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Effects of Phosphorothioate

Oligodeoxynucleotide on Hemoglobin-inducedDamage to Intestinal Mucosa

Ann L. Baldwin, Lucinda DeMaria, and Elizabeth B. Wiley

Department of Physiology, College of Medicine, University of Arizona,Tucson, Arizona, USA

Abstract: Some blood substitutes, such as human diaspirin cross-linked hemo-globin DBBF-Hb, can damage the intestinal mucosa. This response may be due torelease 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 often consecutive cytidines C-10, reduces Hb-induced ROS generation and damagein the mucosa. Anesthetized Sprague-Dawley rats (46 per group) were injectedarterially 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 sal-ine or PS-ODN followed by saline. Either ROS formation was monitored using afluorescence 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 cellcell and cellbasement membrane separation). Pretreatmentwith PS-ODN significantly exacerbated DBBF-Hb-induced ROS formation, and

PS-ODN groups showed significantly more epithelial damage near Peyers patches,(EI of 1:93 0:06 (SEM) at 8 minutes and 1:31 0:04 at thirty minutes), than thenegative 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, mastcell degranulation, eosinophil accumulation and goblet cell secretion were signifi-cantly reduced in the DBBF-Hb groups pre-treated with PS-ODN. Thus, PS-ODN, although an iron chelator, can significantly enhance epithelial damagecaused by DBBF-Hb in the rat intestinal mucosa near Peyers patches, possiblyby formation of the ferryl component of the hemoglobin.

Keywords: Blood substitutes; Iron chelation; Epithelium; Electron microscopy

Artificial Cells, Blood Substitutes, and Biotechnology, 33: 163186, 2005

CopyrightQTaylor & Francis, Inc.

ISSN: 1073-1199 print/1532-4184 online

DOI: 10.1081/BIO-200055892

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INTRODUCTION

Cell-free hemoglobin-based oxygen carriers, such as diaspirin cross-linked hemoglobin (DBBF-Hb), have been proposed as blood substitutesfor transfusions due to their plasma expansion and oxygen transportcapabilities. DBBF-Hb, produced by the US Army, is similar to DCLHb,the commercial analog that was produced by Baxter Health Care, Inc. Anumber of largely unresolved problems were found during pre-clinicaltrials and development of some of these hemoglobin-based substitutes.Baxter has recently terminated its clinical development due to increasedfatalities in the test group [1]. In addition, numerous animal studies havedemonstrated that the administration of extracellular hemoglobin deriva-tives may lead to a variety of undesirable side effects [25].

Previously, we showed that bolus injection of DBBF-Hb and polyethyl-ene glycol (PEG)-conjugated Hb, another Hb-based blood substitute, causetransient ultrastructural alterations in the intestinal epithelium [6, 7]. Thesechanges include detachment of intestinal epithelial cells from each other,and from the basement membrane, degranulation of mucosal mast cellsand the activation of mucin-secreting goblet cells. The cellular mechanismsby which these deleterious effects occur are not known. However, one possi-bility is that the damage is mediated by reactive oxygen species (ROS) that areproduced as a result of release of free iron from the modified hemoglobin. It iswell known that modified hemoglobins spontaneously autooxidize to methe-moglobin in vivo. For example, in guinea pigs it was shown that methemoglo-bin formation increased linearly up to a plateau of 3040% at 12 hoursfollowing infusion of a hemoglobin conjugated to carboxylate dextran [8].Hydrogen peroxide then stimulates the release of free iron from methemoglo-bin[9]. In addition, free hemin is generated during thenormal catabolic degra-dation of administered hemoglobin, and the release of free iron from hemin is

promoted by H2O2[9]. Evidence for a high rate of hemoglobin catabolisminvivo has been presented by Everse and Hsia [10], who used data from Hess [11]regarding the rate of disappearance of injected cross-linked hemoglobin fromthe circulation, taking into account the rate of extravasation.

If free iron is released in the bloodstream it catalyzes production ofROS by the Fenton reaction [12]:

Fe2 H2O2 ! Fe3 OH OH

The products of this reaction are ferric iron, the hydroxyl radical (_OH) andthe hydroxide ion. Fenton-type oxidations of organic substrates proceed viaaddition of OH or via hydrogen ion abstraction as shown below:

164 Ann L. Baldwin et al.

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There are many examples of hemoglobin-induced tissue damage thathave been shown to result, at least partially, from action of ROS producedby free iron. When the free radicals are inactivated with superoxide dismu-tase and=or catalase, or the free iron is chelated, the damage is reduced.For example, Paller [13] showed that the iron chelator, desferroxamine,reduced renal dysfunction and accompanying ROS-mediated lipid peroxi-dation during heme-protein-induced acute renal failure. In an ovine modelof exchange transfusion, metHb concentrations of a gluteraldehyde-polymerized bovine hemoglobin increased from 3% to 40% in only 24hours [14]. Desferrioxamine also prevents oxyHb-induced endothelial andsmooth muscle cell cytoskeletal injury [15]. However, there is little pub-lished information on the effect of iron chelation on the tissue damageinduced by hemoglobin-based blood substitutes. Such information wouldclarify whether or not the Fenton reaction plays a major role in the oxidat-ive tissue damage caused by hemoglobin-based blood substitutes andwould aid in the development of superior products. If the Fenton reactionis responsible for the damage to the intestine produced by modified hemo-globins, then chelation of the free iron should reduce the damage.

For this reason, experiments were designed to determine whetherintravenous injection of an iron chelator, prior to a bolus injection ofDBBF-Hb, reduced the epithelial disruption, mast cell degranulation,eosinophil accumulation and Goblet cell secretion observed in the intes-tinal mucosa of rats [7]. The chelator used in these experiments was aC-10 phosphorothioate oligodeoxynucleotide (PS-ODN) developed byAVI BioPharma, Corvallis, OR. This product has been shown to havea high affinity for iron and facilitates iron excretion [16]. The reason thatPS-ODN was used instead of the more widely used chelator, desferriox-amine, is that treatment with desferrioxamine is costly, requiresparenteral administration and has side effects associated with its use.

MATERIALS AND METHODS

Hemoglobin Solution

The human hemoglobin cross-linked by bis(3,5 dibromosalicyl)fumarate(DBBF-Hb) was a kind gift from the Walter Reed Army Institute ofResearch, Washington, DC. A spectral analysis of hemoglobin oxidation

products was performed using a Beckman DU640 spectrophotometer.The ratio of oxy (Fe2) to met (Fe3) forms of hemoglobin was calculated

PS-ODN and Hemoglobin Damage to Intestine 165

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giving a total molarity of 160.9 mM, and a total osmolarity of 323.8mosmoles. To increase the concentration of oxy to met Hb, the DBBF-Hbwas reduced by adding sodium dithionite (50 mg=100 ml). The samplewas then immediately applied to a Sephadex G-50 column, and the eluant,recognized as reduced Hb by its cherry-red color, was collected. Spectralanalysis of this solution gave a ratio of 6.8 oxy to met forms of Hb. Allhemoglobin solutions were equilibrated with room air during the experi-ments. However, the concentration of hemoglobin used in these experi-ments was less than onetenth of that found in blood, and in addition,oxygen has a low solubility in water. Therefore, the tissues were not exposedto higher concentrations of oxygen than they would experience in vivo.

Pre-Experimental Treatment of Rats

Male Sprague Dawley rats, weighing 350400 g, were obtained fromHarlan. Monthly serology, bacteriology and parasitology evaluationsare performed on animals from each virus-free barrier at Harlan. The ratswere transported to the animal facility at Tucson VAMC by truck. Theanimal facility is small with a low personnel activity, and monthly testsare performed on sentinel rats. On arrival the animals were housed twoper cage in a room (3 m by 4 m) deliberately chosen so as to be remotefrom noisy air vents and cage washers, etc. The cages were 45 cm longand 24 cm wide and contained standard Harlan sanichip bedding. Tento twenty rats were housed in the room at any given time and no otherrats, apart from those participating in this study, were housed with them.A technician entered both rooms once a day to feed and tend to the rats.The temperature ranged between 72 and 74F, and the humidity was keptbetween 55 and 60%. The rats were fed Harlan Tech Lab 485 rat chow,and placed on a light cycle with lights on between 6:00 a.m. and 6:00 p.m.

Anesthesia

Sprague Dawley rats were pre-anesthetized with 1 mg=kg body weight ofthe following mixture: ketamine hydrochloride (5 ml of 100 mg=ml), ace-promazine maleate (2 ml of 10 mg=50 ml) and xylazine (8 ml of20mg=ml). This was followed by an intraperitoneal injection of sodiumpentobarbital (30 mg=kg). In each rat a tracheostomy was performedfor artificial ventilation.

Procedures


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iron released in the circulation in vivo by a bolus injection of DBBF-Hb.Next (procedure #2), this amount of iron was injected into the circu-lation of rats to compare its effects on cells in the intestinal mucosa withthose previously observed following injection of DBBF-Hb. Effects ofpretreatment with PS-ODN on cellular changes induced by DBBF-Hbwere also determined. Next (procedure #3), the effect of pretreatmentwith the iron chelator, PS-ODN, on production of ROS in intestinalmucosa by DBBF-Hb, was examined. Finally (procedure #4), the reac-tions of PS-ODN with DBBF-Hb, in the presence and absence ofH2O2, were evaluated using spectrophotometry to determine the relativeamounts of different hemoglobin derivatives formed in each case.

Procedure 1. To Determine Amount of Iron Released In Vivo

by Injection of DBBF-Hb

a. Surgical Procedures

After anesthesia, a mid-line abdominal incision was made to expose theaorta. The aorta was cannulated just downstream from the superior

mesenteric artery in a retrograde direction. The free end of the cathetertubing was connected to a reservoir of HBS-2% BSA, pH 7.4, at 37C.DBBF-Hb (50 mg in 5 ml HBS-2% BSA) (6 rats) or HBS-2% BSA alone(6 rats) was injected through a 0.2 micron filter via the aortic cannula.Blood samples (1 ml) were collected from the aortic cannula in hepari-nized vials 15 min. and 30 min. after the injection. The samples wereimmediately centrifuged, the hematocrits noted, and the supernatantsaspirated and frozen. Meanwhile, the animal was prepared for a differentstudy before sacrificing it using an intravenous injection of Beuthanasia.

b. Data Acquisition

In order to determine the concentration of free iron in the samples, a totaliron-binding capacity kit (sigma-Aldrich Fine Chemicals, #565-C) wasused. This technique measures total non-hemoglobin-bound iron,whether free or derived from heme. At acid pH and in the presence ofa reducing agent, transferrin-bound iron dissociates and reacts withferrozine, forming a magenta complex. The color intensity at 560 nm

(37C), read using a spectrophotometer, is proportional to the serum totaliron concentration. A standard curve was constructed using a set of iron


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c. Data Analysis

The amount of free iron present per ml of plasma was determined foreach sample, and the data pooled between animals of a given groupfor each time point, to obtain means and standard deviations. PairedStudent t-tests were performed to determine whether there was a sig-nificant difference between the samples obtained from animals injectedwith DBBF-Hb and those injected with HBS-2% BSA, 15 min. and30 min. after injection. The amount of iron present in the circulation30 min. after a bolus injection of DBBF-Hb, as estimated from theseexperiments, was injected into rats, in the form of iron-dextran, in afurther set of experiments described below (procedure #2) to deter-mine whether its effects on epithelial disruption, mast cell degranula-tion and Goblet cell secretion were similar to those produced byDBBF-Hb.

Procedure 2. Microscopy of Intestinal Mucosa to Assess Mast Cell

Degranulation, Goblet Cell Secretion, Eosinophil Accumulation and

Epithelial Damage


The following study was performed to determine the intestinal tissuedamage 30 min. after intravenous injection of iron-dextran (6 rats),and to compare this with the damage caused by 8- and 30-min. treat-ments with 50 mg DBBF-Hb, with and without pretreatment with PS-ODN (46 rats each). Control animals received either HBS-2% BSAfor 30 min. (6 rats), or PS-ODN for 2 min. followed by HBS-2% BSA

for 30 min. (3 rats). After anesthesia, a mid-line abdominal incisionwas made to expose the aorta. The aorta was cannulated just down-stream from the superior mesenteric artery in a retrograde direction.The free end of the catheter tubing was connected to a reservoir ofHBS-2% BSA, pH 7.4, at 37C. A loop of intestinal ileum, close tothe cecum, was pulled outside the body cavity and arranged on a plex-iglas pillar attached to the plastic stage on which the rat was situated. InPS-ODN experiments, 1 mg PS-ODN in 0.5 ml HBS was injectedthrough a 0.2 micron filter via the aortic cannula, followed two minutes

later by 50 mg (10 mg=ml) DBBF-Hb in HBS-2% BSA, or just HBS-2%BSA. In the remaining experiments, the PS-ODN injection was omitted.


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with Karnovskys fixative in phosphate buffer, pH 7.4, at 4C. Whenperfusion was complete, the inlet pressure was dropped to 40 mmHgand the portal vein was clamped. The animal was killed with an intra-venous injection of Beuthanasia. Fixation continued for 60 minutesafter which the intestinal loop was excised and cut into several segments,each containing a Peyers patch; these segments were washed in bufferedsaline.

b. Tissue Preparation for Histology

Tissue squares were immersed in diaminobenzidine (DAB) overnight inthe dark to stain specific granules in eosinphils, and thus make thecells easier to identify. The DAB was prepared as follows [18]: DAB(0.1 g) was added to 50 ml 0.1 M monobasic phosphate buffer andthe pH was adjusted to 7.2 very gradually with concentrated NH4OH.The solution became a light tannish-pink color. Next, the tissuesquares were rinsed in distilled water. Meanwhile, 25 ml DAB solutionwas added to 1.66ml 3% H2O2 to give final concentration of 0.2%.The tissue was placed in this solution for 60 minutes and then rinsed

three times in 0.15 M sodium cacodylate buffer. Finally, the tissue wasdehydrated in increasing concentrations of ethanol and embedded inSpurrs resin. The pieces of tissue were oriented in the resin so thatthe blocks could be sectioned perpendicular to the villus plane. Thicksections (2 mm) were cut for light microscopy, mounted on slides, andstained with toluidine blue. Ultrathin sections were cut for electronmicroscopy (Phillips CM12). Before examining the sections under elec-tron microscopy, the grids were stained with lead citrate and uranylacetate.

c. Data Analysis

Thick sections were examined under light microscopy to count the num-bers of degranulated mast cells secreting goblet cells, and eosinophils pervillus cross section, in 3060 villi per animal taken from four differentregions of the tissue sample. Only villus sections that contained a centrallacteal were included because these villi were centrally sectioned. Mastcells were considered degranulated if they exhibited empty vacuoles.

Eosinophils were recognized by the DAB-stained granules (reddishtinge), together with the toluidine blue stained cytoplasm. For collection


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separation was assigned 2, and epithelial cells detaching from the base-ment membrane scored 3.

Procedure 3. In Vivo Estimation of Reactive Oxygen Species

in Intestinal Mucosa


Experiments were performed on 10 rats (5 with PS-ODN pretreatmentand 5 without). After anesthesia, the femoral artery was cannulated in

a retrograde direction to blood flow. The free end of the catheter tubingwas connected to a syringe of HBS-2% BSA, pH 7.4, at 37C. Next, amid-line abdominal incision was made and a loop of intestinal ileum,close to the cecum and containing a Peyers patch, was pulled outsidethe body cavity. The segment of intestine was arranged on a plexiglaspillar attached to the plastic stage on which the rat was cauterized long-itudinally and the mucosal surface spread over a plexiglass. The mucosalsegment was kept moist with a HBS- 0.5% BSA drip heated to 37C, andautofluorescence from the tissue was recorded from six to eight villi sur-

rounding the Peyers patch. Next, the HBS-BSA was replaced by HBS-BSA containing 0.001 g=dl dihydrorhodamine 123 (DHR). This sub-stance is not fluorescent until oxidized by a high energy radical. The rateof accumulation of the fluorescent product for a constant suffusate con-centration of DHR provides a quantitative measure of oxidant formationin the tissue [19]. An image of fluorescence from the same villi wasrecorded. This pre-heme-protein image served as a backgroud for laterdigital subtraction. Next, a 0.5 ml bolus of 1 mg PS-ODN in HBS-2%BSA (or just HBS-2% BSA for controls) was injected via the femoral

cannula through a 0.2 mm filter, and a timer was started on the videomonitor. Two minutes later, a 5ml bolus of 10mg=ml DBBF-Hb inHBS-2% BSA was injected by the same route. Every 2 minutes, for thenext 30 minutes, several drops of DHR were applied to the preparation,and an epifluorescent image recorded. During acquisition of images, thefollowing precautions were taken:

i. Each fluorescence observation was limited to a few seconds, afterwhich a barrier filter was applied to block the fluorescence and pre-

vent photobleaching.ii. The quantity of DHR that was dripped on the preparation


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b. Data Analysis

The mean intensity of the epithelial fluorescence of the villi containedin each image was measured using NIH-Image, and the backgroundvalue (DHR before injection) subtracted. Data were pooled amongthe five animals in each group for each time point, and the meansand standard deviations calculated. Similar measurements were madeon the villus lamina propria. The data were then analyzed as describedpreviously.

Procedure 4. Spectrophotometry of PS-ODN and DBBF-Hb In Vitro

Stock DBBF-Hb (200 mg=ml) was diluted 80 in 1ml HBS and awavelength scan from 300700 nm was performed using a Beckmanspectrophotometer. Then, the same dilution of DBBF-Hb was mixedwith PS-ODN to a weight ratio of 50:1, as was used in the in vivoexperiments, and wavelength scans were performed 10 min. and20 min. later. The whole procedure was repeated in the presence of100 mM H2O2. It is known that production of H2O2 by activated neu-

trophils can reach concentrations as high as 100600 mM [20]. Next, theWinterbourn equation [17] was used to calculate the percentages of oxyHb, metHb, and ferryl Hb in each case. Ferryl heme, FeIV, is a highlyreactive species that can peroxidize lipids, degrade carbohydrates andcross-link proteins and is produced by the reaction of oxyHb or metHbwith H2O2 [21].

RESULTS

Procedure 1. To Determine Amount of Iron Released In Vivo

by Injection of DBBF-Hb

The concentrations of non-hemoglobin-bound iron detected in theblood samples taken after bolus injection of 50 mg DBBF-Hb in 5 mlHBS-2% BSA, or HBS-2% BSA alone, are shown in Figure 1. After15 min. there was significantly more iron in the samples as a resultof the DBBF-Hb injection compared to control values (88:07 3:75(SEM) mg=dL vs. 61:36 2:00 (SEM) mg=dL. After 30 min. the former

value had risen slightly, and the latter value had fallen slightly, but notsignificantly. Therefore, in experiments to compare the effect of free


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Procedure 2. Microscopy of Intestinal Mucosa to Assess Mast Cell

Degranulation, Goblet Cell Secretion, Eosinophil Accumulation and

Epithelial Damage

a. Degranulated Mast Cells

Mucosal degranulated mast cells (dmc) were easy to identify by light

microscopy because they stained intensely with toluidine blue and demon-strated empty vacuoles. A comparison of the degree of mast cell degranula-

Figure 1. Graph to demonstrate concentration of free iron in plasma derivedfrom blood samples taken from rats at various times after bolus intravascularinjection of 5 ml DBBF-Hb (50 mg) or isotonic saline. Error bars indicateSEM. After 15 min. there was significantly more iron in the samples as a resultof the DBBF-Hb injection compared to control values (88:07 3:75 (SEM)

mg=dL vs. 61:36 2:00 (SEM) mg=dL, p< 0:05.


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iron-dextran than for the HBS-2% BSA control group. Pre-treatment withPS-ODN significantly reduced mast cell degranulation in animals thatreceived DBBF-Hb for 8 or 30 min. in villi both adjacent to, and removedfrom, Peyers patches. Treatment with iron-dextran for 30 min. producedsignificantly less mast cell degranulation than did DBBF-Hb for the sametime duration. These results indicate that mast cell degranulation in thissystem can at least partially be accounted for by the Fenton reaction.

b. Secreting Goblet Cells

Figure 2. Average number of intestinal mucosal degranulated mast cells per villusfor different treatment. The treatments consisted of a 0.5 ml bolus injection of thefirst substance, followed 2 minutes later by a 5 ml bolus injection of the secondsubstance. All experiments were terminated 30 minutes after the second injection.In all cases, the concentrations of DBBF-Hb, iron dextran and PS-ODN were10mg=ml, 0.9 mg=ml and 1 mg in 0.5 ml, respectively; Significantly larger thansaline control group, p< 0:05; #Significantly smaller than saline=DBBF-Hbgroup at same time point, p< 0:05.


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pre-treatment), with DBBF-Hb for 30 min. (with and without pre-treatment with PS-ODN), or with iron-dextran than for the HBS-2%

BSA control group. Similar results were obtained remote from Peyerspatches except for the DBBF-Hb treatment for 8 min. without PS-ODN. Near Peyers patches, pre-treatment with PS-ODN significantlyreduced goblet cell secretion in animals that received DBBF-Hb for8 min., but significantly increased goblet cell secretion after DBBF-Hbtreatment for 30 min. Treatment with iron-dextran for 30 min. producedeither more goblet cell secretion (near Peyers patches) or the sameamount (away from Peyers patches) as DBBF-Hb for the same time dur-ation. These results suggest that goblet cell secretion in villi near Peyers

patches may be stimulated by the Fenton reaction, but later on, in thepresence of PS-ODN, another mechanism comes into play.

Figure 3. Average number of intestinal mucosal secreting goblet cells per villusfor different treatments as described for Figure 2. In all cases, the concentrationsof DBBF-Hb, iron dextran and PS-ODN were 10 mg=ml, 0.9 mg=ml and 1 mg in0.5 ml; Significantly larger than saline control group, p < 0:05; #Significantlydifferent from saline=DBBF-Hb group at same time point, p< 0:05.


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for 8 min. after pretreatment with PS-ODN were 4:0 2:5 (SD) (n 154),9:6 4:2 (n 84) and 5:7 8:8 (n 37), respectively, where n is equal tothe number of villi. The number of villi was lower for the third groupbecause only some of the tissues were stained with diaminobenzoate, whichis needed to demonstrate eosinophils. All three values were significantlydifferent from each other. Thus, bolus injection of DBBF-Hb increasesthe number of eosinophils found within the lamina propria of intestinalmucosal villi, compared to controls, and pretreatment with PS-ODN sig-nificantly reduces this effect. The reduction in eosinophil recruitmentcaused by PS-ODN is consistent with the decrease in mast cell degranula-tion observed under the same conditions because mast cells, on degranula-tion, produce substances that attract eosinophils [22].

d. Epithelial Damage

Electron micrographs of transverse sections through intestinal mucosafrom rats perfused with HBS-2% BSA showed mostly intact epithelium(E), as seen in Figure 4. An intact mast cell (MC) is also visible. Onthe other hand, a bolus injection of iron-dextran (producing an equiva-lent plasma concentration of iron as that released by a bolus injectionof DBBF-Hb after 30 min. in the circulation) caused extensive epithelialdisruption (Figure 5, near Peyers patch). The epithelial cells were some-times separated from each other at the basal aspects of their intercellular

junctions, and were linked only by long, cytoplasmic protuberances. Inaddition, the subepithelial interstitum was edematous, as evidenced bythe large areas of electron-lucent space that were not seen in control pre-parations. Similar pathologies were seen in animals injected with DBBF-Hb for 8 min. (not shown). Pre-treatment with PS-ODN did not improve

matters 8 min. after injection of DBBF-Hb (Figure 6, away from Peyerspatch). In fact, near Peyers patches, the areas of epithelial disruptionwere even more extensive than without the pretreatment. Thirty min.after bolus injection of DBBF-Hb, there was some repair of theepithelium (Figure 7, away from Peyers patch). The epithelial cells drewcloser together and the cytoplasmic protuberances retracted. However, invilli near Peyers patches, pretreatment with PS-ODN inhibited thisrepair (Figure 8, Peyers patch). In villi removed from Peyers patches,the repair was significantly improved by PS-ODN at this timepoint com-

pared to that observed with DBBF-Hb alone. The average EpithelialIntegrity indices for each group are depicted in Figure 9. Near Peyers


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epithelial integrity (lower E.I.) observed 30 min. after DBBF-Hb injectioncompared to that observed without pre-treatment. These results suggestthat the Fenton reaction is partly responsible for epithelial disruptionin this system, but that near Peyers patches, in the presence of PS-ODN, another mechanism comes into play.

Procedure 3. In Vivo Estimation of Reactive Oxygen Species

in Intestinal Mucosa

Figure 4. Electron microscopy of intestinal mucosa to assess epithelial damage.Typical electron micrograph of transverse section through intestinal villus (awayfrom Peyers patch) from rat injected with a 5 ml bolus of HBS-2% BSA (Hepesbuffered saline containing 2% bovine serum albumin). The epithelium (E) ismostly intact and an intact mast cell (MC) is visible. Capillary cross-sections(C) can also be seen. Villi close to Peyers patches were similar in appearance;Scale bar: 5 microns.


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epithelium, and in the lamina propria, rapidly increased within the22 min. observation period (Figure 10). A similar response was observedafter pretreatment with PS-ODN, except that the level of brightness of thefluorescence was significantly greater for both the epithelium and the lam-ina propria at the time of DBBF-Hb injection and 26 min after injection.

Procedure 4. Spectrophotometry of PS-ODN and DBBF-Hb In Vitro

The percentages of oxyHb, metHb and ferrylHb in samples of DBBF-

Figure 5. Electron microscopy of intestinal mucosa to assess epithelial damage.Typical electron micrograph of transverse section through intestinal villus(Peyers patch area) 30 minutes after injection of rat with a 5 ml bolus of iron dex-tran (0.9 mg=ml). The epithelial cells (E) are separating from each other and fromthe basement membrane (arrows) and show extensive cytoplasmic protrusions.The interstitium, (I) is edematous. C: capillary containing two red blood cells;Scale bar: 5 microns.


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Figure 6. Electron microscopy of intestinal mucosa to assess epithelial damage.Typical electron micrograph of transverse section through intestinal villus (awayfrom Peyers patch) from rat that had been injected with 1 mg PS-ODN in 1 ml,followed 2 minutes later by 50 mg DBBF-Hb in 5 ml. The experiment was termi-nated eight minutes after the DBBF-Hb injection. Similar to the results of injec-tion with iron dextran (Figure 5) and with DBBF-Hb alone (not shown), theepithelial cells (E), with extensive cytoplasmic protrusions, are separating fromeach other and from the basement membrane (arrows). Near Peyers patches,the areas of epithelial disruption were even more extensive than with DBBF-Hb

alone. C: capillary containing two red blood cells. Scale bar: 5 microns.


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Figure 7. Electron microscopy of intestinal mucosa to assess epithelial damage.Typical electron micrograph of transverse section through intestinal villus (awayfrom Peyers patch) from rat that had been injected with 0.5 ml HBS-2% BSA,followed 2 minutes later by 50 mg DBBF-Hb. The experiment was terminated30 minutes after the DBBF-Hb injection. The epithelial cells (E) are much closertogether than after 8 minutes perfusion with DBBF-Hb, and the cytoplasmic pro-tuberances are much less evident, indicating some repair of the epithelial lining.

Villi near Peyers patches were even more intact. Scale bar: 5 microns.


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Figure 8. Electron microscopy of intestinal mucosa to assess epithelial damage.Typical electron micrograph of transverse section through intestinal villus(Peyers patch area) from rat that had been injected with 1 mg PS-ODN in0.5 ml, followed 2 minutes later by 50 mg DBBF-Hb in 5 ml. The experimentwas terminated 30 minutes after the DBBF-Hb injection. Unlike the villi fromanimals injected with HBS-2% BSA for 2 minutes followed by DBBF-Hb for

30 minutes (Figure 7), the villous epithelial cells (E) are separated from each otherand show extensive cytoplasmic protuberances. A degranulated mast cell (DMC)is visible Thus pretreatment with PS ODN appears to inhibit epithelial repair of


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DISCUSSION

The results from this study indicate that release of free iron by DBBF-Hb after bolus injection in rats, causes significant mucosal mast celldegranulation in villi adjacent to Peyers patches, eosinophil accumu-lation in the intestinal villus lamina propria, goblet cell secretion, andepithelial disruption. It is probable that the increased accumulation ofeosinophils in the intestinal mucosa was activated by the release of

eosinophil activating factors from the degranulating mast cells [22].Pre-treatment of rats with an iron chelator, the oligonucleotide PS-

Figure 9. The average Epithelial Integrity indices for each group. The scale rangesfrom 1 to 3; a score of 1 means the cells are intact, 2 means the cells show someseparation from each other, and 3 means there is some separation of cells from thebasement membrane; Significantly larger than corresponding saline controlgroup, p< 0:05; #Significantly different from saline=DBBF-Hb group at sametime point, p< 0:05.


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the DBBF-Hb-induced mast cell degranulation, goblet cell secretion andeosinophil accumulation are mainly triggered by the release of free iron,which subsequently catalyzes the Fenton reaction to produce the

Figure 10. In vivo relative estimation of reactive oxygen species in intestinalmucosa. Histogram to show fluorescence intensity of dihydrorhodamine 123 inthe epithelium (upper panel) and lamina propria (lower panel) of intestinal villiafter injection of DBBF-Hb (5 ml bolus at 10 mg=ml), following pre-treatmentwith saline (0.5 ml HBS-2% BSA for 2 minutes) or chelator (0.5 ml PS-ODN(1 mg) for 2 minutes). On the abscissa is plotted the time after injection ofDBBF-Hb in minutes. Asterisks signify value is significantly greater for chelatorpre-treatment than for saline pre-treatment.

Table 1. Percentages of different oxidation states of DBBF-Hb under variousconditions

Condition OxyHb MetHb FerrylHbDBBF-Hb 86.7% 9.0% 4.3%


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hydroxyl radical (OH). The fact that injection of iron dextran did notcause quite as much mast cell degranulation as was evident 30 min. afterinjection of DBBF-Hb suggests that another mechanism may also havebeen involved in the case of the blood substitute. However, this othercomponent was minor because iron chelation reduced mast cell degra-nulation almost to control levels.

Although treatment with PS-ODN significantly reduced mast celldegranulation, goblet cell secretion and eosinophil recruitment, itmarkedly increased epithelial disruption near Peyers patches. Themechanisms responsible for the increased epithelial disruption weresuggested by the in vivo experiments to measure generation of ROSin the epithelium and lamina propria, and by the in vitro spectrophoto-metric measurements. Measurements of the fluorescence intensityproduced by DHR123 showed that generation of ROS for up to6 min. after bolus injection of DBBF-Hb was significantly increasedby pretreatment with PS-ODN, both in the epithelium and in the lam-ina propria. It is known that DHR123 fluoresces in response to H2O2[19]. As explained in a previous publication [24], excess hydrogen per-oxide may accumulate in the intestinal mucosa even in response tointravenous injection of buffered saline, possibly due to dilution ofthe oxidant scavenger, catalase, in the plasma. In addition, H2O2 canbe produced by activated neutrophils [25]. Neutrophils are present inthe lamina propria of intestinal villi and they may be the sourceresponsible for the excess H2O2 that was produced in the presence ofPS-ODN in the lamina propria. In addition, oxidation of Hb to metHbproduces superoxide (O2

), which then reacts with superoxide dismutaseto produce H2O2. A higher rate of auto-oxidation of DBBF-Hb in thepresence of PS-ODN could therefore explain an increased productionof H2O2. In the spectrophotometric measurements, it was found that

metHb production, and hence oxidation of Hb, was increased by PS-ODN in the absence of H2O2. Therefore, perhaps, at the start of thebolus injection of DBBF-Hb when H2O2 in the epithelium is low, thePS-ODN increases the oxidation rate of the DBBF-Hb, forming O2

which is dismutased to H2O2. As the concentration of H2O2 increases,the H2O2 may start reacting with the metHb and oxyHb, transformingthem to ferrylHb, and this may explain why the higher accumulation ofROS in the mucosa of rats pretreated with PS-ODN did not persistpast 6 minutes after injection of DBBF-Hb. This idea is consistent with

the spectrophotometric measurements in which it was found that inthe presence of H2O2, the formation of ferrylHb was enhanced by


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Therefore, injection of DBBF-Hb probably damages the epithelium byproduction of the ferryl form of hemoglobin, and also by release ofOH through the Fenton reaction. On the other hand, the otherresponses to DBBF-Hb injection, such as mast cell degranulation, gob-let cell secretion, and eosinophil accumulation, appear to be mediatedby the Fenton reaction.

Injection of PS-ODN without DBBF-Hb also caused some cellulardamage, although far less than when in the presence of DBBF-Hb. It ispossible that this damage was mediated by activation of complement.In monkeys, high concentrations of oligonucleotides have caused acti-vation of the alternative complement pathway [26]. The complement sys-tem provides the first line of defense against foreign cells or particles,ensuring their phagocytic removals. When complement C3a is formed,it equilibriates rapidly across capillaries due to its low molecular weight(9000). The peak concentration in blood is reached in 15 minutes, andthen it rapidly declines to normal in 60 minutes. This time course is simi-lar to that of the intestinal ultrastructural responses to PS-ODN reportedin the present study.

In summary, it appears that iron chelators may be useful in reduc-ing mast cell degranulation and subsequent eosinophil migration intothe intestinal tissue following bolus injection of DBBF-Hb. Mast celldegranulation is a hallmark of the inflammatory response, and releaseof mast cell mediators, such as histamine and leukotrienes, can increasemicrovascular permeability, resulting in edema and in loss of selectivityof the endothelial barrier, compromising the molecular exchangebetween blood and tissue. In addition, some mast cell mediators recruiteosinophil into the tissue and the eosinophils cause further mast celldegranulation, resulting in a viscous cycle. The observed reduction ofmast cell degranulation and eosinophils recruitment by pre-treatment

with the iron chelator, PS-ODN, should therefore help to inhibit theinflammatory reaction. However, this study has demonstrated that tomaintain epithelial integrity after bolus injection of DBBF-Hb in villinear Peyers patches, iron chelation is insufficient, and it appears tobe necessary to reduce formation of ferrylHb. The villi near Peyerspatches may be more susceptible to epithelial damage than those inother regions because of the presence of large numbers of inflammatoryand immune cells, such as macrophages, neutrophils, eosinophils andlymphocytes. These cells produce H2O2 when activated, which converts

hemoglobin to its ferryl form, particularly in the presence of PS-ODN.Several direct strategies are emerging aimed at cycling ferryl back to ferric


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ACKNOWLEDGEMENTS

We are grateful to BioPharma, Inc., Corvallis, OR, for providing thePS-ODN. This work was supported by National Heart, Lung and BloodInstitute Grant HL-53047.

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