Incidence and potential implications of the toxic metabolite methylglyoxal in cell culture: A review

Methylglyoxal is a toxic metabolite unavoidably produced in mammalian systems as a by-product of glycolysis. Detoxification of this compound occurs principally through the glyoxalase pathway, which consists of glyoxalase I and glyoxalase II, and requires reduced glutathione as a co-enzyme. Recently, it has been demonstrated that variations in glucose, glutamine and fetal bovine serum levels can cause significant changes in the intracellular concentration of methylglyoxal. More importantly, comparative studies involving wild-type Chinese hamster ovary cells and clones overexpressing glyoxalase I indicate that glucose and glutamine, within the range normally found in cell culture media, can cause decreased cell viability mediated solely through increased production of methylglyoxal. In addition, endogenously produced methylglyoxal has been shown to cause apoptosis in cultured HL60 cells. While the exact mechanism of the impact of methylglyoxal on cultured cells is unknown, methylglyoxal is a potent protein and nucleic acid modifying agent at physiological concentrations and under physiological conditions. Protein modification occurs mainly at arginine, lysine and cysteine residues and is believed to be an important signal for the degradation of senescent proteins. Modification of arginine and lysine results in the irreversible formation of advanced glycation endproducts, whereas modification of cysteine results in the formation of a highly reversible hemithioacetal. Methylglyoxal also forms adducts with nucleic acids, principally with guanyl residues. At high extracellular concentrations, it is genotoxic to cells grown in culture. Even at physiological concentrations (100 nM free methylglyoxal), methylglyoxal can modify unprotected plasmid DNA and cause gene mutation and abnormal gene expression. This revised version was published online in June 2006 with corrections to the Cover Date.     

Myeloperoxidase oxidation states involved in myeloperoxidase-oxidase oxidation of thiols.

The human red-blood-cell glyoxalase system was modified by incubation with high concentrations of glucose in vitro. Red-blood-cell suspensions (50%, v/v) were incubated with 5 mM- and 25 mM-glucose to model normal and hyperglycaemic glucose metabolism. There was an increase in the flux of methylglyoxal metabolized to D-lactic acid via the glyoxalase pathway with high glucose concentration. The increase was approximately proportional to initial glucose concentration over the range studied (5-100 mM). The activities of glyoxalase I and glyoxalase II were not significantly changed, but the concentrations of the glyoxalase substrates, methylglyoxal and S-D-lactoylglutathione, and the percentage of glucotriose metabolized via the glyoxalase pathway, were significantly increased. The increase in the flux of intermediates metabolized via the glyoxalase pathway during periodic hyperglycaemia may be a biochemical factor involved in the development of chronic clinical complications associated with diabetes mellitus.     

Production of S-lactoylglutathione by glycerol-adapted Saccharomyces cerevisiae and genetically engineered Escherichia coli cells

Summary The enzymatic production of S-lactoylglutathione was studied by applying glyoxalase I to glycerol-grown cells of Saccharomyces cerevisiae and Escherichia coli cells dosed with Pseudomonas putida glyoxalase I gene. The glyoxalase I in S. cerevisiae cells was markedly induced when the cells were grown on glycerol. The activity of the enzyme in glycerol-grown cells was more than 20-fold higher compared with that of the glucose-grown cells. By using extracts of glycerol-grown yeast cells, about 5 mmol/1 (2 g/l) of S-lactoylglutathione was produced from 10 mM methylglyoxal and 50 mM glutathione within 1 h. The extracts of E. coli cells carrying a hybrid plasmid pGI423, which contains P. putida glyoxalase I gene, showed approximately 170-fold higher glyoxalase I activity than that of E. coli cells without pGI423. The extracts were used for production of S-lactoylglutathione and, under optimal conditions, about 40 mmol/l (15 g/l) of S-lactoylglutathione was produced from 50 mM methylglyoxal and 100mM glutathione within 1 h.     

Induction of sister-chromatid exchanges in Chinese hamster ovary cells by the biotic ketoaldehyde methylglyoxal

The number of sister-chromatid exchanges (SCEs) per metaphase was determined in Chinese hamster ovary cells after 16 h exposure to methylglyoxal (MG) concentrations ranging from 0.1 to 0.75 mM. MG produced an increase of SCE frequency that proved to be dose-dependent, and to reach a maximum of 2 X baseline at the highest nontoxic concentration (0.5 mM).     

Upregulation of Glyoxalase I fails to Normalize Methylglyoxal Levels: A Possible Mechanism for Biochemical Changes in Diabetic Mouse Lenses

Glyoxalase I is the first enzyme in a two-enzyme glyoxalase system that metabolizes physiological methylglyoxal (MGO). MGO reacts with proteins to form irreversible adducts that may lead to crosslinking and aggregation of lens proteins in diabetes. This study examined the effect of hyperglycemia on glyoxalase I activity and its mRNA content in mouse lens epithelial cells (mLE cells) and in diabetic mouse lenses and investigated the relationship between GSH and MGO in organ cultured lenses. mLE cells cultured with 25 mM D-glucose (high glucose) showed an upregulation of glyoxalase I activity and a higher content of glyoxalase I mRNA when compared with either cells cultured with 5 mM glucose (control) or with 20 mM L-glucose + 5 mM D-glucose. MGO concentration was significantly elevated in cells cultured with high D-glucose, but not in L-glucose. GSH levels were lower in cells incubated with high glucose compared to control cells. Glyoxalase I activity and mRNA levels were elevated in diabetic lenses compared to non-diabetic control mouse lenses. MGO levels in diabetic lenses were higher than in control lenses. Incubation of lenses with buthionine sulfoximine (BSO) resulted in a dramatic decline in GSH but the MGO levels were similar to lenses incubated without BSO. Our data suggest that in mouse lenses MGO accumulation may occur independent of GSH concentration and in diabetes there is an upregulation of glyoxalase I, but this upregulation is inadequate to normalize MGO levels, which could lead to MGO retention and chemical modification of proteins.     

Toxicity of methylglyoxal towards rat enterocytes and colonocytes.

Effect of methylglyoxal, a bacterial metabolic product, on protein, DNA, and RNA synthesis in rat enterocytes and colonocytes was investigated. Results showed that 1 mM methylglyoxal inhibited protein, DNA, and RNA synthesis to the extent of 65-85, 65-80, and 10-20 per cent, respectively, in villus and crypt cells and colonocytes. The inhibitory pattern was similar in these various cell types. The inhibitory effect on protein and DNA synthesis was more marked than that on RNA synthesis. Inclusion of thiol compounds up to 4 mM concentration did not protect the cells from the inhibitory effect of methylglyoxal. No alteration in the level of cellular reduced glutathione and glyoxalase enzyme activity was observed when cells were incubated with 2 mM methylglyoxal. These results suggest that the antiproliferative action of methylglyoxal on eukaryotic cells may be through the inhibition of macromolecular synthesis.     

Mutagenesis by simian virus 40. II. Changes in substrate affinities in mutant hypoxanthine-guanine phosphoribosyl transferase enzymes at different pH values.

A number of 8-azaguanine-resistant clones selected from Chinese hamster cells infected with SV 40, and supposed to originate by virus infection was investigated to demonstrate and analyze genetic alterations occurring in the cells after infection. All resistant clones tested showed reduced but detectable activity levels of the enzyme hypoxanthine-guanine phosphoribosyltransferase. The extent of reduction in the activity was not identical for different substrates. In all the clones tested, spontaneous mutants included, the pH optimum for the enzymic reaction with guanine was shifted to lower values. The reduced enzymic activities of resistant clones correlated with their colony-forming ability in corresponding selective media. The results support the suggestion that SV 40 is able to induce gene mutations.     

Characterization of the collagens synthesized by Chinese hamster ovary cells. Effect of colcemid and dibutyryladenosine cyclic monophosphate

The collagens synthesized by Chinese hamster ovary cells have been isolated and characterized. Although these cells produce very small amounts of collagen, at least five distinct collagenous chains could be identified from radiolabeled media and cell extracts after limited pepsin digestion. Two chains were characterized as alpha 1(V) and alpha 2(V), based on electrophoretic mobility, resistance to vertebrate collagenase, chromatographic properties on carboxymethylcellulose, and cyanogen bromide peptide patterns. Two smaller collagenous proteins (Mr 34000 and 37000) were also isolated by carboxymethylcellulose chromatography and characterized by cyanogen bromide digestion patterns. These collagens showed similarities to type IV collagen fragments but may be unique to Chinese hamster ovary cells. A colcemid-resistant mutant of Chinese hamster ovary cells designated CMR795 [Ling, V., Aubin, J.E., Chase, A., & Sarangi, F. (1979) Cell (Cambridge, Mass.) 18, 423-430] was found to synthesize the same collagen chains but in different proportions. In the wild-type cells colcemid (0.05-0.1 microgram/mL) reduced the amount of type V collagen in the culture media but had little effect on the other collagen type, whereas the type V collagen reduction was less pronounced in the CMR795 cells treated with the same concentrations of colcemid. Dibutyryladenosine cyclic monophosphate caused a fibroblast-like "reverse transformation" of the Chinese hamster ovary cells similar to that described previously [Hsie, A.W., & Puck, T. T. (1971) Proc. Natl. Acad. Sci. U.S.A. 68, 358-361]. However, collagen synthesis was increased only slightly. Furthermore, no apparent alteration in the types of collagens synthesized was detected.     

Chlorate Reducing Activity of Spinach Nitrate Reductase

The activities of 2-oxoaldehyde-metabolizing enzymes (glyoxalase I, glyoxalase II, methyl- glyoxal reductase, methylglyoxal dehydrogenase and lactaldehyde dehydrogenase) were found to be widely distributed among microorganisms. One of the enzymes, methylglyoxal reductase, which catalyzes the reductive conversion of methylglyoxal into lactaldehyde, was purified from Escherichia coli cells. The enzyme was judged to be homogeneous on polyacrylamide gel electrophoresis and was a monomer with a molecular weight of 43000. The enzyme was most active at pH 6.5 and 45°C. The enzyme utilized both NADPH and NADH for the reduction of 2- oxoaldehydes (glyoxal, methylglyoxal, phenylglyoxal and 4,5-dioxovalerate) and some aldehydes (glycolaldehyde, D,l-glyceraldehyde, propionaldehyde and acetaldehyde). The Km values of the enzyme for methylglyoxal, NADPH and NADH were 4.0 mm, 1.7 fiM and 2.8 /¿m, respectively. The product of methylglyoxal reduction was identified as lactaldehyde. The enzyme from E. coli cells was different from the yeast and goat liver enzymes in both molecular structure and substrate specificity.     

Protein glycation in Saccharomyces cerevisiae. Argpyrimidine formation and methylglyoxal catabolism

Methylglyoxal is the most important intracellular glycation agent, formed nonenzymatically from triose phosphates during glycolysis in eukaryotic cells. Methylglyoxal-derived advanced glycation end-products are involved in neurodegenerative disorders (Alzheimer's, Parkinson's and familial amyloidotic polyneurophathy) and in the clinical complications of diabetes. Research models for investigating protein glycation and its relationship to methylglyoxal metabolism are required to understand this process, its implications in cell biochemistry and their role in human diseases. We investigated methylglyoxal metabolism and protein glycation in Saccharomyces cerevisiae. Using a specific antibody against argpyrimidine, a marker of protein glycation by methylglyoxal, we found that yeast cells growing on d-glucose (100 mM) present several glycated proteins at the stationary phase of growth. Intracellular methylglyoxal concentration, determined by a specific HPLC based assay, is directly related to argpyrimidine formation. Moreover, exposing nongrowing yeast cells to a higher d-glucose concentration (250 mM) increases methylglyoxal formation rate and argpyrimidine modified proteins appear within 1 h. A kinetic model of methylglyoxal metabolism in yeast, comprising its nonenzymatic formation and enzymatic catabolism by the glutathione dependent glyoxalase pathway and aldose reductase, was used to probe the role of each system parameter on methylglyoxal steady-state concentration. Sensitivity analysis of methylglyoxal metabolism and studies with gene deletion mutant yeast strains showed that the glyoxalase pathway and aldose reductase are equally important for preventing protein glycation in Saccharomyces cerevisiae.     

Complementation of a methotrexate uptake defect in Chinese hamster ovary cells by DNA-mediated gene transfer.

A methotrexate-resistant Chinese hamster ovary cell line deficient in methotrexate uptake has been complemented to methotrexate sensitivity by transfection with DNA isolated from either wild-type Chinese hamster ovary or human G2 cells. Primary and secondary transfectants regained the ability to take up methotrexate in a manner similar to that of wild-type cells, and in the case of those transfected with human DNA, to contain human-specific DNA sequences. The complementation by DNA-mediated gene transfer of this methotrexate-resistant phenotype provides a basis for the cloning of a gene involved in methotrexate uptake.     

Chinese hamster ovary cell mutants defective in the internalization of ricin.

Chinese hamster ovary mutants simultaneously resistant to ricin and Pseudomonas toxin have been isolated. Two mutant cell lines (4-10 and 11-2) were found to retain normal levels of binding of both ricin and Pseudomonas toxin. They were defective in the internalization of [125I]ricin into the mutant cells, as measured by both a biochemical assay for ricin internalization and electron microscopic autoradiographic studies. Although pretreatment of Chinese hamster ovary cells with a Na+/K+ ionophore, nigericin, resulted in an enhancement of the cytotoxicities of ricin and Pseudomonas toxin in the wild-type Chinese hamster ovary cells, preculture of the mutant cells did not alter the susceptibility of the mutant cells to either toxin. These results provide further evidence that there is a common step in the internalization process for ricin and Pseudomonas toxin.     

Effects of growth media on cell cycle progression in CHO cells exposed to the radioprotector WR-1065

Abstract. WR-1065 (2-[(aminopropyl)amino]ethanethiol) reduces cytotoxic and mutagenic effects caused by exposure of cells to radiation and chemotherapeutic drugs, but the mechanisms involved are not fully known. We have observed an accumulation of cells in G, in WR-1065 treated Chinese hamster ovary cells grown in a-minimal essential medium, while others have found no cell cycle effects in WR-1065 treated Chinese hamster ovary cells grown in McCoy's 5A medium. To determine if the two types of media had an effect on cells treated with WR-1065, we examined survival and cell cycle progression. Population doubling times of 12 h were observed for cells grown in both media. Incubation of AA8 cells grown in McCoy's 5A medium with 4 mM WR-1065 30 min prior to and during irradiation with ^13'Cs gamma-rays resulted in a protection factor of 2.2, in close agreement with the value of 2.0 we previously obtained for AA8 cells grown in α-minimal essential medium. Treatment with WR-1065 caused an alteration in the cell cycles of cells grown in both media. An increase in the G₂ population and a decrease in the G₁ population was observed in cells incubated up to 3 h in the presence of 4 mM WR-1065, with a redistribution of the cells throughout the cell cycle occurring following removal of the drug. These data suggest that exposure of cells to WR-1065 is the cause of perturbations in cell cycle progression, and is not affected by the type of medium the cells are grown in.     

Role of GldA in dihydroxyacetone and methylglyoxal metabolism of Escherichia coli K12

The metabolic pathway involving dihydroxyacetone is poorly characterized although novel enzymes associated with this metabolite have recently been demonstrated. The role of GldA in dihydroxyacetone and methylglyoxal metabolism was investigated by purifying the enzyme and characterizing its catalytic ability using nuclear magnetic resonance (NMR) spectroscopy. At neutral pH, the enzyme exhibits much higher affinities towards dihydroxyacetone, methylglyoxal, and glycolaldehyde than glycerol with K(m) values of 0.30, 0.50, 0.85, and 56 mM, respectively. This is consistent with NMR data with crude extracts, showing that the conversion from dihydroxyacetone to glycerol by GldA is far more efficient than the reverse reaction. Dihydroxyacetone was found to be lethal at higher concentration with an LC(50) value of 28 mM compared with 0.4 mM of methylglyoxal, while lactaldehyde was found to exhibit significant growth inhibition in Escherichia coli cells. The toxicity of dihydroxyacetone appears to be due to its intracellular conversion to an aldehyde compound, presumably methylglyoxal, since the glyoxalase mutant becomes sensitive to dihydroxyacetone. Based on information that gldA is preceded in an operon by the ptsA homolog and talC gene encoding fructose 6-phosphate aldolase, this study proposes that the primary role of gldA is to remove toxic dihydroxyacetone by converting it into glycerol.     

Isolation and characterization of Chinese hamster ovary cell mutants defective in glucose transport

Cultured Chinese hamster ovary (CHO) cells possess an insulin-sensitive facilitated diffusion system for glucose transport. Mutant clones of CHO cells defective in glucose transport were obtained by repeating the selection procedure, which involved mutagenesis with ethyl methanesulfonate, radiation suicide with tritiated 2-deoxy-D-glucose, the polyester replica technique and in situ autoradiographic assaying for glucose accumulation. On the first selection, we obtained mutants exhibiting about half the glucose uptake activity of parental CHO-K1 cells and half the amount of a glucose transporter, the amount of which was determined by immunoblotting with an antibody to the human erythrocyte glucose transporter. The second selection, starting from one of the mutants obtained in the first-step selection, yielded a strain, GTS-31, in which both glucose uptake activity and the quantity of the glucose transporter were 10-20% of the levels in CHO-K1 cells, whereas the responsiveness of glucose transport to insulin, and the activities of leucine uptake and several glycolytic enzymes remained unchanged. GTS-31 cells grew slower than CHO-K1 cells at both 33 and 40 degrees C, and in a medium containing a low concentration of glucose (0.1 mM), the mutant cells lost the ability to form colonies. All the three spontaneous GTS-31 cell revertants, which were isolated by growing the mutant cells in medium containing 0.1 mM glucose, exhibited about half the glucose uptake activity and about half the amount of glucose transporter, as compared to in CHO-K1 cells, these characteristics being similar to those of the first-step mutant. These results indicate that the decrease in glucose uptake activity in strain GTS-31 is due to a mutation which induces a reduction in the amount of the glucose transporter, providing genetic evidence that the glucose transporter functions as a major route for glucose entry into CHO-K1 cells.     

Site-directed mutagenesis of GLUT1 in helix 7 residue 282 results in perturbation of exofacial ligand binding.

The structure-function relationship of the HepG2/erythrocyte-type glucose transporter (GLUT1) has been studied by in vitro site-directed mutagenesis. Chinese hamster ovary clones in which glucose transporters were transfected were shown by Western blotting with a GLUT1 anti-COOH-terminal peptide antibody to have expression levels of Gln282----Leu, Asn288----Ile, and Asn317----Ile mutations that were comparable with the wild type. All three mutant GLUT1 clones had high 2-deoxy-D-glucose transport activity compared with a nontransfected clone, suggesting that these residues are not absolutely required for the transport function. We have examined the possibility that the inner and outer portions of the transport pathway are structurally separate by measuring the interaction of the mutant transporters with the inside site-specific ligand cytochalasin B and the outside site-specific ligand 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis(D-mannos-4 -yloxy)-2- propylamine (ATB-BMPA). All three mutant GLUT1 clones showed high levels of cytochalasin B labeling, and the N288I and N317I mutants showed high levels of ATB-BMPA labeling. In contrast to the transport and cytochalasin B labeling results, the transmembrane helix 7 Gln282----Leu mutant was labeled by ATB-BMPA to a level that was only 5% of the level observed in the wild type. We have confirmed that this mutant was defective in the outer site by comparing the inhibition of wild-type and mutant 2-deoxy-D-glucose transport by the outside site-specific ligand 4,6-O-ethylidene-D-glucose. 4,6-O-Ethylidene-D-glucose inhibited wild-type transport with a Ki of approximately 12 mM, but this was increased to greater than 120 mM in the Gln282----Leu mutant. Thus, of the 3 residues mutated in this study, only glutamine 282 substitution causes a major perturbation in function, and this is a specific and striking reduction in the affinity for the outside site-specific ligands ATB-BMPA and 4,6-O-ethylidene-D-glucose.     

Selection of mutant Chinese hamster ovary cells altered glycoproteins by means of tritiated fucose suicide.

Mutant Chinese hamster ovary cells altered in glycoproteins have been isolated by selecting for ability to survive exposure to [6-3H]fucose. Mutagenized wild-type cells were permitted to incorporate [3H]fucose to approximately 1 cpm of trichloroacetic acid-insoluble radioactivity per cell and then frozen for several days to accumulate radiation damage. The overall viability of the population was reduced by 5- to 50-fold. Four consecutive selection cycles were carried out. The surviving cells were screened by replica plating-fluorography for clones showing decreased incorporation of fucose into trichloroacetic acid-insoluble macromolecules. Considerable enrichment for cells deficient in fucose uptake or incorporation into proteins (or both) was found in populations surviving the later selection cycles. Two mutant clones isolated after the fourth selection cycle had the same doubling time as the wild type, but contained only 30 to 40% as much fucose bound to proteins as the wild type. Sialic acid contents of the mutants and the wild type were similar. The mutants differed quantitatively and qualitatively from the wild type and from each other with respect to total glycoprotein profiles as visualized by sodium dodecyl sulfate gel electrophoresis. Differences were also found in resistances to cytotoxicity of lectins such as concanavalin A and wheat germ agglutinin.     

Substitution of leucine for tryptophan 412 does not abolish cytochalasin B labeling but markedly decreases the intrinsic activity of GLUT1 glucose transporter

GLUT1 glucose transporter cDNA was modified to introduce a single amino acid substitution of leucine for tryptophan 412, a putative cytochalasin B photo-affinity labeling site. Although the mutated transporter was expressed into plasma membranes of Chinese hamster ovary cells, glucose transport activity of the mutated transporter was observed to be only 15-30% of that of the wild-type GLUT1 when glucose transport activity was assessed by 2-deoxyglucose uptake at 0.1-10 mM concentrations. Analysis of glucose uptake kinetics depict that a mutation induced a 3-fold decrease in turnover number and a 2.5-fold increase in Km compared with the wild-type GLUT1. Importantly, cytochalasin B labeling was not abolished but decreased by 40%, and cytochalasin B binding was also decreased. In addition, the results obtained with side-specific glucose analogs suggested that the outer glucose binding site of the mutant appeared intact but the inner binding site was modulated. These results indicate 1) tryptophan 412 is not a cytochalasin B labeling site(s), although this residue is located in or close to the inner glucose binding site of the GLUT1 glucose transporter, 2) substitution of leucine for tryptophan 412 decreases the intrinsic activity of GLUT1 glucose transporter, which is definable as the turnover number/Km, to approximately 15% of that of the wild-type.