5-aminosalicylic acid prevents oxidant mediated damage of glyceraldehyde-3-phosphate dehydrogenase in colon epithelial cells.
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 (10(6)/ml phosphate buffered saline; 37 degrees 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) microM for HOCl, 379.8 (21.3) microM for H2O2, and 685.8 (103.8) microM 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.
McKenzie
S M
SM
Division of Molecular Medicine, John Curtin School of Medical Research, Australian National University, Canberra, ACT, Australia.
Doe
W F
WF
Buffinton
G D
GD
D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology*; D018929:Cell Culture Techniques; D003106:Colon / Q000187:drug effects* / Q000201:enzymology; D004305:Dose-Response Relationship, Drug; D004847:Epithelial Cells / Q000187:drug effects / Q000201:enzymology; D016166:Free Radical Scavengers / Q000494:pharmacology; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006801:Humans; D006997:Hypochlorous Acid / Q000494:pharmacology; D015122:Mercaptopurine / Q000494:pharmacology; D019804:Mesalamine / Q000494:pharmacology*; D008775:Methylprednisolone / Q000494:pharmacology; D008795:Metronidazole / Q000494:pharmacology; D016877:Oxidants / Q000494:pharmacology; D018384:Oxidative Stress / Q000187:drug effects*
D000894:Anti-Inflammatory Agents, Non-Steroidal; D016166:Free Radical Scavengers; D016877:Oxidants; D008795:Metronidazole; D019804:Mesalamine; D006997:Hypochlorous Acid; D015122:Mercaptopurine; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D008775:Methylprednisolone