EVIDENCE OF OXIDANT-INDUCED INJURY TO EPITHELIAL-CELLS DURING INFLAMMATORY BOWEL-DISEASE

Evidence of in vivo oxidant-induced injury in inflammatory bowel disea se (IBD) is largely indirect. Colon epithelial crypt cells (CEC) from paired specimens of histologically normal and inflamed bowel from IBD patients with active disease were examined for altered protein thiol r edox status as an indicator of oxidative damage. When CEC preparations from 22 IBD patients were labeled with the reduced-thiol-specific pro be [C-14]-iodoacetamide (IAM), there was decreased labeling of a numbe r of proteins indicating oxidation of thiol groups in CEC from inflame d mucosa compared to paired normal mucosa, especially the loss of thio l labeling of a 37-kD protein which was almost completely lost, The lo ss of reduced protein thiol status for the 37-kD band was paralleled b y loss of epithelial cell glyceraldehyde-3-phosphate dehydrogenase (GA PDH, EC 1.2.1.12) enzyme activity, an enzyme known to contain an essen tial reduced cysteine (Cys(149)) at the active site. The identity of t he 37-kD protein as GADPH monomer was confirmed by NH2-terminal amino acid sequence analysis. To examine whether this type of in vivo injury could be attributed to biologically relevant oxidants produced by inf lammatory cells, CEC prepared from normal mucosa were exposed to H2O2, OCl-, nitric oxide (NO), and a model chloramine molecule chloramine T (ChT) in vitro. Dose-dependent loss of IAM labeling and GAPDH enzyme activity was observed. The efficacy (IC50) against IAM labeling was OC l- much greater than ChT > H2O2 > NO (52+/-3, 250+/-17, 420+/-12, 779/-120 mu M oxidant) and OCl- much greater than ChT > NO > H2O2 (89+/-1 7, 256+/-11, 407+/-105, 457+/-75 mu M oxidant), respectively, for GAPD H enzyme activity.This study provides direct evidence of in vivo oxida nt injury in CEC from inflamed mucosa of IBD patients. Oxidation and i nhibition of essential protein function by inflammatory cells is a pot ential mechanism of tissue injury that may contribute to the pathogene sis of the disease and supports the exploration of compounds with anti oxidant activity as new therapies for IBD.