Implications of the Colonic Deposition of Free Hemoglobin-α Chain: A Previously Unknown Tissue By-product in Inflammatory Bowel Disease

Background: We analyzed inflamed mucosal/submucosal layers of ulcerative colitis (UC = 63) and Crohn's colitis (CC 50), and unexpectedly, we unveiled a pool of free hemoglobin alpha (Hb-alpha) chain. Patients with colitides have increased reactive oxidative stress (ROS), DNA oxidation products, free iron in mucosa, in preneoplastic, and in colitis-cancers and increased risks of developing colorectal cancer. All inflammatory bowel disease-related colorectal cancer lesions are found in segments with colitis. Linking this information, we investigated whether free Hb-alpha is key transformational stepping that increases colitis-related colorectal cancer vulnerability. Methods: UC/CC samples were profiled using matrix-assisted laser desorption/ionization mass spectrometry; protein identification was made by liquid chromatography. Diverticulitis was used as control (Ctrl). The presence of Hb(n) (n = alpha, beta, or hemin)/Hb was validated by Western blotting and immunohistochemistry. We tested for DNA damage (DNAD) by exposing normal colonic epithelial cell line, NCM460, to 10 mu M and 100 mu M of Hb(n)/Hb, individually for 2, 6, and 12 hours. Quantification of Hb-alpha staining was done by Nikon Elements Advance Research Analysis software. ROS was measured by the production of 8-OHdG. DNAD was assessed by Comet assay. Colonic tissue homogenate antioxidants Nrf2-, CAT-, SOD-, and GPx-expressions were analyzed densitometrically/normalized by beta-actin. Results: Immunohistochemistry of CC/UC mucosal/submucosal compartments stained strongly positive for Hb-alpha and significantly higher versus Ctrl. NCM460 exposed to Hb(n)/Hb exhibited steadily increasing ROS and subsequent DNAD. DNAD was higher in 10 mu M than 100 mu M in Hb-beta/hemin the first 2 hours then plateaued followed by DNAD repair. This may be likely due to apoptosis in the later concentration. Nrf2 enzyme activities among UC, CC, and ulcerative colitis-associated colon cancer (UCAC) were observed impaired in all inflammatory bowel disease subjects. Decreased levels of Nrf2 among patients with UC versus patients with CC with active disease were insignificant as well as versus Ctrls but significantly lower in UCAC versus Ctrl. SOD was decreased in UC and UCAC and GPx in CC but statistically not significant. Comparing CC versus UC, SOD was significantly lower in CC (P < 0.05). CAT was observed increased among patients with CC/UC/UCAC and GPx in UC and UCAC versus Ctrl, respectively, and significantly increased in CC versus Ctrl (P < 0.01). Conclusions: In the colitides, mucosal/submucosal tissue microenvironments demonstrated pool of free Hb-alpha chain. In vitro exposure of NCM460 cells to Hb(n)/Hb induced ROS and DNAD. Toxic effect of free Hb-alpha, in colonic epithelial cells, is therefore through production of ROS formation modulated by impairment of antioxidant effects. Targeting reduction-oxidation-sensitive pathways and transcription factors may offer options for inflammatory bowel disease-management and colitis-related cancer prevention.