Effects of phosphorothioate oligodeoxynucleotide on hemoglobin-induced damage to intestinal mucosa.

Some "blood substitutes," such as human diaspirin cross-linked hemoglobin DBBF-Hb, can damage the intestinal mucosa. This response may be due to release of free iron from Hb leading to production of reactive oxygen species (ROS). Phosphorothioate oligodeoxynucleotides can bind and sequester iron. Therefore experiments were performed to test whether PS-ODN, composed of ten consecutive cytidines "C-10," reduces Hb-induced ROS generation and damage in the mucosa. Anesthetized Sprague-Dawley rats (4-6 per group) were injected arterially with 1 mg C-10, followed two minutes later by 50 mg DBBF-Hb. The positive controls received only DBBF-Hb and the negative controls either saline or PS-ODN followed by saline. Either ROS formation was monitored using a fluorescence technique, or the intestine was fixed for microscopy after 8 or 30 min. Sixty villi per rat were assigned an epithelial integrity index (EI), ranging from 1 (intact) to 3 (some cell-cell and cell-basement membrane separation). Pretreatment with PS-ODN significantly exacerbated DBBF-Hb-induced ROS formation, and PS-ODN groups showed significantly more epithelial damage near Peyer's patches, (EI of 1.93 +/- 0.06 (SEM) at 8 minutes and 1.31 +/- 0.04 at thirty minutes), than the negative controls, (1.11 +/- 0.02 at both 8 and 30 minutes), or the positive controls (1.43 +/- 0.05 at 8 minutes and 1.20 +/- 0.03 at 30 minutes) (p < 0.05). However, mast cell degranulation, eosinophil accumulation and goblet cell secretion were significantly reduced in the DBBF-Hb groups pre-treated with PS-ODN. Thus, PS-ODN, although an iron chelator, can significantly enhance epithelial damage caused by DBBF-Hb in the rat intestinal mucosa near Peyer's patches, possibly by formation of the ferryl component of the hemoglobin.

Differential effects of sodium selenite in reducing tissue damage caused by three hemoglobin-based oxygen carriers.

Three "blood substitutes," a diaspirin cross-linked human hemoglobin (DBBF-Hb), a bovine polymerized hemoglobin (PolyHbBv), and a human polymerized hemoglobin (O-R-PolyHbA(0)), that have undergone clinical trials are used in this study. Previously, we showed in the rat that coadministration of sodium selenite (Na(2)SeO(3)) and DBBF-Hb significantly decreased mesenteric venular leakage and epithelial disruption produced by DBBF-Hb alone but did not reduce mast cell degranulation unless given orally. The purpose of this study was to determine whether Na(2)SeO(3) produced similar beneficial responses when used with PolyHbBv and O-R-PolyHbA(0). In anesthetized Sprague-Dawley rats, the mesenteric microvasculature was perfused with PolyHbBv or O-R-PolyHbA(0), with and without Na(2)SeO(3) in the perfusate and suffusate, for 10 min, followed by FITC-albumin for 3 min, and then fixed for microscopy. Na(2)SeO(3) did not reduce leak number or area in preparations perfused with PolyHbBv and only reduced leak number (but not significantly) in preparations perfused with O-R-PolyHbA(0). Na(2)SeO(3) significantly increased mesenteric mast cell degranulation and impaired epithelial integrity in animals treated with PolyHbBv. In vitro, Na(2)SeO(3) significantly reduced the oxidation rate of DBBF-Hb in the presence of oxidants, had little effect on PolyHbBv, and increased the oxidation rate of O-R-PolyHbA(0). These results suggest that Na(2)SeO(3) moderates hemoglobin-induced damage, at least partly, through its redox interactions with the heme sites in the hemoglobin molecules studied and that accessibility of the heme site to Na(2)SeO(3) governs those interactions.

Sodium selenite reduces hemoglobin-induced venular leakage in the rat mesentery.

Modified Hbs are being developed as "blood substitutes," but intravascular injection of diaspirin cross-linked Hb (DBBF-Hb) can produce venular leakage. Hb toxicity may arise from reactive oxygen species, so the antioxidant sodium selenite (Na(2)SeO(3)) was used in an attempt to reduce leak formation. In anesthetized Sprague-Dawley rats, one-half of which received 2 x 10(-6) g/ml Na(2)SeO(3) in their drinking water for 3 wk, the mesenteric microvasculature was perfused with 2 mg/ml DBBF-Hb (N = 8) for 10 min. Controls (N = 7) received saline. This was followed by perfusion with FITC-albumin for 3 min, fixation, and microscopic examination. In rats given DBBF-Hb, Na(2)SeO(3) significantly reduced leak number, leak area, and mast cell degranulation. Venular leakage was also reduced in rats that only received Na(2)SeO(3) locally during DBBF-Hb perfusion. However, Na(2)SeO(3) did not affect animals receiving cyanomet-DBBF-Hb instead of DBBF-Hb and significantly increased leak number and mast cell degranulation in animals receiving saline. In vitro, Na(2)SeO(3) reduced the oxidation rate of DBBF-Hb while in the presence of oxidants. These results suggest that Na(2)SeO(3) reduces DBBF-Hb-induced microvascular leakage partly by retarding the oxidation of its heme iron.

Comparison of effects of two hemoglobin-based O(2) carriers on intestinal integrity and microvascular leakage.

Two "blood substitutes," a diaspirin cross-linked human hemoglobin [bis(3,5 dibromosalicyl)fumarate, DBBF-Hb] and a bovine polymerized hemoglobin (PolyHbBv), advanced to clinical trials, are used in this study. Previously, we have shown that injection of DBBF-Hb into the rat circulation produces venular leakage and intestinal epithelial disruption. The purpose of this study was to determine whether PolyHbBv, currently approved for veterinary use in the United States, shows similar effects. In anesthetized Sprague-Dawley rats, the mesenteric microvasculature was perfused with DBBF-Hb (n = 6), PolyHbBv (n = 5), cyanomet Hb (CNmet-DBBF-Hb), or HEPES-buffered saline with 0.5% bovine serum albumin (HBS-BSA) (controls, n = 7) for 10 min, followed by FITC-albumin for 3 min, and then fixed for microscopy. For DBBF-Hb, the mean leak number per micrometer venule length [2.41 +/- 0.33 (+/-SE) x 10(-3)] was significantly greater than for PolyHbBv (0.53 +/- 0.14 x 10(-3)), CNmet-DBBF-Hb (0.36 +/- 0.14 x 10(-3)), and HBS-BSA (0.12 +/- 0.08 x 10(-3)) (P < 0.01). Corresponding quantities for leak area were 0.10 +/- 0.03, 0.010 +/- 0.003, 0.005 +/- 0.003, and 0.02 +/- 0.02 microm(2)/microm. In rats injected with DBBF-Hb (n = 8), intestinal epithelial integrity was significantly compromised compared with those injected with PolyHbBv (n = 5) or saline (n = 6). These results indicate that intravascular PolyHbBv produces significantly less disruption of the intestinal exchange barrier than does DBBF-Hb, probably because the heme is not so easily oxidized.

Selenium reduces hemoglobin-induced epithelial damage to intestinal mucosa.

Modified hemoglobins are being considered as possible "blood substitutes." Experiments were performed to determine whether diaspirin cross-linked hemoglobin (DBBF-Hb) produces epithelial damage and whether this is reduced by selenium (Se). Anesthetized Sprague-Dawley rats, half of which received 2 x 10(-6) g/ml Se, daily for 3 weeks, in their drinking water, were injected with a 5 ml bolus of 10 mg/ml DBBF-Hb. Control animals received saline (5 animals per group). After 30 minutes, the intestine was perfusion-fixed for light and electron microscopy. Eighty villi per rat were assigned an epithelial integrity index (E.I.), ranging from 1 (intact) to 3 (some cell-cell and cell-basement membrane separation). In non-Se rats, E.I. was significantly compromised by DBBF-Hb, compared to HBS-BSA (2.47+/-0.57 (SD) vs. 1.36+/-0.49, p<0.001). In Se rats, neither injection with DBBF-Hb or HBS-BSA caused epithelial damage (1.03+/-0.17 vs. 1.07+/-0.26). Mast cell degranulation per villus (MCD) was measured in 60 villi per rat. In non-Se rats, MCD was significantly greater after DBBF-Hb than after HBS-BSA injection (1.83+/-1.42 vs. 0.2+/-0.4). Supplementary Se did not reduce this effect. In fact, MCD was significantly increased in both sets of rats compared to their non-Se counterparts (3.27+/-2.40 and 1.48+/-1.70 for DBBF-Hb and HBS-BSA, respectively). Since mast cell mediators damage cells, Se must protect the mucosal epithelium in some way.