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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).
Methylglyoxal, at concentrations ranging from 0.16 to 1.5 mM, caused a dose-dependent increase in the frequency of HGPRT-deficient mutants in V79 cells. Its mutagenic activity was reduced when V79 cells were co-cultured with freshly isolated rat hepatocytes.
Coffee shows direct-acting mutagenicity in Salmonella typhimurium TA100 and most of this mutagenicity is due to the synergistic effects of methylglyoxal and hydrogen peroxide. The modifications of deoxyribonucleosides by methylglyoxal plus hydrogen peroxide were studied in vitro. When 2'-deoxyguanosine (6.25 mumole) was treated with methylglyoxal (125 mumole) and hydrogen peroxide (125 mumole) in 5 ml of 0.1 M phosphate buffer (pH 7.4) at 37 degrees C for 3 h, N2-acetyl-2'-deoxyguanosine was formed with a yield of 1.1%. Its formation increased time-dependently. By contrast, no appreciable modification of other deoxynucleosides was detected after their incubation with methylglyoxal and hydrogen peroxide under similar conditions. N2-Acetyl-2'-deoxyguanosine was also formed during incubation of 2'-deoxyguanosine with instant coffee.
Leishmania braziliensis promastigotes incubated anaerobically produce D-lactate from glucose, ribose, and methylglyoxal, but not from glycerol, alanine, or pyruvate, suggesting the presence of glyoxalases I and II but the absence of D-lactate dehydrogenase. Further support for this is shown by: (1) conversion of methylglyoxal to D-lactate in sonicates of promastigotes in the presence of reduced glutathione, (2) utilization of phenylglyoxal at rates comparable to methylglyoxal, (3) lack of utilization of exogenously supplied D-lactate by promastigotes under aerobic conditions. Sonicates of promastigotes catalyze the conversion of dihydroxyacetone phosphate to methylglyoxal, suggesting the presence of methylglyoxal synthase. Whereas the rate of production of D-lactate from glucose is much greater under anaerobic conditions, the rate from methylglyoxal is independent of oxygen tension, indicating that control of flux through the methylglyoxal pathway occurs at, or before, methylglyoxal synthase.
Methylglyoxal is a toxic metabolite with growth inhibitory properties against Leishmania donovani promastigotes. We have shown in the present study that both log and stationary phase promastigotes of L. donovani can catabolize methylglyoxal to D-lactate as the major end product. The specific activity of methylglyoxal reductase was found to be the highest of all the catabolic enzymes. In contrast, the anabolic pathway for methylglyoxal could not be detected. Moreover, when control promastigotes or promastigotes in which the glycolytic pathway was inhibited were incubated with glucose, glycerol or dihydroxyacetone phosphate as energy source, neither methylglyoxal nor D-lactate could be detected.
The production of D-lactate that accompanies the metabolism of glucose to L-lactate in Plasmodium falciparum was evaluated with erythrocytes that contained either young or mature parasites. Infected cells with ring-stage parasites release L-lactate and D-lactate at rates 1340 and 81 nmol h-1 (10(8) cells)-1, respectively. These rates increase to 2050 and 136 nmol h-1 (10(8) cells)-1, respectively, in infected cells with trophozoite/schizont-stage parasites. D-Lactate represents 6-7% of the total lactate. The formation of D-lactate is by way of a methylgloxal pathway in which methylglyoxal is formed nonenzymatically from dihydroxyacetone phosphate and is then converted into D-lactate by the sequential action of parasite glycoxalase I and glyoxalase II. The kinetic properties of parasite glyoxalase I and glyoxalase II allow these enzymes to be distinguished from those in the host cell. D-Lactate production by the parasite appears to be a defense mechanism to protect the parasite from the toxic effects of methylglyoxal.
Histidine ammonia-lyase from Streptomyces griseus was inactivated by methylglyoxal and phenylglyoxal, dicarbonyl reagents known to react specifically with arginyl residues in proteins. The inactivation showed pseudo-first-order kinetics and could be prevented by protection with histidinol phosphate, a competitive inhibitor of histidine ammonia-lyase. Analysis of the amino acid composition of histidine ammonia-lyase after treatment with phenylglyoxal, together with the kinetics of inactivation, suggested that inactivation was a consequence of specific reaction with one or more essential arginyl residues at or near the active site of the enzyme.
Methylglyoxal is converted to D-lactic acid through a conjugation with glutathione and S-D-lactoylglutathione is an intermediate of this pathway. In isolated hepatocytes prepared from fed mice incubated without nutrients (glucose, pyruvate and amino acids) the formation and release of S-D-lactoylglutathione and also a continuous lowering of cellular glutathione were demonstrated upon addition of methylglyoxal (20 mM). Under these incubation conditions, the glutathione content of the cells decreased in the controls. On the other hand, in hepatocytes incubated in a medium supplemented with the above-mentioned compounds an accumulation of S-D-lactoylglutathione and a transient decrease of glutathione were shown after addition of methylglyoxal. Under these experimental circumstances the glutathione content of the cells was preserved. Buthionine sulfoximine--an inhibitor of glutathione synthesis--prevented the restoration of glutathione level in hepatocytes observed in the presence of methylglyoxal; emetine--an inhibitor of protein synthesis--was ineffective. It is suggested that increased methylglyoxal formation may have a role in alterations of glutathione metabolism under conditions when serum acetone is increased and methylglyoxal production from acetone is elevated.
About 40 coffee aroma constituents belonging to the classes of dicarbonyls, sulphur-containing compounds, furfuryls, N-heterocyclics and others were systematically evaluated in three Ames tester strains. Only aliphatic dicarbonyl compounds showed notable direct mutagenic activity, which mainly affected 'base-pair substitution' in Ames tester strains TA100 and TA102. Very weak effects were also seen with some N-heterocyclics, mainly affecting frameshift tester strain TA98 upon metabolic activation. However, it was shown that these N-heterocyclics do not contribute substantially to the mutagenicity in coffee. The hydrogen peroxide and methylglyoxal contents of coffee were determined up to 26 hr after preparation. Their concentrations tended to decrease whereas mutagenic activity decreased significantly with time in tester strains TA100 and TA102. It is concluded that several highly labile coffee constituents contribute to the bacterial mutagenicity and also that the synergism between hydrogen peroxide and methylglyoxal is not the main factor. The absence of coffee mutagenicity/carcinogenicity in rodents with these highly reactive coffee aroma compounds can be explained in part by detoxification of microsomal enzyme systems.
The potential carcinogenicity of coffee and related compounds was examined using a medium-term liver bioassay based on the induction of glutathione S-transferase placental form (GST-P)-positive foci in F344 rats. A total of 230 males were initially injected with diethylnitrosamine (200 mg/kg body weight, ip) or saline as controls and 2 wk later were fed on diet or drinking water supplemented as follows for 6 wk: 5% regular instant coffee; 5% decaffeinated instant coffee; freshly brewed coffee, 8 g in 140 ml water; 0.1% caffeine, 0.2% methylglyoxal, 0.2% glyoxal; or 0.3% theophylline in the drinking water (w/v); and 0.4% theobromine in the diet (w/w). All rats were subjected to two-thirds partial hepatectomy at wk 3 and killed at wk 8. The resultant values for GST-P-positive hepatic focus induction were slightly increased with methylglyoxal and decreased with glyoxal and theobromine compared with the corresponding controls. Although the increase in number of foci for methylglyoxal was statistically significant at P < 0.05, the value was within the historical control levels. Regular and decaffeinated instant coffee as well as fresh-brewed coffee, caffeine and theophylline exerted no effects on focus development. Thus, the coffee-related compounds examined demonstrated no obvious enhancing potential, and it is therefore concluded that coffee and its main constituents are not carcinogenic for the rat liver.
For yeast glyoxalase I, a stereospecific proton-transfer mechanism requires the formation of either a cis or a trans-enediol intermediate. Analogs of the two possible isometric enediol intermediates, formed from the hemimercaptal due to phenylglyoxal and glutathione, have been synthesized in which the oxygen atoms of the enediol are replaced by protons. Both isomeric analogs are strong linear competitive inhibitors of the enzyme having nearly equal inhibition constants: Ki(cis) = 0.10 mM; Ki(trans) = 0.16 mM. This suggests that while hydrophobic interactions between substrate, enediol intermediate and enzyme may contribute significantly to binding, this type of interaction is not an independent stereochemical determinant of the reaction.
An enzyme fraction which oxidizes lactaldehyde to lactic acid has been purified from goat liver. This enzyme was found to be identical with the cytosolic aldehyde dehydrogenase. Lactaldehyde was found to be primarily oxidized by this enzyme. Almost 90% of the total lactaldehyde-oxidizing activity is located in the cytosol. Methylglyoxal and glyceraldehyde 3-phosphate were found to be strong competitive inhibitors of this enzyme. Aldehyde dehydrogenase from goat liver mitochondria has also been partially purified and found to be strongly inhibited by these metabolites. The inhibitory effects of these metabolites on both these enzymes are highly pH dependent. The inhibitory effects of both the metabolites have been found to be stronger for the cytosolic enzyme at pH values higher than the physiological pH. For the mitochondrial enzyme, the inhibition with methylglyoxal was more pronounced at higher pH values, whereas stronger inhibition was observed with glyceraldehyde 3-phosphate at physiological pH.
The glyoxalase system of human promyelocytic leukaemia HL60 cells was substantially modified during differentiation to neutrophils. The activity of glyoxalase I was decreased and the activity of glyoxalase II was markedly increased relative to the level in control HL60 promyelocytes. There was a decrease in the apparent maximum velocity, Vmax, of glyoxalase I, and an increase in the Vmax of glyoxalase II. The apparent Michaelis constants for both enzymes remained unchanged. The flux of intermediates metabolised via the glyoxalase system increased during differentiation, as judged by the formation of D-lactic acid, whereas the percentage of glucotriose metabolised via the glyoxalase system remained unchanged. The cellular concentrations of the glyoxalase substrates, methylglyoxal and S-D-lactoylglutathione, were markedly decreased during differentiation. The maturation of HL60 promyelocytes is associated with an increased ability to metabolise S-D-lactoylglutathione by glyoxalase II and a concomitant decrease in the mean intracellular concentrations of S-D-lactoylglutathione and methylglyoxal. The maintenance of a high concentration of S-D-lactoylglutathione in HL60 promyelocytes may be related to the status of the microtubular cytoskeleton, since S-D-lactoylglutathione potentiates the GTP-promoted assembly of microtubules.
Treatment of rabbit muscle phosphoglycerate mutase with diketones (2,3-butanedione and 1,2-cyclohexanedione) and with glyoxal derivatives (methylglyoxal and phenylglyoxal) produces the loss of the three activities of the enzyme: phosphoglycerate mutase, glycerate-2,3-P2 synthase and glycerate-2,3-P2 phosphatase. Hydroxylamine reactivates all the activities of the modified enzyme. Inactivated phosphoglycerate mutase is unable to form the functionally active phosphoenzyme when mixed with glycerate-2,3-P2. Both substrate and cofactor protect against inactivation. These results provide additional evidence of the intrinsic character of the three enzymatic activities of phosphoglycerate mutase and favor their location at the same active site. In addition, they suggest that arginine is involved in the binding of the cofactor to the enzyme.
Phosphoglycerate mutase isozymes (types M, B and MB) from pig tissues are inactivated upon treatment with reagents specific for histidyl, arginyl and lysyl residues. Their mutase, 2,3-bisphosphoglycerate synthase and 2,3-bisphosphoglycerate phosphatase activities are concurrently lost, although some differences exist in the rate of inactivation. No significant differences are observed between the isozymes. The reversion of the modifying reactions reactivates the three enzymatic activities. Substrates and cofactors protect against inactivation, the protective effects varying with the modifying reagent. Titration with pCMB shows the existence of two essential thiol groups per subunit type M. These results provide evidence of the intrinsic character of the three enzymatic activities, favor their location at the same active site and suggest the existence of separate binding sites for monophosphoglycerates and bisphophoglycerates. Both type M and B subunit from pig phosphoglycerate mutase are similar to type M subunit from rabbit and to the enzyme from yeast.
1. Methylglyoxal is a reactive alpha-oxoaldehyde and physiological metabolite formed by the fragmentation of triose-phosphates, and by the metabolism of acetone and aminoacetone. 2. Methylglyoxal modifies guanylate residues to form 6,7-dihydro-6,7-dihydroxy-6-methyl-imidazo[2,3-b]purine-9(8)one and N2-(1-carboxyethyl)guanylate residues and induces apoptosis. 3. Methylglyoxal modifies arginine residues in proteins to form N(delta)-(4,5-dihydroxy-4-methylimidazolidin-2-yl) ornithine, N(delta)-(5-hydro-5-methylimidazol-4-on-2-yl)ornithine and N(delta)-(5)methylimidazol-4-on-2-yl)ornithine residues. 4. Methylglyoxal-modified proteins undergo receptor-mediated endocytosis and lysosomal degradation in monocytes and macrophages, and induce cytokine synthesis and secretion. 5. Methylglyoxal is detoxified by the glyoxalase system. Decreased detoxification of methylglyoxal may be induced pharmacologically by glyoxalase I inhibitors which have anti-tumor and anti-malarial activities. 6. The modification of nucleic acids and protein by methylglyoxal is a signal for their degradation and may have a role in the development of diabetic complications, atherosclerosis, the immune response in starvation, aging and oxidative stress.
Despite increased concentration of plasminogen activators in malignant tumors, fibrinolytic potential in blood of cancer patients and tumor tissue is low. At the same time histochemical and microscopic examination revealed the presence of fibrin-like material coating tumor cells. It is postulated in the present paper that the increased concentrations of -SH groups and arginine-rich peptides, and the activation of tissue transglutaminases in the tumor tissue are responsible for the resistance of the fibrin coat to enzymatic degradation. The fibrin coat, in turn, causes neoplastic cells not to be recognized by the immunological system and thus makes them immune to the attack by the natural killer cells. Administration of naturally occurring, but diminished in cancer, derivatives of alpha ketoaldehydes resistant to glyoxalases, reverses the effect of -SH and arginine-rich peptides, and reactivates fibrinolysis. Consequently, removal of the fibrin coat from tumor cells makes them vulnerable to the attack of killer cells and their subsequent elimination.
The effects of a single intraperitoneal injection of methylglyoxal (50-800 mg/kg body wt.) in mice were investigated in the liver after 24 h. The administration of methylglyoxal (400 mg/kg body wt.) resulted in an increase in aniline hydroxylase activity in liver microsomes. At the same time an accumulation of p-amino-phenol, the hydroxylated product of aniline, was observed in isolated hepatocytes upon addition of aniline similarly to conditions (starvation, diabetes mellitus, pyrazole pretreatment) when aniline hydroxylase was induced. Methylglyoxal also decreased the reduced glutathione content in the liver, while the activity of serum glutamate pyruvate transaminase was increased, suggesting the onset of liver injuries. It is assumed that the increased oxidation of aniline hydroxylase combined with decreased glutathione levels after methylglyoxal treatment favours the formation of potentially hazardous phenol derivatives in the liver.