Imidazopurinones are markers of physiological genomic damage linked to DNA instability and glyoxalase 1-associated tumour multidrug resistance

Paul J. Thornalley 1 2 Sahar Waris 2 Thomas Fleming 1 Thomas Santarius 0 4 Sarah J. Larkin 1 2 Brigitte M. Winklhofer-Roob 3 Michael R. Stratton 0 Naila Rabbani 1 2 0 Wellcome Trust Sanger Institute , Hinxton, Cambridge , CB10 1SA 1 Department of Biological Sciences, University of Essex , Colchester, Essex CO4 3SQ 2 Warwick Medical School, Clinical Sciences Research Institute, University of Warwick, University Hospital , Coventry CV2 2DX 3 Human Nutrition and Metabolism Research and Training Center Graz, Institute of Molecular Biosciences, Karl Franzens University , 8010 Graz, Austria 4 Department of Neurosurgery, Addenbrooke's Hospital and University of Cambridge , Hills Road, Cambridge , CB2 0QQ, UK Glyoxal and methylglyoxal are reactive dicarbonyl metabolites formed and metabolized in physiological systems. Increased exposure to these dicarbonyls is linked to mutagenesis and cytotoxicity and enhanced dicarbonyl metabolism by overexpression of glyoxalase 1 is linked to tumour multidrug resistance in cancer chemotherapy. We report herein that glycation of DNA by glyoxal and methylglyoxal produces a quantitatively important class of nucleotide adduct in physiological systemsimidazopurinones. The adduct derived from methylglyoxal-3-(20-deoxyribosyl)-6,7-dihydro6,7-dihydroxy-6/7-methylimidazo-[2,3-b]purine-9(8)one isomerswas the major quantitative adduct detected in mononuclear leukocytes in vivo and tumour cell lines in vitro. It was linked to frequency of DNA strand breaks and increased markedly during apoptosis induced by a cell permeable glyoxalase 1 inhibitor. Unexpectedly, the DNA content of methylglyoxal-derived imidazopurinone and oxidative marker 7,8-dihydro-8-oxo20-deoxyguanosine were increased moderately in glyoxalase 1-linked multidrug resistant tumour cell lines. Together these findings suggest that imidazopurinones are a major type of endogenous DNA damage and glyoxalase 1 overexpression in tumour cells strives to counter increased imidazopurinone formation in tumour cells likely linked to their high glycolytic activity. - Guanyl bases of nucleotides and nucleosides are susceptible to modification by glyoxal and methylglyoxal (1). Glyoxal and methylglyoxal react with deoxyguanosine under physiological conditions to form mainly imidazopurinone derivatives, 3-(20-deoxyribosyl)-6,7dihydro-6,7-dihydroxyimidazo[2,3-b]purin-9(8)one (GdG) and 3-(20-deoxyribosyl)-6,7-dihydro-6,7-dihydroxy-6/7methylimidazo-[2,3-b]purine-9(8)one (MGdG)a 6- and 7-methyl structural isomeric mixture, respectively (2) (Figure 1a). Glyoxal and methylglyoxal also form N2-carboxymethyl-deoxyguanosine (CMdG) (3) and N2-(1,R/S-carboxyethyl)-deoxyguanosine (CEdG)the latter a stereoisomeric mixture of R/S-epimers at the N2-1-carboxyethyl chiral centre (4). Glyoxal and methylglyoxal are formed in physiological systems: glyoxal is formed by lipid peroxidation and also by degradation of glycated proteins and monosaccharides; methylglyoxal is formed mainly by non-enzymatic degradation of triosephosphates and is also formed by ketone body metabolism and threonine catabolism. Increased methylglyoxal formation occurs in cells with high glycolytic activity. Many tumours have high 6,7-Dihydro-6,7-dihydroxy-7-methylimidazo-[2,3-b]purin-9(8)one 6,7-Dihydro-6,7-dihydroxy-6-methylimidazo-[2,3-b]purin-9(8)one MeCH(OH)CO-SG S-D-lactoylglutathione glycolytic activity which is thought to be a survival adaptation to growth under hypoxic conditions (5,6). Detection of imidazopurinones derived from glyoxal and methylglyoxal in cellular DNA in vitro and in vivo has proven generally elusive to date. There has been extensive research quantifying the level of the dG-derived oxidative marker 8-oxo-7,8-dihydro20-deoxyguanosine (8-OxodG) nucleotides in DNA and related nucleosides released into plasma and excreted in urine (7), as well as other trace endogenous dG adducts (8). There have also been some reports on the minor methylglyoxal-derived nucleotide adduct CEdG (9) and a recent report on the minor glyoxal-derived adduct CMdG (10). Failure to detect imidazopurinones in cell systems may have been due to poor adduct stability and recovery in pre-analytic processing of analytical protocols. Dicarbonyl adducts of DNA are of likely functional importance because glyoxal and methylglyoxal are both weak mutagens. Diseases associated with high plasma levels of dicarbonylsdiabetes and renal failureare also associated with increased mutagenicity, cancer risk and vascular cell apoptosis (11). Protection against dicarbonyl mutagenicity and cytotoxicity is provided mainly by the glutathione-dependent cytosolic glyoxalase system. The glyoxalase system is comprised of glyoxalase 1 (Glo1), glyoxalase 2 (Glo2) and a catalytic amount of glutathione. Glo1 catalyses the detoxification of glyoxal and methylglyoxal to S-glycolylglutathione and S-Dlactoylglutathione, respectively, and Glo2 catalyses the hydrolysis of these glutathione thioesters to glycolate and D-lactate, reforming glutathione consumed by Glo1 (Figure 1b) (12). In 2000, Tsuruo and co-workers (13) discovered overexpression of Glo1 as a novel factor producing multidrug resistance (MDR) in tumours. Glo1-linked MDR was found in tumour cells of lung, colorectal, breast and prostate origin, was acquired by experimental Glo1 overexpression and could be countered by the cell permeable Glo1 inhibitor S-p-bromobenzylglutathione cyclopentyl diester (BBGD) (14)an experimental cancer chemotherapeutic agent (15). In this report, we describe the concurrent quantitation of imidazopurinones, GdG and MGdG, and CEdG and 8-OxodG by stable isotopic dilution analysis liquid chromatography with tandem mass spectrometric detection (LC-MS/MS) in physiological samples and explore the link of levels of imidazopurinone adducts to DNA strand breaks and Glo1-associated MDR in human tumour cell lines. MATERIALS AND METHODS 20-Deoxyguanosine monohydrate and glyoxal and methylglyoxal solutions (40%), ribonuclease (RNase) A from bovine pancreas, RNase T1 from Aspergillus oryzae, deoxyribonuclease (DNase) II from porcine spleen, phosphodiesterase (PDE) II from bovine spleen, acid phosphatase from potato were purchased from Sigma (Poole, Dorset, UK). Protease was from Qiagen. [13C10,15N5]-20-deoxyguanosine (all >98% isotopic purity) was purchased from Cambridge Isotope Laboratories (Andover, MA, USA). GdG, MGdG and related stable isotopic standards were prepared as described earlier (16). CEdG was conveniently prepared from the crude product mixture of MGdG by addition of 10 mM phosphate buffer, adjustment of the pH to7.4 and continued incubation at 37 C for 6 daysthe MGdG therein degrading in part to CEdG. CEdG was purified by preparative anion exchange HPLC on DEAE Protein-Pak, formate form (2 10 cm column; Waters); sample loading 20 mg. The column was equilibrated and eluted isocratically for 10 min with 5 mM ammonium formate buffer, pH 4.0, and then with a linear gradient of 5100 mM of the same (...truncated)