Protein carbonyl formation on mucosal proteins in vitro and in dextran sulfate-induced colitis

Reactive oxygen and nitrogen species have been implicated as mediators of m ucosal injury in inflammatory bowel disease, but few studies have investiga ted protein oxidation in the inflamed mucose. In this study, protein carbon yl formation on colonic mucosal proteins from mice was investigated followi ng in vitro exposure of homogenates to iron/ascorbate, hydrogen peroxide, h ypochloric acid HOCl), or nitric oxide ((NO)-N-.). Total carbonyl content w as measured spectrophotometrically by derivatization with dinitrophenylhydr azine (DNPH), and oxidation of component proteins within the tissue was exa mined by Western blotting for DNPH-derivatized proteins using anti-dinitrop henyl DNP antibodies. These results were compared with protein carbonyl for mation found in the acutely inflamed mucose from mice with colitis induced by dextran sulfate sodium (DSS) administered at 5% w/v in the drinking wate r for 7 d. In vitro, carbonyl formation was observed after exposure to iron /ascorbate, HOCl and (NO)-N-.. Iron/ascorbate (20 mu M/20 mM) exposure for 5 h increased carbonyl groups by 80%, particularly on proteins of 48, 75-10 0, 116, 131, and 142 kDa. Oxidation by 0.1 and 0.5 mM HOCl did not increase total carbonyl levels, but Western blotting revealed carbonyl formation on many proteins, particularly in the 49-95 kDa region. After exposure to 1-1 0 mM HOCI, total carbonyl levels were increased by 0.5 to 12 times control levels with extensive cross-linking and fragmentation of proteins rich in c arbonyl groups observed by Western blotting. In mice with acute colitis ind uced by DSS, protein carbonyl content of the inflamed mucosa was not signif icantly different from. control mucose, (7.80 +/- 1.05 vs. 8.43 +/- 0.59 nm ol/mg protein respectively, p =.16 n = 8, 10); however, Western blotting an alysis indicated several proteins of molecular weight 48, 79, 95, and 131 k Da that exhibited increased carbonyl content In the inflamed mucosa. These proteins corresponded to those observed after in vitro oxidation of normal intestinal mucosa with iron/ ascorbate and HOCI, suggesting that both HOCl and metal ions may be involved in protein oxidation in DSS-induced colitis. Identification and further analysis of the mucosal proteins susceptible to carbonyl modification may lead to a better understanding of the contributi on of oxidants to the colonic mucosa tissue injury in inflammatory bowel di sease. (C) 1999. Elsevier Science Inc.