA were diluted : in NaCl ⁄ Pi, and samples of the incubation mixtures containing mm and mm DHA were diluted : prior to ELISA measurement To remove DHA, plasmid DNA was precipitated with isopropanol and resolved in H2O to a final concentration of lgỈlL)1 The unmodified plasmid was treated in the same way, but in the absence of DHA Competitive ELISA for CEdG The formation of CEdG modifications of plasmid DNA was monitored by ELISA Ninety-six-well microtiter plates were coated with 100 lL per well of a carboxyethylguanine ⁄ BSA solution (0.2 lgỈmL)1 BSA conjugate in 0.2 m sodium carbonate buffer, pH 9.7) at °C overnight The plates were washed twice with washing buffer [1 mm KH2PO4, mm K2HPO4, 15 mm NaCl, 0.02 mm potassium sorbate, and 0.05% (v ⁄ v) Tween-20 (Sigma-Aldrich)] after each step Unspecific binding was minimized by blocking the wells for 1.5 h at room temperature with 150 lL per well of skimmed milk powder (Fluka) in water (3%) Aliquots of 50 lL of the sample as well as 50 lL of the mAb M-5.1.6 [13] diluted : 100 in diluting buffer [0.2% BSA (Sigma-Aldrich) and 0.05% Tween-20 in NaCl ⁄ Pi] were added per well and incubated for h at room temperature Labeling was performed with anti-(mouse IgG) horseradish peroxidase conjugate (Sigma-Aldrich) diluted : 2500 in NaCl ⁄ Pi containing mgỈmL)1 BSA The plates were incubated for 45 at room temperature After the plates had been washed three times, antibody binding was detected using 100 lL of 3,3¢,5,5¢-tetramethylbenzidine dihydrochloride solution (TMB) (Sigma-Aldrich) The reaction was stopped after 15 by adding 25 lL of m sulfuric acid The absorbance was measured at 450 nm The concentrations of carboxyethyl-modified nucleobases were calculated from a calibration curve using CEdGA,B as a standard DNA advanced glycation end-products in cells Transient transfection All transient transfection experiments were carried out in sixwell multi-dishes HEK 293 T cells were seeded 24 h before transfection at · 105 cells per well Transfection of CEdGmodified plasmid DNA was performed using calcium phosphate precipitation [34] Cells were transfected with lg per well of DHA-treated plasmid DNA and cotransfected with lg per well of an unmodified pSV–b-galactosidase (Promega) plasmid to determine the transfection efficiency After transfection, cells were grown for 48 h at 37 °C For the determination of b-galactosidase and luciferase activity in transfected cells, a b-galactosidase and a luciferase reporter gene assay were used (both Roche Applied Science, Mannheim, Germany) Both assays were conducted following the manufacturer’s instructions Relative luciferase activities were defined as the ratio of the firefly luciferase to the b-galactosidase value of each sample relative to the mean value of unmodified pGL3 Control vector All assays were carried out in triplicate Data were reported as mean ± standard deviation In all cases, statistical comparison was done between the plasmids just after adding DHA (0 h) and the plasmid after an incubation time of h, h, or h, respectively Statistical analyses were performed using the unpaired Student’s t-test The significance level was set to P < 0.05 Cytotoxic effect on transfected HEK 293 T cells CEdG-modified and unmodified plasmid DNA was transfected into HEK 293 T cells using calcium phosphate precipitation After 24 h, Tryptan blue was added, and the number of viable cells was determined using a Neubauer counting chamber Transformation of DNA to a bacterial host CEdG adducts were introduced into the vector pGL3 Control by incubation of 0.1 lgỈmL)1 plasmid and mm DHA for 0, 3, and h at 70 °C At each point, aliquots of 50 lL were taken for further analysis The samples were purified by isopropanol precipitation To ensure equal amounts in the electroporation mixtures, the concentration after isopropanol precipitation was determined by UV spectrometry Quantities of 100 ng of each sample as well as 100 ng of the unmodified control vector were added to 50 lL of the electrocompetent bacterium E coli JM 109 Electroporation was performed at 240 V An aliquot of mL of · tryptan yeast medium (TY; Roth, Karlsruhe, Germany) was then added per cuvette and incubated for 45 at 37 °C Finally, 10 lL, 20 lL and 50 lL of each sample was plated on ampicillin-supplemented · TY plates Colonies were counted after an overnight incubation at 37 °C In parallel, transformation assays were plated on · TY plates (without ampicillin) to analyze cell viability FEBS Journal 275 (2008) 914–925 ª 2008 The Authors Journal compilation ª 2008 FEBS 923 DNA advanced glycation end-products in cells V Breyer et al Restriction digestion of CEdG-modified plasmids References CEdG-modified pGL3 Control vector was produced by a h treatment with mm DHA Aliquots of 0.5 lg of CEdG-modified and unmodified vector were incubated in parallel with restriction endonucleases NcoI, AfeI, MluI, BspMI, BspHI, and PvuI, respectively (NEB, Ipswich, USA) The digests were incubated overnight at 37 °C, and fragments were separated by 1% agarose gel electrophoresis Maillard LC (1912) Action des acides amines sur les ` ´ sucres; formation de melanoides par voie methodique C R Acad Sci 154, 66–68 Koening R, Blobstein S & Cerami A (1977) Structure of carbohydrate of hemoglobin A1c J Biol Chem 252, 2992–2997 Vlassara H, Bucala R & Striker L (1994) Pathogenic effects of advanced glycosylation: biochemical, biological, and clinical implications for diabetes and aging Lab Invest 70, 138–151 Park L, Raman KG, Lee KJ, Lu Y, Ferran LJJ, Chow WS, Stern D & Schmidt AM (1998) Suppression of accelerated diabetic atherosclerosis by the soluble receptor for advanded glycation endproducts Nat Med 4, 1025–1031 Singh R, Barden A, Mori T & Beilin L (2001) Advanced glycation end-products: a review Diabetologia 44, 129–146 Knerr T & Severin T (1993) Reaction of glucose with guanosine Tetrahedron Lett 34, 7389–7390 Lee A & Cerami A (1987) The formation of reactive intermediate(s) of glucose 6-phosphate and lysine capable of rapidly reacting with DNA Mutat Res 179, 151– 158 Ochs S & Severin T (1994) Reaction of 2¢-deoxyguanosine with glyceraldehyde Liebigs Ann Chem 851–853 Seidel W & Pischetsrieder M (1998) DNA-glycation leads to depurination by the loss of N2-carboxyethylguanine in vitro Cell Mol Biol (Noisy-le-grand) 44, 1165–1170 10 Larisch B, Pischetsrieder M & Severin T (1997) Formation of guanosine adducts from L-ascorbic acid under oxidative conditions Bioorg Med Chem Lett 7, 2681– 2686 11 Seidel W & Pischetsrieder M (1998) Immunochemical detection of N2-[1-(1-carboxy)ethyl]guanosine, an advanced glycation end product formed by the reaction of DNA and reducing sugars or L-ascorbic acid in vitro Biochim Biophys Acta 1425, 478–484 12 Frischmann M, Bidmon C, Angerer J & Pischetsrieder M (2005) Identification of DNA adducts of methylglyoxal Chem Res Toxicol 18, 1586–1592 13 Schneider M, Thoss G, Hubner-Parajsz C, Kientschă Engel R, Stahl P & Pischetsrieder M (2004) Determination of glycated nucleobases in human urine by a new monoclonal antibody specific for N2-carboxyethyl-2¢deoxyguanosine Chem Res Toxicol 17, 1385–1390 14 Li H, Nakamura S, Miyazaki S, Morita T, Suzuki M, Pischetsrieder M & Niwa T (2006) N2-carboxyethyl-2¢deoxyguanosine, a DNA glycation marker, in kidneys and aortas of diabetic and uremic patients Kidney Int 69, 388–392 RNA isolation and RT-PCR HEK 293 T cells were transfected with CEdG-modified and unmodified pGL3 Control vector, respectively, and cotransfected with pSV–b-galactosidase plasmid for normalization As control, mock transfections were performed by transfecting the b-galactosidase plasmid only RNA was isolated 24 h after transfection using a peqGOLD RNAPureTM extraction kit (Peqlab, Erlangen, Germany) The isolated RNA was dissolved in 30 lL of nuclease-free water (Biorad, Munich, Germany) RNA concentrations were determined by UV measurement RNA was treated with DNAse I (Fermentas) and purified with phenol ⁄ chloroform prior to reverse transcription Five micrograms of RNA were reverse transcribed using an iScriptTM cDNA Synthesis Kit (Biorad) according to the manufacturer’s instructions cDNA was diluted : and used as a template for PCR to quantify luciferase and b-galactosidase expression levels cDNA quality was assayed by a b-actin PCR The primers used were as follows: b-actin – hsbact-S3-391 (5¢-TGA GAC CTT CAA CAC CCC AG-3¢) and hsbact-A5-1046 (5¢-CAT CTG CTG GAA GGT GGA CA-3¢); b-galactosidase – b_Gal_F (5¢AAT CGT CTG ACC GAT GAT CC-3¢) and b_Gal_R (5¢-CGG ATA AAC GGA ACT GGA AA-3¢); and luciferase – Luci_F (5¢-TAT CCG CTG GAA GAT GGA AC-3¢) and Luci_1R (5¢-TTT CTT GCG TCG AGT TTT CC-3¢) Samples of 250 ng were amplified using 30 cycles of denaturing for 30 s at 96 °C, annealing for 30 s at 60 °C, and extension for at 72 °C PCR products were analyzed on 1.5% agarose gels Expression levels were determined by semiquantification of gel bands using image j (http://rsb info.nih.gov/ij/) Luciferase levels were normalized to the b-galactosidase value Experiments were carried out in duplicate Acknowledgements We thank Dr Kristina Becker, Dr Kseniya Kashkevich, Barbara Orlicz-Welcz and Rosa Weber for technical support and helpful advice AS was supported by the Interdisciplinary Center for Clinical Research (IZKF) at the University Hospital of the University of Erlangen-Nuremberg Project D3 (Prof Dr C.-M Becker, Dr Strissel) 924 FEBS Journal 275 (2008) 914–925 ª 2008 The Authors Journal compilation ª 2008 FEBS V Breyer et al 15 Nakamura S, Li H, Adijiang A, Pischetsrieder M & Niwa T (2007) Pyridoxal phosphate prevents progression of diabetic nephropathy Nephrol Dial Transplant 22, 2165–2174 16 Pischetsrieder M, Seidel W, Munch G & Schinzel R (1999) N(2)-(1-Carboxyethyl)deoxyguanosine, a nonenzymatic glycation adduct of DNA, induces single-strand breaks and increases mutation frequencies Biochem Biophys Res Commun 264, 544–549 17 Eriksson UJ, Wentzel P, Minhas HS & Thornalley PJ (1998) Teratogenicity of 3-deoxyglucosone and diabetic embryopathy Diabetes 47, 1960–1966 18 Shires TK, Tresnak J, Kaminsky M, Herzog SL & Truc-Pham B (1990) DNA modification in vivo by derivatives of glucose: enhancement by glutathione depletion FASEB J 4, 3340–3346 19 Vaca CE, Nilson JA, Fang JL & Grafstrom RC (1998) ă Formation of DNA adducts in human buccal epithelial cells exposed to acetaldehyde and methylglyoxal in vitro Chem Biol Interact 108, 197–208 20 Schneider M, Georgescu A, Bidmon C, Tutsch M, Fleischmann EH, Popov D & Pischetsrieder M (2006) Detection of DNA-bound advanced glycation endproducts by immunoaffinity chromatography coupled to HPLC-diode array detection Mol Nutr Food Res 50, 424–429 21 Bidmon C, Frischmann M & Pischetsrieder M (2007) Analysis of DNA-bound advanced glycation endproducts by LC and mass spectrometry J Chromatogr B Analyt Technol Biomed Life Sci 855, 51–58 22 Rao KS (1993) Genomic damage and its repair in young and aging brain Mol Neurobiol 7, 23–48 23 Rao KS & Loeb LA (1992) DNA damage and repair in brain: relationship to aging Mutat Res 275, 317–329 24 Randerath K, Zhou GD, Hart RW, Turturro A & Randerath E (1993) Biomarkers of aging: correlation of DNA I-compound levels with median lifespan of calorically restricted and ad libitum fed rats and mice Mutat Res 295, 247–263 DNA advanced glycation end-products in cells 25 Thornalley PJ (2003) Protecting the genome: defence against nucleotide glycation and emerging role of glyoxalase I overexpression in multidrug resistance in cancer chemotherapy Biochem Soc Trans 31, 1372–1377 26 Baynes JW (2002) The Maillard hypothesis on aging: time to focus on DNA Ann NY Acad Sci 959, 360–367 27 Bucala R, Lee A, Rourke L & Cerami A (1993) Transposition of an Alu-containing element induced by DNA-advanced glycosylation endproducts Proc Natl Acad Sci USA 90, 2666–2670 28 Murata-Kamiya N, Kamiya H, Kaji H & Kasai H (1997) Glyoxal, a major product of DNA oxidation, induces mutations at G:C sites on a shuttle vector plasmid replicated in mammalian cells Nucleic Acids Res 25, 1897–1902 29 Murata-Kamiya N, Kamiya H, Kaji H & Kasai H (2000) Methylglyoxal induces G:C to C:G and G:C to T:A transversions in the supF gene on a shuttle vector plasmid replicated in mammalian cells Mutat Res 468, 173–182 30 Seidel W & Pischetsrieder M (1998) Reaction of guanosine with glucose under oxidative conditions Bioorg Med Chem Lett 8, 2017–2022 31 Levy MS, Lotfian P, O’Kennedy R, Lo-Yim MY & Shamlou PA (2000) Quantitation of supercoiled circular content in plasmid DNA solutions using a fluorescencebased method Nucleic Acids Res 28, E57 32 Ravanat JL, Douki T, Duez P, Gremaud E, Herbert K, Hofer T, Lasserre L, Saint-Pierre C, Favier A & Cadet J (2002) Cellular background level of 8-oxo-7,8-dihydro-2¢-deoxyguanosine: an isotope based method to evaluate artefactual oxidation of DNA during its extraction and subsequent work-up Carcinogenesis 23, 1911–1918 33 Crain P (1990) Preparation and enzymatic hydrolysis of DNA and RNA for mass spectrometry Methods Enzymol 193, 783–790 34 Chen C & Okayama H (1987) High-efficiency transformation of mammalian cells by plasmid DNA Mol Cell Biol 7, 2745–2752 FEBS Journal 275 (2008) 914–925 ª 2008 The Authors Journal compilation ª 2008 FEBS 925 ... Breyer et al DNA advanced glycation end-products in cells 300 Glycation rate (%) 250 200 150 100 50 0 days of incubation 14 days of incubation Fig Increase in the glycation rate of genomic DNA from... for the analysis of the CEdG formation rate in genomic DNA of cultured cells Furthermore, the in? ??uence of CEdG DNA advanced glycation end-products in cells formation on cellular protein expression... we investigated whether CEdG accumulates in resting HSC-T6 cells After 14 days of incubation, a 1.7-fold increase of CEdG content in the genomic DNA of the cells was measured These results indicated