Detoxification of Methylglyoxal by the Nucleophilic Bidentate

350 Community Drive, Manhasset, New York 11030. Received January 15, 1999. Dicarbonyl-containing compounds such as methylglyoxal (MG) are toxic to ...
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Chem. Res. Toxicol. 1999, 12, 617-622

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Detoxification of Methylglyoxal by the Nucleophilic Bidentate, Phenylacylthiazolium Bromide Gail P. Ferguson,† Sonya VanPatten,‡ Richard Bucala,‡ and Yousef Al-Abed*,‡ Department of Molecular & Cell Biology, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, U.K., and The Picower Institute for Medical Research, 350 Community Drive, Manhasset, New York 11030 Received January 15, 1999

Dicarbonyl-containing compounds such as methylglyoxal (MG) are toxic to cells since they can interact with the nucleophilic centers of macromolecules. MG has been found to accumulate during hyperglycemia, and it has been suggested that this reactive dicarbonyl may contribute to the tissue damage and long-term complications of diabetes. A sensitive bacterial assay for investigating the ability of nucleophilic agents to interact with and detoxify MG has been developed. This assay utilizes the sensitivity of exponential phase cells of an Escherichia coli double mutant lacking the KefB and KefC potassium channels toward MG. The bidentate nucleophile, phenylacylthiazolium bromide (PTB), was found to protect and allow the growth of E. coli cells in the presence of either externally added or endogenously produced MG. In the absence of PTB, growth was completely inhibited and rapid cell death occurred under these conditions. PTB protected E. coli against MG almost as well as aminoguanidine, a compound shown previously to be involved in detoxification. The level of protection by PTB against MG was much greater than for the endogenous nucleophile, glutathione. These data suggested that PTB could interact with and detoxify MG. The mechanism of this interaction was characterized by NMR and mass spectroscopy.

Introduction Toxic dicarbonyl-containing compounds such as MG1 (R-oxopropanal or pyruvaldehyde) have been found to accumulate during hyperglycemia, and it has been suggested that these may contribute to the tissue damage and long-term complications of diabetes (1). MG is produced either by the elimination of phosphate from dihydroxyacetone phosphate and glyceraldehyde 3-phosphate or as one of the reactive intermediates produced in the Maillard reaction [also known in vivo as advanced glycoslyation end products (AGEs)] (2, 3). In the Maillard reaction, the formation of MG occurs via degradation of the Amadori product, the reaction product of the amino groups of proteins with glucose (4). The production of MG is thought to be toxic to cells since it can interact with the nucleophilic centers of macromolecules. We have shown previously that the nucleotide base guanine is modified when incubated in the presence of either MG or glucose (4). MG also cross-links proteins and reacts with the side chains of arginine, lysine, and cysteine (57). The reaction of MG with the guanidino moiety of arginine leads to the formation of imidazol-4-one and pyrimidinium adducts (7, 8), whereas reaction with lysine produces an imidazolium cross-link across two amino groups (8, 9). In addition to MG, the Maillard reaction * To whom correspondence should be addressed. Telephone: (516) 562-9461. Fax: (516) 365-5090. E-mail: [email protected]. † University of Aberdeen. ‡ The Picower Institute for Medical Research. 1 Abbreviations: AGE, advanced glycosylation end product; APdione, alkylamino-2,3-hexodiulose; AG, aminoguanidine; 3-DG, 3-deoxyglucosone; DNPH, (2,4-dinitrophenyl)hydrazine; ESI-MS, electrospray ionization mass spectrometry; GSH, glutathione; HTA, hemithiolacetal; MG, methylglyoxal; PTB, phenylacylthiazolium bromide.

can also generate other reactive products such as alkylamino-2,3-hexodiulose (AP-dione). The AP-dione also has the potential to cross-link proteins via the dione functional group. Recently, we have demonstrated that APdione was the precursor of a novel AGE cross-link that exhibits immunological cross-reactivity with an in vivoformed AGE (10). Thus, from these studies it is clear how elevated levels of reactive dicarbonyls in human cells could cause major damage. Nucleophiles such as glutathione (GSH) can interact with toxic dicarbonyl compounds such as MG, and it is thought that this results in their detoxification (11-13). GSH is a tripeptide that is synthesized by all cell types, with the exception of Gram-positive bacteria (14). Conjugation of MG to GSH occurs spontaneously and results in the production of hemithiolacetal (HTA) (11). HTA is then further detoxified to D-lactate, via the formation of S-lactoylglutathione, by the actions of the glyoxalase I and II enzymes (15, 16). In addition to the role of GSH in detoxification, the formation of S-lactoylglutathione in E. coli cells activates two potassium transport systems, KefB and KefC (16-18). The activation of the KefB and KefC systems by S-lactosylglutathione results in the rapid loss of potassium by the cell. It has been demonstrated previously that the activity of KefB and KefC protects cells against the toxic effects of MG and other electrophiles (18, 19). Mutant cells lacking both these systems were highly sensitive toward MG and rapidly lost viability upon exposure (18, 20). Growth of Escherichia coli cells on a “poor” carbon source such as D-xylose in the presence of cAMP results in high levels of endogenously produced MG such that millimolar quantities of this toxic metabolite are produced (18, 21, 22). Under

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these conditions, cells of the mutant lacking KefB and KefC died faster than cells of the parent strain (18). In addition to KefB and KefC, bacteria also employ other strategies for protection against electrophiles. Stationary phase bacteria are more resistant to electrophiles than their exponential phase counterparts, and this is due, at least in part, to the accumulation of an alternative sigma factor, RpoS (23). To design more effective drugs to protect human cells against the toxic effects of the accumulation of dicarbonyl compounds, it is essential to understand the mechanisms by which nucleophiles interact with and detoxify them. It has been shown previously that aminoguanidine (AG), an inhibitor of AGE formation, reacts with dicarbonyl compounds to form stable adducts such as a triazine (24). Thiazolium compounds such as phenylacylthiazolium bromide (PTB), which act as bidentate nucleophiles, also attack dicarbonyl systems to effect a carbon-carbon breaking reaction (25). In this paper, we have utilized the sensitivity of exponential phase cells of an E. coli double mutant, lacking KefB and KefC, toward MG to develop a model system for studying the protection by AG, PTB, and GSH. These data presented show that PTB is almost as effective as AG in protecting E. coli cells against externally added and endogenously produced MG. Both PTB and AG were found to be more effective than the endogenous nucleophile, GSH, in mediating protection. We propose a possible mechanism for the protective effect of PTB on MG.

phosphate buffer (pH 7.4) was added MG (10.8 µL, 6.5 M). The reaction mixture was stirred at 37 °C for 24 h followed by HPLC analysis. The flow rate was initially 3.0 or 1.0 mL/min (depending on the column size) with 100% buffer A (0.05% trifluoroacetic acid/ddH2O) and reached 50% buffer B (acetonitrile)/50% buffer A at 30 min following a linear gradient. By 35 min, the gradient reached 100% buffer B, and at 38 min, the mobile phase was reset to 100% buffer A. The wavelengths that were monitored were 214 and 254 nm. Two major fractions have been isolated and characterized by MS and NMR. 1H NMR and ESI-MS data for each distinct fraction are listed as follows. For fraction 1, diastereoisomers 1 and 2 (1:1): 1H NMR δ 1.72, 1.74 (CH3), 4.68 (1H, d, J ) 4.2 Hz), 5.15 (1H, d, J ) 5.7 Hz), 6.42 (1H, J ) 4.4 Hz), 6.94 (1H, d, J ) 5.7 Hz), 7.61-8.22 (m, Ph), 8.29-8.36 (m, thiazol-H4 and -H5); ESI-MS m/z 276 (M+, 100% relative intensity). For fraction 2, diastereoisomers 3 and 4 (1:1): 1H NMR δ 1.62, 1.78 (CH3), 4.62 (1H, d, J ) 4.0 Hz), 5.10 (0.5H, d, J ) 5.0 Hz), 6.57 (1H, d, J ) 3.7 Hz), 6.75 (1H, d, J ) 5.2 Hz), 7.62-8.20 (5H, m, Ph), 8.27-8.32 (2H, m, thiazol-H4 and -H5); ESI-MS m/z 276 (M+, 100% relative intensity). Reproducibility. Variations in the actual viability data were observed between different days, although the trends were always the same. Therefore, it was important to test all compounds for comparison on the same day. Each graph represents data obtained from experiments conducted on the same day only and is representative of at least two experiments. For the viability measurements, the error bars represent the standard deviation from the mean for one experiment.

Experimental Procedures

PTB Protects E. coli Cells against Externally Added MG. We have shown previously that cells of E. coli strain MJF276, which lacks both the KefB and KefC systems, have an increased sensitivity toward both externally added and endogenously produced MG compared with cells that possess these systems (18, 20). This is due to the inability of MJF276 cells to transport potassium ions and lower their intracellular pH upon addition of MG (20). Exponential phase cells of MJF276 also were more sensitive to electrophile-induced cell death compared with stationary phase cells (23). To investigate the effect of PTB on MG toxicity, we utilized the sensitivity of exponential phase cells of MJF276 to MG. Exponential phase cells of MJF276 were grown in K0.2 medium and exposed to 0.7 mM MG in either the presence or absence of 0.7 mM PTB, and the effect on growth and viability was determined (panels a and b of Figure 1, respectively). Consistent with previous findings (20), cells lacking the KefB and KefC systems were unable to grow in the presence of 0.7 mM MG and rapidly lost viability. In contrast, the inclusion of 0.7 mM PTB enabled growth in the presence of MG and completely prevented cell death (panels a and b of Figure 1, respectively). In fact, PTB aided the growth and survival of MJF276 cells in the presence of MG to an extent similar to that of AG, a compound previously shown to react with and detoxify MG (panels a and b of Figure 1). It is known that GSH plays a central role in protection of E. coli cells against MG (12, 13). However, unlike that with PTB and AG, the addition of 0.7 mM GSH to exponential phase cells of MJF276 did not enable growth in the presence of 0.7 mM MG (Figure 1a). GSH did confer some protection against MG-induced cell death; however, this effect was unlike that of PTB and AG which completely prevented cell death (Figure 1b). In the absence of MG, 0.7 mM AG, PTB, or GSH had no effect

Analytical Methods. NMR spectra were recorded in D2O on a General Electric Q-300 (270 MHz) spectrometer. Electrospray ionization mass spectrometry (ESI-MS) samples were run on a Quattro triple-quadrupole mass spectrometer. Loop injection samples were taken using an ABI model 140B syringe pump employing H2O/CH3CN at a flow rate of 15 µL/min, a Rheodyne model 7125 valve with a 10 µl loop, and a Micromass Megaflow ESI probe using nitrogen as the nebulizer/drying gas. HPLC was performed using a Waters instrument (Waters 626 pump and Waters 490 E multiwavelength detector), Millenium Software, and a Vydac protein C4 column (250 mm × 10 mm or 250 mm × 4.6 mm). Bacterial Growth and Viability. The bacterial strain MJF276 (F- kdp thi rha lacI lacZ kefB151 kefC::Tn10) used in this study was a derivative of E. coli K12 and lacks both the KefB and KefC systems. All cultures were grown in K0 minimal medium supplemented with 0.2 mM KCl (K0.2 medium) (26). The growth medium contained either 0.2% (w/v) glucose or xylose as the sole carbon source as defined in the text. Exponential phase cultures were prepared by diluting an overnight culture 15-fold into fresh medium and incubating at 37 °C (300 rpm) until the OD650 reached approximately 0.4. The exponential phase cultures then were diluted 10-fold into fresh prewarmed medium (37 °C), and MG was added from an aqueous solution to the concentration defined in the text. For the endogenous production of MG, cAMP was added at this stage to a final concentration of 2 mM. MG production was assayed colorimetrically by reaction with (2,4-dinitrophenyl)hydrazine (DNPH) exactly as described previously (18, 22). PTB, AG, and GSH were all prepared as 100 mM aqueous stock solutions and were added to the concentrations defined in the text. Viability experiments were conducted exactly as described previously (18, 20). Cells were serially diluted into K0 medium lacking a carbon source, and the recovery was conducted on K10 plates. Colonies could be counted after incubation of the plates at 37 °C for 2436 h. Preparation and Isolation of PTB-MG Derivatives. To a solution of PTB (20.0 mg) in 100 mL of aqueous 0.20 M

Results

Phenylacylthiazolium Scavenges MG

Figure 1. PTB enables growth and prevents loss of cell viability in the presence of exogenously added MG. Exponential phase cells of MJF276 were grown in K0.2 minimal medium supplemented with 0.2% (w/v) glucose and the growth and viability experiments conducted exactly as described in Experimental Procedures. (a) Cells were grown in the presence of 0.7 mM MG added at time zero. The following compounds were also added to a final concentration of 0.7 mM along with the MG: (b) PTB, (9) AG, (2) GSH, and (O) control (no compound). (b) Cell viability (average of three experiments) (symbols as for panel a). (c) MG assay (symbols as for panel a). The added compounds did not affect either cell growth or viability in the absence of MG (data not shown).

on cell growth or viability (data not shown). These data provide evidence that AG and PTB protect E. coli cells against MG to a greater extent than the endogenous nucleophile, GSH. They also suggest that PTB is able to interact with and detoxify MG. The ability of PTB to interact with MG is further supported from the findings of the MG assay (Figure 1c). The concentration of MG in the medium is assayed by reaction with DNPH (18, 20). In the presence of 0.7 mM PTB, the concentration of reactive MG in the medium was immediately reduced from 0.7 to 0.25 mM and then

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slowly declined over time (Figure 1c). This result suggested that MG could react with PTB such that it was no longer available to react with DNPH in the assay. AG (0.7 mM) also reduced the amount of reactive MG to the same extent as PTB, whereas 0.7 mM GSH had only a slight effect (Figure 1c). These data correlate with our findings for the growth and viability experiments and provide evidence that PTB can react with MG. PTB Protects E. coli Cells against Endogenously Produced MG. It has been shown previously that cells of E. coli accumulate high concentrations of MG when grown on a poor carbon source such as D-xylose in the presence of millimolar concentrations of cAMP (18, 21, 22). To confirm that PTB could protect E. coli cells against endogenously produced MG, exponential phase cells of MJF276 grown on D-xylose were treated with and without 0.7 mM PTB in the presence of 2 mM cAMP (Figure 2). MG starts to accumulate after cAMP addition, and ultimately cell growth becomes inhibited (panels c and a of Figure 2, respectively). Cells grown in the presence of 0.7 mM PTB took longer to become completely inhibited compared to cells without PTB (Figure 2a). Once again, the inclusion of 0.7 mM AG in the presence of cAMP resulted in a growth profile similar to that with PTB, whereas GSH was only slightly better than in the absence of any compounds (Figure 2a). The addition of 2 mM cAMP to cells of MJF276, growing on D-xylose minimal medium, results in the accumulation of MG such that it is excreted into the medium (Figure 2c). However, in the presence of either 0.7 mM PTB or AG, the amount of DNPH-reactive MG in the medium was 6-fold lower than in the control, 2.5 h after cAMP addition (Figure 2c); however, by 3.5 h there was only 1.6-fold less, and by 4.5 h the levels were the same, independent of the presence or absence of PTB. In contrast, GSH (0.7 mM) only slightly reduced the level of DNPH-reactive MG (Figure 2c). These data suggest that PTB can react with the MG that is generated by the E. coli cells after cAMP treatment. However, 0.7 mM PTB could only provide limited protection to cells against MG-induced cell death (Figure 2b). Increasing the PTB concentration from 0.7 to 1.4 mM allowed cells of MJF276 to grow and survive for longer in the presence of 2 mM cAMP (panels a and b of Figure 3, respectively). Under these conditions, the amount of DNPH-reactive MG remained at a low level until 4.5 h after cAMP addition (Figure 3c). Reducing the PTB concentration from 0.7 to 0.3 mM only slightly delayed growth inhibition, cell death, and DNPH-reactive MG accumulation in the medium in comparison to the control (Figure 3). These data provide further evidence that PTB can interact with and protect E. coli cells against MG. Reaction of PTB with MG. The synthesis of PTB was performed as described previously using thiazol and bromoacetophenone (27). PTB has two nucleophilic centers at the thiazolium-2 and R-position of the N-substituent, and these are thought to attack the carbonyl groups of MG, resulting in the reduction of the carbonyl moieties and fused ring formation (Figure 4). PTB reacts with MG in phosphate buffer (0.20 M, pH 7.4) to yield two major adducts and other minor fractions as indicated by HPLC analysis after the reaction mixture was stirred for 24 h. The distinct fractions were isolated, lyophilized, and subjected to NMR and mass spectrometry. Each fraction was composed of a mixture of diastereoisomers as determined by 1H NMR. For example, the C-4 and 5H resonate

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Figure 2. PTB delays growth inhibition and cell death in the presence of endogenously produced MG. Exponential phase cells of MJF276 were grown in K0.2 minimal medium supplemented with 0.2% (w/v) D-xylose, and the MG assay and growth and viability experiments were conducted exactly as described in Experimental Procedures. (a) Cells were grown in the presence of 2 mM cAMP added at time zero. The following compounds were also added to a final concentration of 0.7 mM along with the MG: (b) PTB, (9) AG, (2) GSH, and (O) control (no compound). (b) Cell viability (average of three experiments) (symbols as for panel a). (c) MG assay (symbols as for panel a). The added compounds did not affect either cell growth or viability in the absence of cAMP (data not shown). The arrow means there were no viable cells by the next time point.

Figure 3. Increasing the concentration of PTB further delays growth inhibition and cell death in the presence of endogenously produced MG. Exponential phase cells of MJF276 were grown in K0.2 minimal medium supplemented with 0.2% (w/v) D-xylose and the growth, viability, and MG assay conducted exactly as described in Experimental Procedures. (a) Cells were grown in the presence of 2 mM cAMP added at time zero. PTB was also added to a final concentration of (b) 0.3, (9) 0.7, (2) 1.4, and (O) 0 mM (no compound) (b) Cell viability (average of three experiments) (symbols as for panel a). (c) MG assay (symbols as for panel a). The added compounds did not affect either cell growth or viability in the absence of cAMP (data not shown). The arrow means there were no viable cells by the next time point.

at 8.29-8.36 ppm, similar to the starting material; however, the disappearance of the C-2 proton, which resonates at 10.21 ppm in PTB, indicates the involvement of this nucleophilic site in the formation of the new adduct. Moreover, the disappearance of the methylene proton, which resonates at 6.46 ppm as a singlet in PTB, is accompanied by the appearance of two new resonances at 6.42 (J ) 4.4 Hz) and 6.94 ppm (J ) 5.7 Hz) in a ratio of 1:1 in one fraction. To determine the positions of the

nucleophilic attack of PTB on MG, the 1H NMR data of a single isomer were extracted on the basis of coupling constants analysis. The two protons which resonate each as doublets (J ) 5.7 Hz) at 5.15 and 6.94 ppm indicate that these two protons are vicinal. On the basis of these data, we conclude that the C-2 and R-position of the phenylacyl attack the R-keto and aldehydic groups of MG, respectively (Figure 4). These data also show that the diastereomers are five-membered rings fused to PTB. The

Phenylacylthiazolium Scavenges MG

Figure 4. Reaction of PTB with MG. The two nucleophilic centers of PTB react with the carbonyl groups of MG to form a five-membered ring structure.

mass spectra of the two fractions display an identical molecular ion at 276 (M+, 100% relative intensity), supporting diastereoisomer formation. It should be noted that the results obtained from the reaction of PTB with MG in the presence of MJF276 cells (data not shown) were similar to those in the absence of bacteria.

Discussion MG has been found to accumulate in people during hyperglycemia, and it is also thought to be responsible for tissue damage in diabetic patients. It has been reported that approximately 189 ( 37 nM MG accumulates in the plasma of diabetic patients compared with 123 ( 37 nM in nondiabetic control patients (28). The association of dicarbonyl compounds in diabetic complications is not limited to MG since other Maillard reaction products such as 3-deoxyglucosone (3-DG), APdione, and glyoxal and lipid peroxidation products such as malondialdehyde are also thought to be involved. Thus, there is a clinical need to develop drugs that can interact with and detoxify these toxic dicarbonyl compounds. The presented data demonstrate that we have developed a sensitive assay system for analyzing the ability of compounds to interact with and detoxify MG. Exponential phase cells of the E. coli double mutant lacking the KefB and KefC systems were highly sensitive toward MG. However, in the presence of PTB, the cells survived micromolar quantities of MG and were able to grow. MG has been found to cause severe damage to DNA in E. coli cells,2 and hence, our results suggest that PTB can interact with and detoxify MG, thereby limiting these lethal effects. At present, the major drug being developed to detoxify dicarbonyl compounds is AG (24). However, in our experiments, we have found that PTB was almost as effective as AG in protecting bacteria against MG. Both PTB and AG were nontoxic to the E. coli cells at micromolar concentrations. However, it is important to determine the toxicity of PTB to human cells if this compound is going to be of clinical importance. In diabetic patients, nanomolar quantities of MG are produced (28), and hence, PTB would be required at a lower concentration than in the bacterial system. PTB was found to be far more effective than the endogenous nucleophile, GSH, in protecting E. coli cells against MG. It has been shown previously that GSH plays an important role in the detoxification and protection of E. coli cells against MG (12, 13). Cells of an E. coli mutant unable to synthesize glutathione could not metabolize MG and were highly sensitive to MG-induced killing (13). Evidence supporting the interaction of PTB with MG has come from the DNPH assay and NMR and mass 2 G. P. Ferguson, D. Reiss, I. R. Booth, J. Batistta, and A. Lee, unpublished data.

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spectroscopy. In the presence of PTB, MG could no longer interact with DNPH, suggesting a PTB-MG interaction. This interaction was further characterized by NMR and mass spectroscopy and showed the formation of a fivemembered ring structure. On the basis of our bacterial assay, we propose that PTB scavenges MG and forms nontoxic products. This reaction must be rapid since we have shown previously that for E. coli cells to survive exposure to MG, protection must occur within a few minutes of MG addition (20). If the protection was delayed, then a proportion of cells that were already predisposed to death could not be rescued upon activation of the defense mechanism. Therefore, our finding that 0.7 mM PTB was able to completely prevent the death of E. coli cells against 0.7 mM MG provides evidence that the reaction of PTB with MG must be rapid. In conclusion, we have developed a sensitive bacterial assay for investigating the ability of nucleophiles to detoxify MG. Reactions involving other clinically relevant toxic dicarbonyls such as 3-DG, AP-dione, malondialdehyde, and glyoxal are currently being investigated.

Acknowledgment. G.P.F. is a Wellcome Trust Toxicology Fellow. The visit of G.P.F. to the Picower Institute was funded by the Wellcome Trust and a SHERT Medical Research Foreign Travel Grant. We thank Professor Ian Booth and Dr. Debbie McLaggan for the critical reading of the manuscript. Thanks also to Prof. Booth for strain MJF276.

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622 Chem. Res. Toxicol., Vol. 12, No. 7, 1999 (13) Ferguson, G. P., and Booth, I. R. (1998) The importance of glutathione for the growth and survival of Escherichia coli cells: detoxification of MG and maintenance of intracellular K+. J. Bacteriol. 180, 4314-4318. (14) Fahey, R. C., Brown, W. C., Adams, W. B., and Worsham, M. B. (1978) Isolation and initial characterization of gluthathionedeficient mutants of Escherichia coli K12. J. Bacteriol. 133, 11261129. (15) Cooper, R. A., and Anderson, A. (1970) The formation and catabolism of methylglyoxal during glycolysis in Escherichia coli. FEBS Lett. 11, 273-276. (16) MacLean, M. J., Ness, L. S., Ferguson, G. P., and Booth, I. R. (1998) The role of glyoxalase I in the detoxification of methylglyoxal and in activation of the KefB K+ efflux system in Escherichia coli. Mol. Microbiol. 27, 563-571. (17) Elmore, M. J., Lamb, A. J., Ritchie, G. Y., Douglas, R. M., Munro, A., Gajewska, A., and Booth, I. R. (1990) Activation of potassium efflux from Escherichia coli. Mol. Microbiol. 4, 405-412. (18) Ferguson, G. P., Munro, A. W., Douglas, R. M., McLaggan, D., and Booth, I. R. (1993) Activation of potassium channels during metabolite detoxification in Escherichia coli. Mol. Microbiol. 9, 1297-1303. (19) Ferguson, G. P., Nikolaev, Y., McLaggan, D., MacLean, M., and Booth, I. R. (1997) Survival during exposure to the electrophilic reagent N-Ethylmaleimide in Escherichia coli: role of KefB and KefC potassium channels. J. Bacteriol. 179, 1007-1012. (20) Ferguson, G. P., McLaggan, D., and Booth, I. R. (1995) Potassium channel activation by glutathione-S-conjugates in Escherichia

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