Effect of methylglyoxal on glucose formation, drug oxidation and glutathione content in isolated murine hepatocytes

The first stage in the formation of glucose from acetone involves two oxidation steps catalyzed by isozymes of the cytochrome P-450 II E1 gene subfamily; methylglyoxal formed this way is further converted to pyruvate by a reversible conjugation with reduced glutathione. The effect of methylglyoxal on glucose formation, oxidation of aminopyrine, aniline and on reduced glutathione content was investigated in isolated hepatocytes prepared from (i) fasted or (ii) fasted and acetone (known to induce isozymes of P-450 II E1 gene subfamily) pretreated mice. Glucose formation and drug oxidation were increased by methylglyoxal at concentrations below 1 mM, but were severely decreased above 1 mM. Methylglyoxal also decreased protein synthesis at concentrations above 1 mM. If the addition of methylglyoxal was combined with that of other gluconeogenic precursors and glucose the initial increasing effect on drug oxidation was moderated or diminished and the decreasing effect (at high concentrations) was enhanced. The glutathione content of the cells was decreased by methylglyoxal in a concentration dependent manner. Acetone pretreatment of mice also resulted in a decreased glutathione content of the liver. Based on these observations it is assumed that methylglyoxal has contrasting effects in hepatocytes, and can contribute to the disturbed metabolism under circumstances when the acetone production is elevated.     

The tandem of free radicals and methylglyoxal

Methylglyoxal is an alpha-oxoaldehyde inevitably produced from triose-phosphate intermediates of phosphorylating glycolysis, and also from amino acids and acetone. Recently, the attention has been focused on the involvement of free radicals in methylglyoxal toxicity. In this review, a summary of the relationship between methylglyoxal metabolism and free radical production is presented, extending discussion from the possible metabolic routes to the toxicological events by reviewing the role of free radicals in both generation and degradation of this 1,2-dicarbonyl as well as in the modification of biological macromolecules, and focusing on the action of methylglyoxal upon cellular glutathione content. Methylglyoxal-provoked free radical generation involving reactive oxygen species (ROS), reactive nitrogen species (RNS) as well as organic radicals like methylglyoxal radial or crosslinked protein radical as potential risk factors to tissue damage propagation, is thoroughly discussed. Special attention is paid to the potential therapeutic interventions. The paper arrives at the conclusion that a tight junction exists between methylglyoxal toxicity and free radical (particularly ROS) generation, though the toxicity of 1,2-dicarbonyl evolves even under anaerobic conditions, too. The events follow a sequence beginning with carbonyl stress essential for the toxicity, leading to free radical formation and finally ending in either apoptosis or necrosis. Both oxidative and nitrosative stress play important but not indispensable role in the development of methylglyoxal toxicity.     

Reduction of trioses by NADPH-dependent aldo-keto reductases. Aldose reductase, methylglyoxal, and diabetic complications.

The substrate specificities of human aldose reductase and aldehyde reductase toward trioses, triose phosphates, and related three-carbon aldehydes and ketones were evaluated. Both enzymes are able to catalyze the NADPH-dependent reduction of all of the substrates used. Aldose reductase shows more discrimination among substrates than does aldehyde reductase and is generally the more efficient catalyst. The best substrate for aldose reductase is methylglyoxal (kcat = 142 min-1, kcat/Km = 1.8 x 10(7) M-1 min-1), a toxic 2-oxo-aldehyde that is produced nonenzymatically from triose phosphates and enzymatically from acetone/acetol metabolism. D- and L-glyceraldehyde and D- and L-lactaldehyde are also good substrates for aldose reductase. The aldose reductase-catalyzed reduction of methylglyoxal produces 95% acetol, 5% D-lactaldehyde. Further reduction of acetol produces only L-1,2-propanediol. Acetol and propanediol are two products that accumulate in uncontrolled diabetes. Both acetol and methylglyoxal were compared with glucose for their abilities to produce covalent modification of albumin. All three of these carbonyl compounds reacted with albumin to produce modified proteins with new absorption and emission bands that are spectrally similar. Both methylglyoxal and acetol are much more reactive than glucose. A new integrative model of diabetic complications is proposed that combines the aldose reductase/polyol pathway theory and the nonenzymatic glycation theory except that emphasis is placed both on methylglyoxal/acetol metabolism and on glucose metabolism.     

Organocatalysis with endogenous compounds: Towards novel non-enzymatic reactions

The aldol reaction of the endogeneous compounds acetone and methylglyoxal has been studied using organocatalysis in relation to biologically relevant non-enzymatic reactions. Under preparative conditions, 3-hydroxy-2,5-hexadione, known as Henze’s ketol, is formed in high yield and with enantioselectivities up to 88% ee. Furthermore, Henze’s ketol is also formed under simulated physiological conditions at micromolar scale, indicating that this reaction might take place in living organisms.     

Kinetics of alpha-dicarbonyls reduction by L-glycol dehydrogenase from hen muscle

Michaelis constants of L-glycol dehydrogenase from hen muscle (isozyme of pI 7.2) for the alpha-dicarbonyls tested (glyoxal, 2,3-pentanedione, methylglyoxal, and diacetyl) range from 35 microM for pentanedione to 0.41 mM for glyoxal. The enzyme shows a high affinity for NADPH, Km (2.2-3.1 microM), and Ks (1.2-1.9 microM) being so much lower than its tissue concentration that L-glycol dehydrogenase has to operate in vivo saturated with the coenzyme; this condition is very unfavorable to play a role in regulating the equilibrium oxidized/reduced forms of the pyridine nucleotides, as it has been proposed for some similar enzymes. Convergence of the double reciprocal plots and the pattern of inhibition by products and by acetone, a substrate analog, demonstrate that glyoxal reduction--and most likely that of diacetyl--proceeds via an ordered Bi-Bi mechanism in which NADPH is fixed before the addition of the carbonyl; the reduction of methylglyoxal and 2,3-pentanedione could follow the same model, but our experimental results are also consistent with that of Theorell-Chance.     

Acidic acetone extract from pregnant sow ovaries is a rich source of substances affecting uterine contractility in vitro

Acidic acetone extract of pregnant sow ovaries was subjected to Sephadex G-25 chromatography. The solution coming from column was analysed for UV absorption, molecular weight, and also for its biological effect on a myometrium strip in vitro. This biodetection system has made it possible continuously to determine the biologically active fractions eluted from the Sephadex G-25 column. The reference materials to calibrate the Sephadex G-25 column were Blue dextran and acetone, while for calibration of the biodetection system, synthetic oxytocin was used. The extract of ovaries of pregnant sows was separated chromatographically into 8 different, biologically active fractions with distinct UV absorption and molecular weight. One of these fractions showed elution characteristics and biological effect similar to those of synthetic oxytocin in the same biodetection system. The results indicated that acidic acetone extract originating from ovaries of pregnant sows is a rich source of biologically active substances with effects on the myometrium strips in pregnancy. Partial identification of oxytocin-like substances in the ovarian extract verified the effectiveness of the biodetection system in the first steps of research to obtain new, biologically active substances from different unpurified extracts.     

Scavenging system efficiency is crucial for cell resistance to ROS-mediated methylglyoxal injury

Methylglyoxal is a reactive dicarbonyl compound endogenously produced mainly from glycolytic intermediates. Recent research indicates that methylglyoxal is a potent growth inhibitor and genotoxic agent. The antiproliferative activity of methylglyoxal has been investigated for pharmacological application in cancer chemotherapy. However, various cells are not equally sensitive to methylglyoxal toxicity. Therefore, it would be important to establish the cellular factors responsible for the different cell-type specific response to methylglyoxal injury, in order to avoid the risk of failure of a therapy based on increasing the intracellular level of methylglyoxal. To this purpose, we comparatively evaluated the signaling transduction pathway elicited by methylglyoxal in human glioblastoma (ADF) and neuroblastoma (SH-SY 5Y) cells. Results show that methylglyoxal causes early and extensive reactive oxygen species generation in both cell lines. However, SH-SY 5Y cells show higher sensitivity to methylglyoxal challenge due to a defective antioxidant and detoxifying ability that, preventing these cells from an efficient scavenging action, elicits extensive caspase-9 dependent apoptosis. These data emphasize the pivotal role of antioxidant and detoxifying systems in determining the grade of sensitivity of cells to methylglyoxal.     

Methylglyoxal production in bacteria: suicide or survival?

Methylglyoxal is a toxic electrophile. In Escherichia coli cells, the principal route of methylglyoxal production is from dihydroxyacetone phosphate by the action of methylglyoxal synthase. The toxicity of methylglyoxal is believed to be due to its ability to interact with the nucleophilic centres of macromolecules such as DNA. Bacteria possess an array of detoxification pathways for methylglyoxal. In E. coli, glutathione-based detoxification is central to survival of exposure to methylglyoxal. The glutathione-dependent glyoxalase I-II pathway is the primary route of methylglyoxal detoxification, and the glutathione conjugates formed can activate the KefB and KefC potassium channels. The activation of these channels leads to a lowering of the intracellular pH of the bacterial cell, which protects against the toxic effects of electrophiles. In addition to the KefB and KefC systems, E. coli cells are equipped with a number of independent protective mechanisms whose purpose appears to be directed at ensuring the integrity of the DNA. A model of how these protective mechanisms function will be presented. The production of methylglyoxal by cells is a paradox that can be resolved by assigning an important role in adaptation to conditions of nutrient imbalance. Analysis of a methylglyoxal synthase-deficient mutant provides evidence that methylglyoxal production is required to allow growth under certain environmental conditions. The production of methylglyoxal may represent a high-risk strategy that facilitates adaptation, but which on failure leads to cell death. New strategies for antibacterial therapy may be based on undermining the detoxification and defence mechanisms coupled with deregulation of methylglyoxal synthesis. Received: 30 March 1998 / Accepted: 22 June 1998     

Extracellular methylglyoxal toxicity in Saccharomyces cerevisiae: role of glucose and phosphate ions

Aim: The purpose of this study was to investigate the behaviour of Saccharomyces cerevisiae in response to extracellular methylglyoxal. Methods and Results: Cell survival to methylglyoxal and the importance of phosphates was investigated. The role of methylglyoxal detoxification systems and methylglyoxal‐derived protein glycation were studied and the relation to cell survival or death was evaluated. Extracellular methylglyoxal decreased cell viability, and the presence of phosphate enhanced this effect. d ‐glucose seems to exert a protective effect towards this toxicity. Methylglyoxal‐induced cell death was not apoptotic and was not related to intracellular glycation processes. The glyoxalases and aldose reductase were important in methylglyoxal detoxification. Mutants lacking glyoxalase I and II showed increased sensitivity to methylglyoxal, while strains overexpressing these genes had increased resistance. Conclusions: Extracellular methylglyoxal induced non‐apoptotic cell death, being unrelated to glycation. Inactivation of methylglyoxal‐detoxifying enzymes by phosphate is one probable cause. Phosphate and d ‐glucose may also act through their complex involvement in stress response mechanisms. Significance and Impact of the Study: These findings contribute to elucidate the mechanisms of cell toxicity by methylglyoxal. This information could be useful to on‐going studies using yeast as a eukaryotic cell model to investigate methylglyoxal‐derived glycation and its role in neurodegenerative diseases.     

Purification and partial characterization of a methylglyoxal reductase from goat liver

An enzyme which catalyzes the reduction of methylglyoxal to lactaldehyde has been isolated and purified from goat liver to apparent homogeneity. NADH was found to be a better substrate than NADPH for methylglyoxal reduction. Stoichiometrically equivalent amounts of lactaldehyde and NAD are formed from methylglyoxal and NADH. Enzyme activity was located only in the soluble supernatant fractions of liver cells. Of the various carbonyl compounds tested, methylglyoxal was found to be the best substrate. The pH optimum of the enzyme was found to be 6.5, and Km for methylglyoxal was 0.4 mM. The molecular weight of the enzyme was found to be 89000 by gel filtration on a Sephadex G-200 column. Electrophoresis on sodium dodecyl sulfate-polyacrylamide gel revealed that the enzyme is composed of two subunits. The enzyme is highly sensitive to sulfhydryl group reagents. The inactivation by p-chloromercuribenzoate could be substantially protected by methylglyoxal in combination with NADH, indicating a possible involvement of one or more sulfhydryl group(s) at the active site of the enzyme.     

Methylglyoxal can completely replace the requirement of kinetin to induce differentiation of plantlets from some plant calluses

It is well known that cytokinins, a group of plant hormones are absolutely required for the differentiation of calluses for the regeneration of plantlets through organogenesis. In the present work, it had been observed that methylglyoxal could completely replace kinetin to initiate differentiation of plantlets from calluses of Solanum nigrum and Daucus carota. Moreover, the effect of methylglyoxal was more pronounced compared to that of kinetin and the optimum concentration for methylglyoxal had been determined to be 0.5Â mM. Parallel with the differentiation of calluses to plantlets, the chlorophyll contents increased whereas the endogenous level of methylglyoxal remained unchanged. This remarkable effect of methylglyoxal in plant differentiation had been found out to be specific because some related compounds such as pyruvate and -lactate could not replace the requirement for methylglyoxal in the differentiation process. The activities of several enzymes were monitored during both methylglyoxal and kinetin-induced differentiation. The activity of the enzyme glyceraldehyde-3-phosphate dehydrogenase (NADP-dependent) involved in energy generation process in photosynthesis, increased as the differentiation proceeded. Whereas, the activities of both glucose-6-phosphate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase (NAD-dependent) decreased with differentiation. The activity of glyoxalase I, which catalyzes the conversion of methylglyoxal and glutathione to S--lactoylglutathione, decreased with differentiation. The endogenous level of glutathione showed an initial decrease followed by an increase. It appears from the results presented above, that the effect of kinetin and methylglyoxal are similar in nature, the significance of which has been discussed.     

Similar nature of inhibition of mitochondrial respiration of heart tissue and malignant cells by methylglyoxal. A vital clue to understand the biochemical basis of malignancy

The effect of methylglyoxal on the oxygen consumption of mitochondria of heart and of several other organs of normal animals of different species has been tested. The results indicate that methylglyoxal (3.5 mM) strongly inhibits ADP-stimulated -oxoglutarate and malate plus pyruvate-dependent respiration of exclusively heart mitochondria of normal animals of different species. Whereas, with the same substrates, but at a higher concentration of methylglyoxal (7.5 mM), the respiration of mitochondria of other organs of normal animals is not inhibited. Methylglyoxal also inhibits the respiration of slices of rat and toad hearts. But this inhibition is less pronounced. However, methylglyoxal (15 mM) fails to have any effect on perfused toad heart. Using rat heart mitochondria as a model, the effect of methylglyoxal on the oxygen consumption was also tested with different respiratory substrates, electron donors at different segments of the mitochondrial respiratory chain and site-spe inhibitors to identify the specific respiratory complex which might be involved in the inhibitory effect of methylglyoxal. The results strongly suggest that methylglyoxal inhibits the electron flow through complex I of rat heart mitochondrial respiratory chain. Moreover, lactaldehyde (0.6 mM), a catabolite of methylglyoxal, can exert a protective effect on the inhibition of rat heart mitochondrial respiration by methylglyoxal (2.5 mM). The effect of methylglyoxal on heart mitochondria as described in the present paper is strikingly similar to the results of our previous work with mitochondria of Ehrlich ascites carcinoma cells and leukemic leukocytes. We have recently proposed a new hypothesis on cancer which suggests that excessive ATP formation in cells may lead to malignancy. The above mentioned similarity apparently provides a solid experimental foundation for the proposed hypothesis which has been discussed.     

High-performance liquid chromatographic determination of glyoxal and methylglyoxal in urine by prederivatization to lumazinic rings using in serial fast scan fluorimetric and diode array detectors

Glyoxal and methylglyoxal are two important markers of oxidative stress and both are involved in the evaluation of several diseases. A new HPLC method for determining glyoxal and methylglyoxal in urine was developed. The method is based on the reaction of alpha-dialdehydes, glyoxal and methylglyoxal, with 5,6-diamino-2,4-hydroxypyrimidine sulfate in basic medium to form highly fluorescent lumazine derivatives. Creatinine was also included in the method even though it does not react with the reagent. The derivatives and creatinine are separated on a C(18) reversed-phase column with a mobile phase consisting of acetonitrile:citrate buffer, pH 6.0 (3:97 v/v). The flow rate was 1.0mLmin(-1) and the effluent was monitored photometrically at 250 nm for determination of creatinine and fluorimetrically at 500 nm (exciting at 330 nm) for determination of glyoxal and methylglyoxal derivatives. Recording time of the separation is less than 10 min. Determination of the analytes is performed in urine after incubation of the sample, with the reagent in alkaline medium, for 30 min at 60 degrees C. Urinary levels of glyoxal and methylglyoxal, expressed as glyoxal/creatinine and methylglyoxal/creatinine ratios, in healthy young women and men were determined. For women, values of 0.80+/-0.37 and 0.60+/-0.22 microg/mg of creatinine were found for glyoxal and methylglyoxal, respectively. For men, values of 0.63+/-0.15 and 0.49+/-0.05 microg/mg of creatinine were found for glyoxal and methylglyoxal, respectively. These results were also related to the body mass index of each individual.     

Rat acetoacetate decarboxylase: its role in the disposal of 4C-ketone bodies by the fetus

21-day pregnant rats show high tissular and plasmatic acetone concentrations when submitted to a 48-hr fast. This rise is, in fact, associated with an enhanced placental and fetal acetoacetate decarboxylase activity. We propose that acetone formation by the fetus could be a mechanism for pH maintenance and that acetoacetate decarboxylase can play a significant role in the handling of 4C-ketone bodies under conditions in which the substrate concentration cannot easily be controlled by other physiological mechanisms.     

Protection of the DNA during the exposure of Escherichia coli cells to a toxic metabolite: the role of the KefB and KefC potassium channels

The effect of the toxic metabolite methylglyoxal on the DNA of Escherichia coli cells has been investigated. Exposure of E. coli cells to methylglyoxal reduces the transformability of plasmid DNA and results in the degradation of genomic DNA. The activity of the KefB and KefC potassium channels protects E. coli cells against methylglyoxal and limits the amount of DNA damage. In mutants lacking KefB and KefC, methylglyoxal-induced DNA damage was reduced by incubation with a weak acid that lowers the pHi to the same extent as through KefB and KefC activation. This provides evidence that acidification of the cytoplasm protects E. coli DNA against methylglyoxal. By the analysis of cells lacking UvrA, we demonstrate that this repair protein is required for the degradation of the DNA upon methylglyoxal exposure. However, protection by KefB and KefC occurred independently of UvrA. Although we present evidence that exposure of E. coli cells to methylglyoxal results in DNA degradation, our results suggest this event is not essential for methylglyoxal-induced death. The implications of these findings will be discussed.     

Substrate specificity of bovine liver formaldehyde dehydrogenase

Formaldehyde dehydrogenases isolated from several different biological sources have been reported to catalyze the NAD+-dependent oxidative acylation of glutathione by methylglyoxal to form S-pyruvylglutathione, suggesting the involvement of this enzyme in the metabolism of methylglyoxal. However, formaldehyde dehydrogenase from bovine liver is found not to use methylglyoxal or related alpha-ketoaldehydes as substrates. Using methylglyoxal with the enzyme under conditions favoring the forward reaction did not result in the formation of S-pyruvylglutathione. Using independently synthesized S-pyruvylglutathione with the enzyme under conditions favoring the reverse reaction did not result in the production of methylglyoxal. In addition, methylglyoxal and several related alpha-ketoaldehydes did not exhibit detectable activity with formaldehyde dehydrogenase partially purified from human liver, contrary to a previous report. Some, if not all, past reports that methylglyoxal serves as a substrate for the dehydrogenase may be due to the demonstrated presence of contaminating formaldehyde in some commercially available preparations of methylglyoxal. In a related study, S-hydroxymethylglutathione, formed by pre-equilibrium addition of formaldehyde to glutathione, is concluded to be direct substrate for the dehydrogenase. This follows from the observation that the catalytic turnover number of the enzyme in the forward direction exceeds by a factor of approximately 20 the first order rate constant for decomposition of S-hydroxymethylglutathione to glutathione and formaldehyde (k = 5.03 +/- 0.30 min-1, pH 8, 25 degrees C).     

Potassium channel activation by glutathione-S-conjugates in Escherichia coli: protection against methylglyoxal is mediated by cytoplasmic acidification

Escherichia coli possesses two glutathione-gated potassium channels, KefB and KefC, that are activated by glutathione-S-conjugates formed with methylglyoxal. We demonstrate that activation of the channels leads to cytoplasmic acidification and that this protects cells during electrophilic attack. Further, we demonstrate that mutants lacking the channels can be protected against the lethal effects of methylglyoxal by acidification of the cytoplasm with a weak acid. The degree of protection is determined by the absolute value of the pH_i and the time at which acidification takes place. Alterations in the pH_i do not accelerate the rate of detoxification of methylglyoxal. The mechanism by which methylglyoxal causes cell death and the implications for pH_i-mediated resistance to methylglyoxal are discussed.     

2-Oxoaldehyde metabolism in microorganisms

The properties of methylglyoxal-metabolizing enzymes in prokaryotic and eukaryotic microorganisms were studied systematically and compared with those of mammalian enzymes. The enzymes constitute a glycolytic bypass and convert methylglyoxal into pyruvate via lactate. The first step in this conversion is catalyzed by glyoxalase I, methylglyoxal reductase, or methylglyoxal dehydrogenase. The regulation of the yeast glyoxalase system was analyzed. The system was closely related to the proliferative states of yeast cells, the activity of the system being high in dividing cells and low in nondividing ones. The gene for the glyoxalase I of Pseudomonas putida and the genes responsible for the activity of glyoxalase I and methylglyoxal reductase in Saccharomyces cerevisiae were cloned and their structural and phenotypic characters studied.