L. Kruidenier

Intestinal oxidative damage in inflammatory bowel disease: semi-quantification, localization, and association with mucosal antioxidants

Intestinal inflammation is accompanied by excessive production of reactive oxygen and nitrogen metabolites. In order to counteract their harmful effects, the intestinal mucosa contains an extensive system of antioxidants. It has previously been shown that the levels of and the balance between the most important antioxidants are seriously impaired within the intestinal mucosa from inflammatory bowel disease (IBD) patients compared with normal mucosa. The present study investigated the consequences of this antioxidative imbalance by evaluating parameters of oxidative stress‐related mucosal damage in the same tissue samples. The extent of apoptosis, peroxynitrite‐mediated protein nitration (3‐NT), and lipid peroxidation were assessed in relation to the expression of nitric oxide synthase (NOS) and the superoxide‐producing enzyme xanthine oxidase (XO). In addition, bi‐ and multi‐variate regression analyses were performed to associate these parameters with the levels of the antioxidants assessed previously. Apoptotic cell death was visualized by TUNEL staining in luminal epithelium of normal controls, and in IBD additionally in the inflammatory infiltrate and in deeper parts of the crypts, but its frequency was unrelated to the severity of inflammation. In Crohns disease (CD), epithelial apoptosis levels were strongly associated with the expression of XO, implying a role for this enzyme in the regulation of epithelial cell homeostasis, although its levels were unaffected by intestinal inflammation and were comparable to those in normal control mucosa. 3‐NT immunoreactivity was substantially increased in luminal crypt cells, neutrophils, and mononuclear cells in the inflamed mucosa of ulcerative colitis (UC) patients. The inflamed IBD luminal epithelium, but not the inflammatory cells, also contained increased amounts of NOS. The immunoreactivity of both 3‐NT and NOS was significantly higher in UC than in CD. Unexpectedly, the increased 3‐NT expression in UC was associated with neutrophilic myeloperoxidase and not with NOS, which suggests that 3‐NT is formed in areas with a dense neutrophilic infiltrate via a peroxynitrite‐independent oxidation pathway. Lipid peroxidation, as estimated by the malondialdehyde (MDA) concentration, was elevated in both the inflamed CD and the inflamed UC mucosa, and was identified in the luminal epithelium using a histochemical technique. In CD, lipid peroxidation was independently associated with the concentration of metallothionein and with Mn‐superoxide dismutase activity, suggesting the involvement of hydroxyl radicals and superoxide anions. In UC, however, the amount of MDA was associated with epithelial catalase expression and neutrophilic myeloperoxidase activity, suggesting a hydrogen peroxide‐ and/or hypochlorous acid‐mediated mechanism. The present study underlines the importance of oxidative stress in the pathogenesis of IBD and provides clues regarding the (anti)oxidants involved which indicate that this process evolves through diverging pathways in CD and UC. Copyright

Imbalanced secondary mucosal antioxidant response in inflammatory bowel disease

Intestinal mucosal damage in the inflammatory bowel diseases (IBD) Crohns disease (CD) and ulcerative colitis (UC) involves reactive oxygen metabolites (ROMs). ROMs are neutralized by endogenous antioxidant enzymes in a carefully balanced two‐step pathway. Superoxide dismutases (SODs) convert superoxide anion to hydrogen peroxide (H2O2), which is subsequently neutralized to water by catalase (CAT) or glutathione peroxidase (GPO). Remarkably changed expression levels of the three isoforms of SOD in paired non‐inflamed and inflamed mucosae from CD and UC patients have been previously reported in comparison to normal control mucosa. Most notable was the strong up‐regulation of Mn‐SOD in inflamed epithelium. It was hypothesized that in order to provide optimal protection against ROM‐mediated damage, these changes should be coordinately counterbalanced by an increased H2O2‐neutralizing capacity. Therefore, the same tissue samples were used to assess the levels, activities, and/or localization of the most prominent mucosal H2O2‐related antioxidants CAT, GPO, glutathione (GSH), myeloperoxidase (MPO), and metallothionein (MT). Quantitative measurements showed that in both CD and UC patients, intestinal inflammation was associated with increased activities of CAT, GPO, and MPO, whereas the mucosal GSH content was unaffected and the concentration of MT was decreased. Despite this overall increase in mucosal H2O2‐metabolizing enzyme capacity, immunohistochemical analysis revealed a differentially disturbed antioxidant balance in IBD epithelium and lamina propria. In the lamina propria, the risk of direct H2O2‐mediated damage seemed to be restrained by the increasing numbers of CAT‐ and MPO‐positive monocytes/macrophages and neutrophils that infiltrated the inflamed areas. On the other hand, MPO overexpression might increase the lamina propria levels of hypochlorous acid, a stable ROM with multiple pro‐inflammatory effects. In the epithelium, the number of cells that expressed CAT remained unchanged during inflammation and GPO was found in only a very low and constant number of epithelial cells. In addition, the inflamed epithelium displayed decreased expression of the hydroxyl radical (OH•) scavenger MT. In view of the high epithelial SOD levels in inflamed IBD epithelium, it is speculated that the efficient removal of excess H2O2 is hampered in these cells, thereby increasing not only the risk of detrimental effects of H2O2 directly, but also those of its extremely reactive derivatives such as OH•. Taken together, the results suggest an imbalanced and inefficient endogenous antioxidant response in the intestinal mucosa of IBD patients, which may contribute to both the pathogenesis and the perpetuation of the inflammatory processes. Copyright

Differential mucosal expression of three superoxide dismutase isoforms in inflammatory bowel disease.

Mucosal tissue damage and dysfunction in chronic inflammatory bowel disease (IBD) are partly caused by an enduring exposure to excessive amounts of reactive oxygen metabolites (ROMs). Although the three human isoforms of superoxide dismutase (SOD), copper/zinc (Cu/Zn)‐SOD, manganese (Mn)‐SOD, and extracellular (EC)‐SOD, form the primary endogenous defence against ROMs, their expression levels and cellular localization in IBD mucosa are largely unknown. The present study used enzyme‐linked immunosorbent assays (ELISAs), spectrophotometric activity assays, and immunohistochemistry to evaluate the protein concentration, enzymatic activity, and distribution of Cu/Zn‐, Mn‐, and EC‐SOD in paired inflamed and non‐inflamed mucosal resection specimens of patients with Crohns disease (CD) or ulcerative colitis (UC) and compared these with the levels obtained in normal control mucosa. Gut mucosal SOD isoform expression was found to be differentially affected in IBD patients, without major differences between CD and UC. A marked step‐wise increase in Mn‐SOD protein levels was observed in non‐inflamed and inflamed IBD mucosae, whereas the Cu/Zn‐SOD content decreased with inflammation. EC‐SOD was only found in low amounts, which tended to be decreased in IBD patients. Immunohistochemical evaluation confirmed these observations. Mn‐SOD and Cu/Zn‐SOD were both predominantly expressed in intestinal epithelial cells and the percentage of epithelial cells positive for Mn‐SOD was considerably increased in IBD, whereas epithelial Cu/Zn‐SOD expression was much less affected. Within the lamina propria, SOD expression was much lower. Cu/Zn‐SOD and Mn‐SOD were prominently present in neutrophils and macrophages, and EC‐SOD was mainly localized in small vessels, stromal cells, and neutrophils. The percentage of lamina propria cells positive for Cu/Zn‐, Mn‐, or EC‐SOD was not affected by inflammation. Enzyme activity measurements showed consistent results for Cu/Zn‐SOD and EC‐SOD, but the activity of Mn‐SOD did not concordantly increase with the immunological assessments, which may indicate that a proportion of the Mn‐SOD in IBD is present in an enzymatically inactive form. This study reveals remarkable changes in the expression levels of the three SOD isoforms in IBD, particularly in the epithelium. Disturbances in the carefully orchestrated mucosal antioxidant cascade may contribute to the induction and perpetuation of intestinal inflammation in IBD, and may have important implications for the development of antioxidant treatment of IBD patients. Copyright

Attenuated mild colonic inflammation and improved survival from severe DSS-colitis of transgenic Cu/Zn-SOD mice

Mucosal tissue damage in chronic inflammatory bowel disease (IBD) is partly caused by an enduring exposure to excessive amounts of reactive oxygen metabolites (ROM). To protect themselves from the toxic effects of ROM, most intestinal cell types constitutively express the highly specific, key ROM-neutralizing cytosolic enzyme Cu/Zn-superoxide dismutase (SOD). Under inflammatory conditions, however, its protein and activity levels have consistently been reported as being decreased. To elucidate a direct functional relationship between intracellular Cu/Zn-SOD expression and intestinal inflammation, we investigated the effects of transgenic human Cu/Zn-SOD overexpression in acute and chronic murine dextran sodium sulfate (DSS)-induced colitis. When subjected to a mild form of acute colitis, the Cu/Zn-SOD overexpressing mice showed a significantly lower colonic activity of neutrophilic myeloperoxidase (MPO) than their nontransgenic littermates. This difference was particularly evident in the male animals. In contrast, a severe acute colitis did not lead to any differences in MPO activity between both groups. Yet, when the animals were subsequently allowed to recover, MPO levels were again significantly lower in the transgenes, suggesting an involvement of Cu/Zn-SOD in, particularly, the clearance of neutrophils. Specific, immunohistochemical identification of neutrophils confirmed the validity of the MPO activity measurements. In addition, transgenic animals showed a remarkable survival benefit from severe DSS colitis over their nontransgenic littermates, particularly during or shortly after the acute inflammatory phase. During the chronic inflammatory phase, which was not characterized by massive neutrophil infiltration, no effects of Cu/Zn-SOD overexpression were noted. Paradoxically, overexpression of Cu/Zn-SOD did not obviously improve the colitis-related (oxidative) injury or symptoms at any stage of the experiment. Surprisingly, however, we did observe a pronounced male gender preference for DSS susceptibility that was reflected by increased male colitis mortality. Our findings provide direct in vivo evidence for a protective, neutrophil-related role for Cu/Zn-SOD in intestinal inflammation. As such, they support the concept of SOD-based (adjunct) antioxidant treatment strategies for inflammatory bowel disease.

Superoxide dismutases in the human colorectal cancer sequence

Abstract Purpose: The oxidant-antioxidant balance within tissues is thought to contribute to the development and progression of cancer. Previous investigations have indicated changes in this balance during the colorectal oncogenic process that merit further investigation. The aim of the present study was to evaluate whether the human colorectal cancer sequence is accompanied by changes in the protein and activity levels of the antioxidant enzymes manganese- and copper/zinc-superoxide dismutase (Mn-SOD and Cu/Zn-SOD). Patients and methods: SOD levels were assessed in colorectal adenomas, carcinomas, and liver metastases and were compared with those in the corresponding normal tissues (n=35 in each group). Mn- and Cu/Zn-SOD expression was first evaluated semiquantitatively by electrophoretic activity analysis, immunoblotting, and immunohistochemistry and was subsequently quantified by enzyme-linked immunosorbent assays (ELISAs) and spectrophotometric activity assays. Results: The semiquantitative analyses showed enhanced Mn-SOD levels, primarily localized in (neoplastic) epithelial cells, in carcinomas, and in liver metastases as compared with adenomas and normal mucosa, whereas no consistent pattern was observed for Cu/Zn-SOD. Normal liver tissue expressed the highest levels of both SODs. The quantitative SOD analyses confirmed these observations and revealed that carcinomas and liver metastases expressed 2–4 times more Mn-SOD protein and enzymatic activity (0.0005 < P ≤ 0.01) than did the normal mucosa. Adenomas expressed intermediate Mn-SOD levels, which increased significantly with the diameter and tended to increase with the grade of dysplasia and presence of a villous component. In contrast, adenomas, carcinomas, and the corresponding normal mucosa were found to have a similar Cu/Zn-SOD content, whereas liver metastases contained significantly (P < 0.02) more Cu/Zn-SOD as compared with these tissues. In addition, the Cu/Zn-SOD content was not related to any histopathological characteristic of the carcinomas or adenomas. Conclusions: Our study indicates that the development of neoplasia in the human colorectum is accompanied by major changes in the level and activity of Mn-SOD. This observation illustrates that Mn-SOD might have a functional role in human colorectal carcinogenesis.