The relationship of hydrogen peroxide to the inhibition of the glyoxalase activity of intact erythrocytes by x-radiation

The x-irradiation of a dilute suspension of erythrocytes results in a decrease in the glyoxalase activity of the cells as a result of a fall in the reduced glutathione level. The present paper deals with the possible role of H₂O₂ in this reaction. The addition of intact erythrocytes to physiological saline previously irradiated with 150,000 r or 225,000 r results in a fall in the glyoxalase activity of the cells. The inhibition is prevented by the preincubation of the irradiated saline with catalase and is reversed by the addition of plasma, glucose, adenosine, and inosine to the cell suspension. An inhibition of the glyoxalase activity is also produced by the addition of H₂O₂ to the suspension of erythrocytes. The inhibitory effect of H₂O₂ can be prevented and largely reversed by plasma, glucose, adenosine, and inosine. Methylglyoxal is also protective under these conditions. Hydrogen peroxide formed continuously and in low concentrations by enzyme systems appears to be more effective than added H₂O₂ in inhibiting the glyoxalase system. The inhibition by H₂O₂-producing enzyme systems is minimized by the addition of catalase, plasma, glucose, methylglyoxal, and to a lesser extent, by adenosine and inosine, and is accentuated by the addition of sodium azide. The results are discussed in relation to the role of H₂O₂ and catalase in the toxicity of ionizing radiations.     

Production of Inosine from Hydrocarbons by Corynebacterinm petrophilum

Using the adenine auxotroph of hydrocarbonoclastic microorganism, Corynebacterium petrophilum, the effects of glucose on the inosine productivity were investigated. The mutant did not produce inosine from glucose as the sole source of carbon. Production of inosine in n-C₁₆ medium was found to be inhibited by the addition of glucose. To obtain information on such effect of glucose, several characters were compared between the cells grown in glucose medium and those grown in n-C₁₆ medium. Intracellular content of UV-absorbing materials of the glucose-cells was higher than that of hydrocarbon-cells. The glucose-celle could not grow in media containing adenosine or 5′-AMP. On the other hand, hydrocarbon-cells were able to achieve growth, with adenine, adenosine and 5′-AMP contained in the hydrocarbon medium, but, in the case of glucose medium, the cells could grow only in the presence of adenine. Furthermore, the growth of this mutant in n-C₁₆ medium was found to be inhibited by a larger amount of adenine than that required for the maximum growth, and this inhibition was overcome by the addition of guanine. The significance of the effect of guanine was discussed.     

Rat adipocyte lipoprotein lipase activity is inhibited by theophylline and stimulated by inosine through adenosine-independent mechanisms

Incubation of rat adipose tissue or isolated rat adipocytes with high (50 mM) but not with low concentrations (0.5 mM) of theophylline results in a decrease of lipoprotein lipase (LPL) activity. This effect is not altered by the addition of adenosine deaminase, indicating that the decrease of adipose LPL activity by theophylline is not due to the competition of theophylline with adenosine. On the contrary, incubation of isolated fat cells with adenosine (0.1 – 100 μM) results in an increase of the intracellular form of LPL activity. As this effect is also observed in cells incubated with adenosine deaminase (40 mU/ml) or with inosine (0.1 – 100 μM) but not in cells incubated with the adenosine analog N⁶-phenylisopropyladenosine, it is concluded that the increase in the intracellular form of LPL found after incubation with adenosine is not due to adenosine $\text{per se}$ but to inosine generated from the breakdown of endogenous adenosine by adenosine deaminase.     

Adenosine transport by cultured glial cells from chick embryo brain

The transport of adenosine was studied in pure cultures of glial cells from chick embryo brain. In order to avoid complications in uptake measurements due to adenosine metabolism, cultures were depleted of ATP by incubation with cyanide and iodoacetate prior to addition of [³H]adenosine. Under the 5- to 25-s periods used for the transport assay, no adenosine metabolism could be detected. Initial rates of adenosine transport under these conditions obeyed the Michaelis-Menten relationship with K_m = 370 μM and V_max = 10.3 nmol/min/mg cell protein. ATP depletion or elimination of Na⁺ from the assay medium had no significant effect on initial rates of adenosine uptake. However, when assays were carried out under conditions of significant adenosine metabolism (10-min uptake in the absence of metabolic inhibitors), a high-affinity incorporation process could be demonstrated in the glial cells (K_m = 12 μM; V_max = 0.34 nmol/ min/mg protein). The transport activity expressed in ATP-depleted glial cells was most sensitive to inhibition by nitrobenzylthioinosine, dipyridamole, and N⁶-benzyladenosine. In decreasing order of potency, N⁶-methyladenosine, 2-chloroadenosine, inosine, and thymidine also blocked adenosine translocation in glial cultures. Thus, adenosine transport by cultured glial cells occurs by means of a low-affinity, facilitated diffusion system which is similar to the nucleoside transporter in cells of nonneural origin.     

Purine catabolism in plants : purification and some properties of inosine nucleosidase from yellow lupin (lupinus luteus L.) seeds

Inosine nucleosidase (EC 3.2.2.2), the enzyme which hydrolyzes inosine to hypoxanthine and ribose, has been partially purified from Lupinus luteus L. cv. Topaz seeds by extraction of the seed meal with low ionic strength buffer, ammonium sulfate fractionation, and chromatography on aminohexyl-Sepharose, Sephadex G-100, and hydroxyapatite.

Molecular weight of the native enzyme is 62,000 as judged by gel filtration. The inosine nucleosidase exhibits optimum activity around pH 8. Energy of activation for inosine hydrolysis estimated from Arrhenius plot is 14.2 kilocalories per mole. The K_m value computed for inosine is 65 micromolar.

Among the inosine analogs tested, the following nucleosides are substrates for the lupin inosine nucleosidase: xanthosine, purine riboside (nebularine), 6-mercaptopurine riboside, 8-azainosine, adenosine, and guanosine. The ratio of the velocities measured at 500 micromolar concentration of inosine, adenosine, and guanosine was 100:11:1, respectively. Specificity (V_max/K_m) towards adenosine is 48 times lower than that towards inosine.

In contrast to the adenosine nucleosidase activity which is absent from lupin seeds and appears in the cotyledons during germination (Guranowski, Pawełkiewicz 1978 Planta 139: 245-247), the inosine nucleosidase is present in both lupin seeds and seedlings.

     

Inhibition of Adenosine Uptake by Platelets

ADENOSINE inhibits platelet aggregation induced by adenosine diphosphate both in vivo and in vitro^1,2. In platelet-rich plasma, inhibition by adenosine first increases and then decreases^1–6, presumably because adenosine is either incorporated into the platelet and converted to nucleotides, or degraded in the plasma by adenosine deaminase (ADA) to inosine and then hypoxanthine^6,7.     

Erythrocyte acid phosphatase (ACP1) activity

Summary Erythrocyte acid phosphatase (ACP₁) activity was determined in the absence of modulators and in the presence of either adenosine or inosine as modulators in 154 samples of red blood cells collected from adult donors. Adenosine and inosine showed modulating effects (activation), that were genotype dependent in the allele order p^bac; the activation by inosine was much higher than by adenosine. The modulating effect was dependent on adenosine deaminase (ADA) genotype: In carriers of ADA² allele the activation with ACP₁ phenotype A was lower and that with phenotypes CA and CB was higher than in ADA¹/ADA¹ subjects. In addition, the basic ACP₁ activity (i.e., without modulators) also appeared to be dependent on ADA genotype: The lowest ACP₁ activity was observed in A and BA subjects carrying the ADA² allele. Since the deamination of adenosine to inosine associated with ADA2-1 phenotype is slower than that associated with ADA1, the interaction of ADA on ACP₁ activity may in fact be explained by a lower intracellular concentration of inosine in ADA² carriers and, therefore, by a lower modulating effect of this on acid phosphatase activity.     

Enhancement of inosine-mediated A2AR signaling through positive allosteric modulation

Inosine is an endogenous nucleoside that is produced by metabolic deamination of adenosine. Inosine is metabolically more stable (half-life 15 h) than adenosine (half-life < 10 s). Inosine exerts anti-inflammatory and immunomodulatory effects similar to those observed with adenosine. These effects are mediated in part through the adenosine A_2A receptor (A_2AR). Relative to adenosine inosine exhibits a lower affinity towards the A_2AR. Therefore, it is generally believed that inosine is incapable of activating the A_2AR through direct engagement, but indirectly activates the A_2AR upon metabolic conversion to higher affinity adenosine. A handful of studies, however, have provided evidence for direct inosine engagement at the A_2AR leading to activation of downstream signaling events and inhibition of cytokine production. Here, we demonstrate that under conditions devoid of adenosine, inosine as well as an analog of inosine 6-S-[(4-Nitrophenyl)methyl]-6-thioinosine selectively and dose-dependently activated A_2AR-mediated cAMP production and ERK1/2 phosphorylation in CHO cells stably expressing the human A_2AR. Inosine also inhibited LPS-stimulated TNF-α, CCL3 and CCL4 production by splenic monocytes in an A_2AR-dependent manner. In addition, we demonstrate that a positive allosteric modulator (PAM) of the A_2AR enhanced inosine-mediated cAMP production, ERK1/2 phosphorylation and inhibition of pro-inflammatory cytokine and chemokine production. The cumulative effects of allosteric enhancement of adenosine-mediated and inosine-mediated A_2AR activation may be the basis for the sustained anti-inflammatory and immunomodulatory effects observed in vivo and thereby provide insights into potential therapeutic interventions for inflammation- and immune-mediated diseases.     

Counter-regulation of insulin-stimulated glucose transport by catecholamines in the isolated rat adipose cell

The interaction between catecholamines and insulin in regulating glucose transport in isolated rat adipose cells has been evaluated. In the absence of insulin, 1 microM isoproterenol stimulates 3-O-methylglucose transport approximately 2-fold. However, isoproterenol in combination with adenosine deaminase inhibits glucose transport activity approximately 60%. N6-Phenylisopropyladenosine, a nonmetabolizable adenosine analogue, substantially reverses this inhibitory effect and actually stimulates glucose transport activity approximately 2-fold in the absence of isoproterenol. Dibutyryl cAMP inhibits glucose transport activity approximately 75% regardless of adenosine deaminase. While none of these agents significantly influences the basal concentration of plasma membrane glucose transporters, as assessed by specific D-glucose-inhibitable cytochalasin B binding, isoproterenol or dibutyryl cAMP in combination with adenosine deaminase reduces that in the low density microsomes 19 and 58%, respectively. In the presence of insulin, both isoproterenol and adenosine deaminase alone inhibit glucose transport activity approximately 25%. However, only the latter is accompanied by a corresponding decrease in the insulin-stimulated concentration of plasma membrane glucose transporters. Together, isoproterenol and adenosine deaminase inhibit insulin-stimulated glucose transport activity approximately 75%, even in the presence of 5 mM glucose to maintain cellular ATP levels. A similar inhibition is observed with dibutyryl cAMP. However, these agents decrease the insulin-stimulated concentration of plasma membrane glucose transporters only approximately 45%. Nevertheless, all of these inhibitory effects occur through decreases in the transport Vmax. In addition, N6-phenylisopropyladenosine partially reverses the inhibitory effects induced by the presence of adenosine deaminase. These results suggest that catecholamines counter-regulate basal and insulin-stimulated glucose transport in rat adipose cells through a cAMP-mediated mechanism, but only in part by modulating the translocation of glucose transporters.     

Alleviation of osmotic stress in Brassica napus,B. campestris,and B. juncea by ascorbic acid application

The roles of ascorbic acid (AsA, 1 mM) under an osmotic stress [induced by 15 % (m/v) polyethylene glycol, PEG-6000] were investigated by examining morphological and physiological attributes in Brassica species. The osmotic stress reduced the fresh and dry masses, leaf relative water content (RWC), and chlorophyll (Chl) content, whereas increased the proline (Pro), malondialdehyde (MDA), and H₂O₂ content, and lipoxygenase (LOX) activity. The ascorbate content in B. napus, B. campestris, and B. juncea decreased, increased, and remained unaltered, respectively. The dehydroascorbate (DHA) content increased only in B. napus. The AsA/DHA ratio was reduced by the osmotic stress in all the species except B. juncea. The osmotic stress increased the glutathione (GSH) content only in B. juncea, but increased the glutathione disulfide (GSSG) content and decreased the GSH/GSSG ratio in all the species. The osmotic stress increased the activities of ascorbate peroxidase (APX) (except in B. napus), glutathione reductase (GR) (except in B. napus), glutathione S-transferase (GST) (except in B. juncea), and glutathione peroxidase (GPX), and decreased the activities of catalase (CAT) and monodehydroascorbate reductase (MDHAR) (only in B. campestris). The osmotic stress decreased the glyoxalase I (Gly I) and increased glyoxalase II (Gly II) activities. The application of AsA in combination with PEG improved the fresh mass, RWC, and Chl content, whereas decreased the Pro, MDA, and H₂O₂ content in comparison with PEG alone. The AsA addition improved AsA-GSH cycle components and improved the activities of all antioxidant and glyoxalase enzymes in most of the cases. So, exogenous AsA improved physiological adaptation and alleviated oxidative damage under the osmotic stress by improving the antioxidant and glyoxalase systems. According to measured parameters, B. juncea can be recognized as more drought tolerant than B. napus and B. campestris.     

Photoaffinity labelling by S-(p-azidophenacyl)-glutathione of glyoxalase II and glutathione S-transferase

S-(p-azidophenacyl)-glutathione, $\text{l}$, is a linear competitive inhibitor at pH 7.40 of beef liver glyoxalase II with K_i = 7.96 × 10^?4 M. On irradiation at 340 nm it covalently inhibits glyoxalase II to a level of 42 ± 5% inhibition. This photoaffinity labelling is prevented by the presence of a glyoxalase II competitive inhibitor (the hemimercaptal of glutathione and methylglyoxal). A crude preparation of sheep liver glutathione S-transferases is also irreversibly inactivated (86% ± 5% inhibition) by irradiation at 320 nm in the presence of $\text{l}$.     

Metabolism of the 2-oxoaldehyde methylglyoxal by aldose reductase and by glyoxalase-I: roles for glutathione in both enzymes and implications for diabetic complications

Numerous physiological aldehydes besides glucose are substrates of aldose reductase, the first enzyme of the polyol pathway which has been implicated in the etiology of diabetic complications. The 2-oxoaldehyde methylglyoxal is a preferred substrate of aldose reductase but is also the main physiological substrate of the glutathione-dependent glyoxalase system. Aldose reductase catalyzes the reduction of methylglyoxal efficiently (k_cat=142 min⁻¹ and k_cat/K_m=1.8×10⁷ M⁻¹ min⁻¹). In the presence of physiological concentrations of glutathione, methylglyoxal is significantly converted into the hemithioacetal, which is the actual substrate of glyoxalase-I. However, in the presence of glutathione, the efficiency of reduction of methylglyoxal, catalyzed by aldose reductase, also increases. In addition, the site of reduction switches from the aldehyde to the ketone carbonyl. Thus, glutathione converts aldose reductase from an aldehyde reductase to a ketone reductase with methylglyoxal as substrate. The relative importance of aldose reductase and glyoxalase-I in the metabolic disposal of methylglyoxal is highly dependent upon the concentration of glutathione, owing to the non-catalytic pre-enzymatic reaction between methylglyoxal and glutathione.     

Adenosine aminohydrolase inhibition in cosolvent--buffer mixtures

Adenosine aminohydrolase from calf intestinal mucosa is sensitive to changes in the cooperative water structure of its environment as induced by the cosolvent dioxane. When dioxane is added to lower the dielectric constant from that of 78 of neat water to about 74, V is approximately halved, competitive inhibition by N⁶-(Δ²-isopentenyl)adenosine is virtually abolished, and competitive inhibition by the product of the reaction, i.e., inosine, is significantly decreased (K_i changes from 0.2 to 0.5 mm inosine). Yet K_m remains unaltered at 40 μm adenosine even to a dielectric constant of 66.Since both N⁶-(Δ²-isopentenyl)adenosine and inosine are competitive inhibitors, they cannot be bound by the enzyme at the same time as adenosine. The fact that substrate binding remains unaltered at dielectric constants where these inhibitors are impotent indicates that binding of these inhibitors by portions of the enzyme not directly involved in substrate binding is important. The degree of alteration of binding with increasing dioxane concentration is different for these two inhibitors, with appreciable inosine binding at mole fractions dioxane where N⁶-(Δ²-isopentenyl)-adenosine binding cannot be demonstrated. Because of this differential effect of dioxane on inosine and N⁶-(Δ²-isopentenyl)adenosine binding, it is apparent that two substances can be competitive inhibitors kinetically and yet be bound differently by an enzyme. Cosolvents may thus be useful probes for the study of enzyme inhibitor interactions. It is proposed that studies of cosolvent effects on enzyme catalysis and substrate and inhibitor binding are capable of revealing the sensitivities of these various sites to alterations in the dielectric constant of the medium and thus may be considered as models for enzyme behavior near cytoplasmic membranes in vivo.     

Modulation by gibberellic acid and adenosine-3′,5′-cyclic monophosphate of starch hydrolysing activity of cowpea seedlings

In cowpea seedlings starch hydrolysing activity increases 35–50 fold on germination for 4 days. This increase in enzyme activity was inhibited by the in vivo addition of 1% glucose but this inhibition was completely overcome by the addition of gibberellic acid (GA₃) (10^?5 M) and adenosine-3′,5′-cyclic monophosphate (cAMP) (10^?5 M). At 5% glucose, GA₃ and cAMP were only partially effective. Structural analogues of cAMP failed to relieve the inhibitory effect of glucose. The inhibition by glucose is not direct but RNA and protein synthesis may be involved. Glucose appears to reduce the internal pool of cAMP which causes inhibition of RNA synthesis and decrease in starch hydrolysing activity. Exogenous application of cAMP may replenish the endogenous pool of cyclic nucleotide and thus overcome inhibition of RNA synthesis and enzyme activity.     

The influence of adenosine on intermediary metabolism of isolated hepatocytes

The effect of adenosine was tested on the energetic metabolism of fed rat liver cells after isolation. The cells were incubated in a buffered saline medium with glucose (5 mM) and adenosine (1 mM) for 30 minutes at 37°C. This increased the concentration of the adenylic nucleotides ATP (+ 57 per cent), ADP (+ 39 per cent). Cyclic AMP was increased (+ 50 per cent) and the intracellular inorganic phosphate decreased (− 22 per cent). These changes were accompanied by a decrease of glycogenolysis, glucose consumption and lactate production. Measurement of glycolytic intermediates showed decreased concentrations of fructose 1,6-bisphosphate and 3-phosphoglycerate proportional to the increase in ATP concentration. The near-equilibrium of the glyceraldehyde 3-phosphate dehydrogenase-phosphoglycerate kinase system was not modified by adenosine. The decrease of the NAD⁺/NADH ratio along with the increase of the ATP/ADP × PO₄ ratio explains the decrease of 3-phosphoglycerate. The decrease in glucose consumption can be explained by the cross over at the phosphofructokinase stage with the decrease of fructose 1,6-bisphosphate. The major part of adenosine was deaminated as indicated by an increase in the production of ammonia and urea. The effects of inosine, or adenosine along with an inhibitor of adenosine deaminase (pentostatin) suggest that adenosine acts on the glucose consumption through adenylic nucleotides. However the increase of the adenylic nucleotide level cannot totally explain the other metabolic changes: decrease of the NAD⁺/NADH cytoplasmic ratio, constancy of this ratio in mitochondria, decrease of gluconeogenesis from lactate. A direct action of adenosine can therefore be expected.     

Adenosine Inhibition of Human Platelet Aggregation by ADP

WE have shown that in human platelet-rich plasma, inosine is a concentration-dependent inhibitor of adenosine incorporation into platelets^1,2 and at high concentrations inosine inhibits adenosine decomposition³. This prompted us to investigate the effect of inosine and other adenosine decomposition products on aggregation of human platelets in vivo by ADP.     

Substitution of inosine for yeastolate in the culture medium forDrosophila cells

Summary The nutritional requirement ofDrosophila cells (GM₁ and GM₂) was studied. TC Yeastolate contained in the medium forDrosophila cell culture was found to be replaceable with adenosine or inosine without appreciable changes in the generation time of cells. The optimal concentration of either adenosine or inosine was 0.01 mM. Whereas adenosine manifested cell toxicity at concentrations higher than 0.1 mM, in the case of inosine, such an inhibitory effect was not observed up to and at the concentration of 1.0 mM. Further-more, the plating efficiency at cell densities as low as 2×10³ cells per cm² was raised from 0 to 10% by supplementing inosine (0.1 mM) for the TC Yeastolate. Therefore inosine is in practice more useful than adenosine. Experiments using radioactive nucleosides suggested that both adenosine and inosine were exclusively incorporated into RNA as adenosine-monophosphate.     

Inhibition of Platelet Activation and Thrombus Formation by Adenosine and Inosine: Studies on Their Relative Contribution and Molecular Modeling

Background

The inhibitory effect of adenosine on platelet aggregation is abrogated after the addition of adenosine-deaminase. Inosine is a naturally occurring nucleoside degraded from adenosine.

Objectives

The mechanisms of antiplatelet action of adenosine and inosine in vitro and in vivo, and their differential biological effects by molecular modeling were investigated.

Results

Adenosine (0.5, 1 and 2 mmol/L) inhibited phosphatidylserine exposure from 52±4% in the control group to 44±4 (p<0.05), 29±2 (p<0.01) and 20±3% (p<0.001). P-selectin expression in the presence of adenosine 0.5, 1 and 2 mmol/L was inhibited from 32±4 to 27±2 (p<0.05), 14±3 (p<0.01) and 9±3% (p<0.001), respectively. At the concentrations tested, only inosine to 4 mmol/L had effect on platelet P-selectin expression (p<0.05). Adenosine and inosine inhibited platelet aggregation and ATP release stimulated by ADP and collagen. Adenosine and inosine reduced collagen-induced platelet adhesion and aggregate formation under flow. At the same concentrations adenosine inhibited platelet aggregation, decreased the levels of sCD40L and increased intraplatelet cAMP. In addition, SQ22536 (an adenylate cyclase inhibitor) and ZM241385 (a potent adenosine receptor A_2A antagonist) attenuated the effect of adenosine on platelet aggregation induced by ADP and intraplatelet level of cAMP. Adenosine and inosine significantly inhibited thrombosis formation in vivo (62±2% occlusion at 60 min [n = 6, p<0.01] and 72±1.9% occlusion at 60 min, [n = 6, p<0.05], respectively) compared with the control (98±2% occlusion at 60 min, n = 6). A_2A is the adenosine receptor present in platelets; it is known that inosine is not an A_2A ligand. Docking of adenosine and inosine inside A_2A showed that the main difference is the formation by adenosine of an additional hydrogen bond between the NH₂ of the adenine group and the residues Asn253 in H6 and Glu169 in EL2 of the A_2A receptor.

Conclusion

Therefore, adenosine and inosine may represent novel agents lowering the risk of arterial thrombosis.     

Analysis of the energy metabolism after incubation of Saccharomyces cerevisiae with sulfite or nitrite

After addition of 5 mM sulfite or nitrite to glucose-metabolizing cells of Saccharomyces cerevisiae a rapid decrease of the ATP content and an inversely proportional increase in the level of inorganic phosphate was observed. The concentration of ADP shows only small and transient changes. Cells of the yeast mutant pet 936, lacking mitochondrial F₁ATPase, after addition of 5 mM sulfite or nitrite exhibit changes in ATP, ADP and inorganic phosphate very similar to those observed in wild type cells. They key enzyme of glucose degradation, glyceraldehyde-3-phosphate dehydrogenase was previously shown to be the most sulfiteor nitrite-sensitive enzyme of the glycolytic pathway. This enzyme shows the same sensitivity to sulfite or nitrite in cells of the mutant pet 936 as in wild type cells. It is concluded that the effects of sulfite or nitrite on ATP, ADP and inorganic phosphate are the result of inhibition of glyceraldehyde-3-phosphate dehydrogenase and not of inhibition of phosphorylation processes in the mitochondria. Levels of GTP, UTP and CTP show parallel changes to ATP. This is explained by the presence of very active nucleoside monophosphate kinases which cause a rapid exchange between the nucleoside phosphates. The effects of the sudden inhibition of glucose degradation by sulfite or nitrite on levels of ATP, ADP and inorganic phosphate are discussed in terms of the theory of Lynen (1942) on compensating phosphorylation and dephosphorylation in steady state glucose metabolizing yeast.Abbreviations ATP adenosine triphosphate - ADP adenosine diphosphate - AMP adenosine monophosphate - P_i inorganic orthophosphate Dedicated to Prof. Dr. Hans Grisebach on the occasion of his sixtieth birthday     

Effect of endogenous methylglyoxal on Chinese hamster ovary cells grown in culture

Methylglyoxal is a ketoaldehyde that reacts readily under physiological conditions with biologically relevant ligands, such as amine and sulfhydryl groups. It is produced in mammalian cells primarily as a by-product of glycolysis. The level of glucose, L-glutamine and fetal bovine serum in culture media was found to significantly affect levels of intracellular methylglyoxal in Chinese hamster ovary cells. Medium with 25 mM glucose and 5 mM L-glutamine caused an increase in free methylglyoxal levels of 90 to 100% relative to medium containing 5 mM glucose and 2 mM L-glutamine. Both of these media compositions are representative of those found in commercially available media. Pseudomonas putida glyoxalase I was expressed in Chinese hamster ovary cells to enhance methylglyoxal detoxification. The Chinese hamster ovary cell clones showed an 80 to 90% decrease in free methylglyoxal levels. The colony-forming ability of these cells was compared to wild-type Chinese hamster ovary cells under conditions found to cause elevated methylglyoxal levels. The wild-type cells showed a 10% decrease in colony-forming ability relative to the clones. This decrease was found to be statistically significant (P>0.99) by analysis of variance. The variation in colony-forming ability amongst the clones was statistically insignificant. More importantly, the clones shoed increased colony-forming ability relative to the wild-type cells under conditions of higher methylglyoxal production with fair to good statistical significance (P>0.75 to P>0.95). This result is the first quantifiable evidence that endogenously produced methylglyoxal can negatively affect cell function under conditions found in animal cell culture.Abbreviations ANOVA analysis of variance - CHO Chinese hamster ovary cells - CFA colony-forming ability - dhfr gene for dihydrofolate reductase - DHAP dihydroxyacetone phosphate - FBS fetal bovine serum - G-3-P glyceraldehyde-3-phosphate - GloI glyoxalase I - GloII glyoxalase II - GSH reduced glutathione - HPLC high-performance liquid chromatography - IMDM Iscove's modified Dulbecco's medium - MTX methotrexate - 2-MQ 2-methylquinoxaline - 5-MQ 5-methylquinoxaline - MEM minimal essential medium - P_i inorganic phosphate - PCA perchloric acid - o-PD o-phenylenediamine