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. 1999 Feb;44(2):180–185. doi: 10.1136/gut.44.2.180

5-Aminosalicylic acid prevents oxidant mediated damage of glyceraldehyde-3-phosphate dehydrogenase in colon epithelial cells

S McKenzie 1, W Doe 1, G Buffinton 1
PMCID: PMC1727397  PMID: 9895376

Abstract

Background—Reactive oxygen and nitrogen derived species produced by activated neutrophils have been implicated in the damage of mucosal proteins including the inhibition of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the active inflammatory lesion in patients with inflammatory bowel disease (IBD). This study investigated the efficacy of currently used IBD therapeutics to prevent injury mediated by reactive oxygen and nitrogen derived species. 
Methods—GAPDH activity of human colon epithelial cells was used as a sensitive indicator of injury produced by reactive oxygen and nitrogen derived species. HCT116 cells (106/ml phosphate buffered saline; 37°C) were incubated in the presence of 5-aminosalicylic acid (5-ASA), 6-mercaptopurine, methylprednisolone, or metronidazole before exposure to H2O2, HOCl, or NO in vitro. HCT116 cell GAPDH enzyme activity was determined by standard procedures. Cell free reactions between 5-ASA and HOCl were analysed by spectrophotometry and fluorimetry to characterise the mechanism of oxidant scavenging. 
Results—GAPDH activity of HCT116 cells was inhibited by the oxidants tested: the concentration that produced 50% inhibition (IC50) was 44.5 (2.1) µM for HOCl, 379.8 (21.3) µM for H2O2, and 685.8 (103.8) µM for NO (means (SEM)). 5-ASA was the only therapeutic compound tested to show efficacy (p<0.05) against HOCl mediated inhibition of enzyme activity; however, it was ineffective against H2O2 and NO mediated inhibition of GAPDH. Methylprednisolone, metronidazole, and the thiol-containing 6-mercaptopurine were ineffective against all oxidants. Studies at ratios of HOCl:5-ASA achievable in the mucosa showed direct scavenging to be the mechanism of protection of GAPDH activity. Mixing 5-ASA and HOCl before addition to the cells resulted in significantly greater protection of GAPDH activity than when HOCl was added to cells preincubated with 5-ASA. The addition of 5-ASA after HOCl exposure did not restore GAPDH activity. 
Conclusions—Therapies based on 5-ASA may play a direct role in scavenging the potent neutrophil oxidant HOCl, thereby protecting mucosal GAPDH from oxidative inhibition. These findings suggest that strategies for the further development of new HOCl scavenging compounds may be useful in the treatment of IBD. 



Keywords: 5-aminosalicylic acid; 6-mercaptopurine; prednisolone; metronidazole; oxidants; glyceraldehyde-3-phosphate dehydrogenase

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Figure 1 .

Figure 1

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Efficacy of 5-aminosalicylic acid (5-ASA) and 6-mercaptopurine (6-MP) against oxidant mediated inhibition of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity. HCT116 cells were exposed to H2O2, HOCl, or NO after preincubation (A) in the absence or presence of 5-ASA or incubated with 5-ASA after exposure to HOCl or (B) in the absence or presence of 6-mercaptopurine. Oxidant and therapeutic concentrations were as described in the Materials and methods section. Data represent mean (SEM) (n = 3). *p<0.05 compared with no 5-ASA. 


Figure 2 .

Figure 2

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Efficacy of methylprednisolone and metronidazole against oxidant mediated inhibition of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity. HCT116 cells were exposed to H2O2, HOCl, or NO after preincubation (A) in the absence or presence of methylprednisolone or (B) in the absence or presence of metronidazole (MTZ). Oxidant and therapeutic concentrations were as described in the Materials and methods section. Data represent mean (SEM) (n = 3). 


Figure 3 .

Figure 3

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Dose-dependence of 5-aminosalicylic acid (5-ASA) prevention of the inhibition of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity mediated by HOCl. HCT116 cells were exposed to increasing ratios of 5-ASA:HOCl according to the following protocol: 5-ASA alone, 5-ASA after exposure to 125 µM HOCl, 5-ASA before exposure to 125 µM HOCl, or the spent reaction resulting from the premixing of 5-ASA with 125 µM HOCl. Data represent mean (SEM) (n = 3). 


Figure 4 .

Figure 4

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Reaction of 5-aminosalicylic acid (5-ASA) with HOCl. Increasing concentrations of HOCl were allowed to react with 5-ASA as described in the Materials and methods section and differential absorbance spectrums determined. The reference cuvette contained 250 µM 5-ASA in HBSS, and the sample cuvette increasing ratios of 5-ASA:HOCl; 37°C. Absorbance traces of 5-ASA:HOCl were as follows: A, 1:0; B, 1:0.25; C, 1:0.5; D, 1:0.75; E, 1:1; F, 1:2; G, 1:3; H, 1:4; I, 1:5. Insert, 5-ASA fluorescence. 


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