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Original Article Iron chelation prevents lung injury after major hepatectomyKonstantinos Kalimeris, 1 Constantinos Nastos, 2 Nikolaos Papoutsidakis, 2 Marianna N. Xanthopoulou, 3 George Defterevos, 2 Aliki Tympa, 2 Agatha Pafiti, 4 Ioanna Andreadou, 5 Georgia Kostopanagiotou, 1 Vassilios Smyrniotis 2 and Nikolaos Arkadopoulos 2 1 2nd Department of Anesthesiology, Attikon Hospital, 2 2nd Department of Surgery and 4 1st Department of Pathology, Aretaieion Hospital, University of Athens School of Medicine, 3 Department of Science of Nutrition-Dietetics, Harokopio University, and 5 Department of Pharmaceutical Chemistry, University of Athens School of Pharmacy, Athens, Greece Aim: Oxidative stress has been implicated in lung injury fol- lowing ischemia/reperfusion and resection of the liver. We tested whether alleviating oxidative stress with iron chelation could improve lung injury after extended hepatectomy. Methods: Twelve adult female pigs subjected to liver ischemia for 150 min, 65–70% hepatectomy and reperfusion of the remnant liver for 24 h were randomized to a desfer- rioxamine (DF) group (n = 6) which received i.v. desferriox- amine to a total dose of 100 mg/kg during both ischemia and reperfusion, and a control (C) group (n = 6). We re- corded hemodynamic and respiratory parameters, plasma interleukin-6 and malondialdehyde levels, as well as liver malondialdehyde and protein carbonyls content. Total non- heme iron was measured in lung and liver. Pulmonary tissue was evaluated histologically for its nitrotyrosine and protein carbonyls content and for superoxide dismutase (SOD) and platelet-activating factor acetylhydrolase (PAF- AcH) activities. Results: Reperfusion of the remnant liver resulted in gradual deterioration of gas-exchange and pulmonary vascular abnor- malities. Iron chelation significantly decreased the oxidative markers in plasma, liver and the lung and lowered activities of pulmonary SOD and PAF-AcH. The improved liver function was followed by improved arterial oxygenation and pulmo- nary vascular resistance. DF also improved alveolar collapse and inflammatory cell infiltration, while serum interleukin-6 increased. Conclusion: In an experimental pig model that combines liver resection with prolonged ischemia, iron chelation during reperfusion of the remnant liver is associated with improve- ment of several parameters of oxidative stress, lung injury and arterial oxygenation. Key words: iron regulation, liver resection, liver–lung interactions, remote lung injury INTRODUCTION R EPERFUSION OF THE remnant liver after hepatec- tomy and the subsequent liver regeneration are known to release oxygen free radicals and inflammatory mediators in systemic circulation. 1–3 During this period, the lung, along with other remote organs, is not only exposed to the accompanying oxidative stress and inflammation, but also to the systemic effects of impaired liver function. 3,4 Imposing such challenges to the usually already compromised respiratory system of the patient with liver failure can readily complicate the postoperative course and prove detrimental for the res- piratory reserves and the patient themselves. Previous work from our laboratory showed that inflammation and depletion of surfactant contribute to lung injury following surgical acute liver failure. 5 Inhibiting free radical generation through iron chela- tion with desferrioxamine (DF) in this setting resulted in consistently improved pulmonary parameters and increased survival. 6,7 Administration of DF before liver ischemia/reperfusion (IR) in rats yielded an Correspondence: Professor Vassilios Smyrniotis, 2nd Department of Surgery, University of Athens, “Aretaieion” Hospital, 76 Vassilisis Sofias, Athens 11528, Greece. Email: [email protected] Received 23 November 2009; revision 11 April 2010; accepted 21 April 2010. Hepatology Research 2010; 40: 841–850 doi: 10.1111/j.1872-034X.2010.00682.x © 2010 The Japan Society of Hepatology 841

Iron chelation prevents lung injury after major hepatectomy

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Page 1: Iron chelation prevents lung injury after major hepatectomy

Original Article

Iron chelation prevents lung injury aftermajor hepatectomyhepr_682 841..851

Konstantinos Kalimeris,1 Constantinos Nastos,2 Nikolaos Papoutsidakis,2

Marianna N. Xanthopoulou,3 George Defterevos,2 Aliki Tympa,2 Agatha Pafiti,4

Ioanna Andreadou,5 Georgia Kostopanagiotou,1 Vassilios Smyrniotis2 andNikolaos Arkadopoulos2

12nd Department of Anesthesiology, Attikon Hospital, 22nd Department of Surgery and 41st Department ofPathology, Aretaieion Hospital, University of Athens School of Medicine, 3Department of Science ofNutrition-Dietetics, Harokopio University, and 5Department of Pharmaceutical Chemistry, University of AthensSchool of Pharmacy, Athens, Greece

Aim: Oxidative stress has been implicated in lung injury fol-lowing ischemia/reperfusion and resection of the liver. Wetested whether alleviating oxidative stress with iron chelationcould improve lung injury after extended hepatectomy.

Methods: Twelve adult female pigs subjected to liverischemia for 150 min, 65–70% hepatectomy and reperfusionof the remnant liver for 24 h were randomized to a desfer-rioxamine (DF) group (n = 6) which received i.v. desferriox-amine to a total dose of 100 mg/kg during both ischemiaand reperfusion, and a control (C) group (n = 6). We re-corded hemodynamic and respiratory parameters, plasmainterleukin-6 and malondialdehyde levels, as well as livermalondialdehyde and protein carbonyls content. Total non-heme iron was measured in lung and liver. Pulmonarytissue was evaluated histologically for its nitrotyrosine andprotein carbonyls content and for superoxide dismutase(SOD) and platelet-activating factor acetylhydrolase (PAF-AcH) activities.

Results: Reperfusion of the remnant liver resulted in gradualdeterioration of gas-exchange and pulmonary vascular abnor-malities. Iron chelation significantly decreased the oxidativemarkers in plasma, liver and the lung and lowered activities ofpulmonary SOD and PAF-AcH. The improved liver functionwas followed by improved arterial oxygenation and pulmo-nary vascular resistance. DF also improved alveolar collapseand inflammatory cell infiltration, while serum interleukin-6increased.

Conclusion: In an experimental pig model that combinesliver resection with prolonged ischemia, iron chelation duringreperfusion of the remnant liver is associated with improve-ment of several parameters of oxidative stress, lung injuryand arterial oxygenation.

Key words: iron regulation, liver resection, liver–lunginteractions, remote lung injury

INTRODUCTION

REPERFUSION OF THE remnant liver after hepatec-tomy and the subsequent liver regeneration are

known to release oxygen free radicals and inflammatorymediators in systemic circulation.1–3 During this period,the lung, along with other remote organs, is not onlyexposed to the accompanying oxidative stress and

inflammation, but also to the systemic effects ofimpaired liver function.3,4 Imposing such challenges tothe usually already compromised respiratory system ofthe patient with liver failure can readily complicate thepostoperative course and prove detrimental for the res-piratory reserves and the patient themselves.

Previous work from our laboratory showed thatinflammation and depletion of surfactant contributeto lung injury following surgical acute liver failure.5

Inhibiting free radical generation through iron chela-tion with desferrioxamine (DF) in this setting resultedin consistently improved pulmonary parametersand increased survival.6,7 Administration of DF beforeliver ischemia/reperfusion (IR) in rats yielded an

Correspondence: Professor Vassilios Smyrniotis, 2nd Department ofSurgery, University of Athens, “Aretaieion” Hospital, 76 VassilisisSofias, Athens 11528, Greece. Email: [email protected] 23 November 2009; revision 11 April 2010; accepted 21April 2010.

Hepatology Research 2010; 40: 841–850 doi: 10.1111/j.1872-034X.2010.00682.x

© 2010 The Japan Society of Hepatology 841

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improvement in pulmonary microvascular leakage,associated with decreased tumor necrosis factor (TNF)release.8

The encouraging results from a fairly simple treat-ment to chelate iron in liver failure, prompted us tofurther test the efficacy of iron chelation in preventinglung injury after extended hepatectomy. The hypoth-esis behind our study is that oxidative damage mayconfer significantly to lung injury during liver IR. Forthis reason, we examined systemic, hepatic and pulmo-nary indices of oxidative stress and pulmonary bio-chemical and functional parameters in the first 24 hof liver reperfusion after liver resection in pigs, usingcontinuous i.v. infusion of DF as the antioxidativetreatment.

METHODS

THE PROTOCOL WAS approved by the AnimalResearch Committee of the University of Athens. For

care of animals we followed the national and Europeanguidelines for ethical animal research.

Induction and maintenance of anesthesiaTwelve young adult female domestic pigs (22–25 kg,3–4 months old) were used. General anesthesia wasinduced and maintained according to our previouslypublished protocol,6 except that ventilator settingswere as follows: fraction of inspired oxygen (FiO2) of0.4, tidal volume of 8 mL/kg and respiratory rate of10–15 breaths/min. Ketamine, fentanyl and vecuro-nium bromide were used for maintenance of anesthe-sia as previously described.6 After placement of anasogastric tube, the right external jugular vein wasexposed and a sheath 8.0 Fr (Arrow International,Reading, PA, USA) was introduced to allow insertionof a 7.0-Fr Swan-Ganz thermodilution catheter (Opti-cath; Abbott Critical Care Systems, North Chicago, IL,USA). The right carotid artery was cannulated with an18-G catheter for blood sampling and arterial pressuremeasurement.

Surgical procedure and blood samplingA midline abdominal incision was followed by cystec-tomy and insertion of a Foley catheter. In all animals aside-to-side portacaval anastomosis was performed andthe hepatoduodenal ligament with its contents wasclamped to allow resection of the middle and left

hepatic lobes (65–70% of the liver mass). Ischemia ofthe liver was maintained for 150 min, followed byunclamping of the hepatoduodenal ligament to allowreperfusion of the liver and clamping of the portacavalanastomosis to restore normal portal circulation. Bloodsamples were obtained at 0 h, 6 h, 12 h and 24 h ofreperfusion (beginning from unclamping of the porta-caval anastomosis). Animals were then killed with KCl2 g, propofol 1% 20 mL and vecuronium bromide20 mg, and lung biopsies were obtained from the leftlower lobe for histological analysis and wet/dry mea-surement. Samples from the right liver lobe were alsosnap-frozen in liquid nitrogen and stored at -80°C untilanalysis.

Desferrioxamine protocolAnimals were randomly allocated (closed envelopemethod) to either the control (group C, n = 6) or des-ferrioxamine group (group DF, n = 6). A total dose of100 mg/kg desferrioxamine was diluted in 500 mL dex-trose 5%. Two-thirds of the dose were administratedduring ischemia and the first 6 h of reperfusion and theremaining dose was given in the next 18 h in group DF.Animals in group C received 500 mL dextrose 5% withthe same regimen but without desferrioxamine.

Nitrotyrosine and interleukin-6 (IL-6) analysisFrozen lung tissues samples were homogenized in50 mM HEPES pH 7.5, 150 mM NaCl, 15 mMb-mercaptoethanol, 0.5 mM phenylmethylsulfonylfluoride, 0.1% NP40 (Sigma-Aldrich, St Louis, MO,USA). The homogenate was centrifuged at 1000 g) at4°C for 5 min. The supernatant was collected andadjusted to 1 mg/mL aprotinin, 1 mg/mL leupeptin and1 mg/mL pepstatin A (Merck, Whitehouse Station, NJ,USA). Nitrotyrosine was measured with enzyme-linkedimmunoassay (ELISA) using a Nitrotyrosine kit byHycult Biotechnology (Uden, the Netherlands) accord-ing to manufacturer’s instructions. IL-6 in serumsamples was measured with ELISA using a commerciallyavailable porcine kit (R&D Systems, Minneapolis, MN,USA).

Total non-heme iron assayThe lung and liver non-heme iron content wasmeasured according to Rebouche et al.9 The tissues(~100 mg) were homogenized in cold ultrapure water(1:10 w/v) by sonication (4 ¥ 10 s, 40% maximumpower). The homogenate was centrifuged at 500 g for

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10 min at 4°C and 100 mL of the supernatants, water(blank) and iron standards (2, 4, 6, 8, 10 mg iron/mLultrapure water) were incubated with 100 mL of 1N HCl/10% trichloroacetic acid (v/v) at 95°C for 60 min. Afterthe incubation, the mixture was centrifuged at 10 000 gfor 10 min at room temperature. Eighty microliters ofthe supernatant were mixed with either 80 mL of sampleblank solution (1.5 M sodium acetate and 0.1% [v/v]thioglycolic acid) or 80 mL of chromogen solution(0.508 mM ferrozine, 1.5 M sodium acetate, 0.1% [v/v]thioglycolic acid). After 30 min incubation, the absor-bance was read by a microplate reader at 562 nm. Theiron concentration was calibrated against the iron stan-dard curve and calculated as mg iron per gram of wettissue.

Malondialdehyde analysis in plasma andliver tissuePlasma malondialdehyde (MDA) content was mea-sured as a marker of lipid oxidation according to ourpreviously described protocol.7 Liver tissues were sus-pended in an ice-cold buffer containing 100 mM NaCl,0.5 mM KCl, 3.1 mM CaCl2, 1 mM MgSO4, 0.55 mMKH2PO4 and 50 mM Tris-HCl pH 7.4. The tissues werehomogenized by sonication and the resulting suspen-sion was centrifuged at 500 g for 10 min. The pelletswere discarded and the supernatants were centrifugedat 20 000 g for 20 min. The supernatant was discardedand the pellet (membrane fraction) was suspended inthe aforementioned buffer to a final concentration of10% w/v. The total protein of the membrane fractionwas determined and the MDA content of the livertissue was determined using 100 mg of membraneprotein.

Determination of protein carbonyls in lungand liver tissuesThe lung and liver tissues (~200 mg) were homogenizedin cold 50 mM phosphate buffer, pH 7.2 containing1 mM ethylene diamine tetra acetate (1:10 v/w) by soni-cation (4 ¥ 10 s, 40% maximum power). The homoge-nates were centrifuged at 10 000 g for 15 min at 4°Cand the supernatant was collected. For the eliminationof nucleic acids which interfere with the assay the super-natants were incubated with 1% streptomycin sulfate for15 min at room temperature and then centrifuged at6 000 g for 10 min at 4°C. Protein carbonyls were deter-mined in the supernatants by the method of Levineet al.10

Determination of total superoxidedismutase (SOD), Mn-SOD and Cu/Zn-SODactivity in lung tissueThe lung tissues (~200 mg) were homogenized incold 20 mM HEPES buffer, pH 7.2, containing 1 mMethylene glycol tetraacetic acid, 210 mM mannitol and70 mM sucrose (1:10 v/w) by sonication (4 ¥ 10 s, 40%maximum power). The homogenates were centrifugedat 1500 g for 5 min at 4°C and the supernatant was usedfor the determination of the SOD activities according toMcCord.11

Platelet-activating factor acetylhydrolase(PAF-AcH) in lung tissueThe lung tissues (~200 mg) were homogenized incold 0.1 M Tris-HCl, pH 7.2 (1:10 v/w) by sonication(4 ¥ 10 s, 40% maximum power). The homogenateswere centrifuged at 10 000 g for 15 min at 4°C and thesupernatant was collected for the determination of PAF-AcH. Briefly, 200 mg of the 10 000 g supernatant wereincubated with 150 mM 2-thio-PAF (Cayman Chemi-cal, Ann Arbor, MI, USA) in 100 mM Tris-HCl, pH 7.2(final volume: 160 mL) for 20 min at 37°C. The reactionwas terminated with the addition of 40 mL of 1 mM5,5′-dithiobis-(2-nitrobenzoic acid) which reacts withthe free-sulfhydryl group of the reaction product(2-lyso-thio-PAF) releasing 5-thio-2-nitrobenzoic acidwhose absorbance was read at 405 nm. The absorbanceof proper blanks (without the addition of 2-thio-PAFor supernatant) was subtracted from the absorbance ofthe assay and the DA/min was calculated. The reactionrate of the enzyme was calculated from the formula:PAF-AcH activity (mM/min per mL) = ([DA/min]/10.00 mM-1) ¥ (0.200 mL/Vsample). The specific activ-ity (pM/min per mg protein) was calculated afterdetermining the protein concentration of the superna-tant by the Bradford assay.12

Lung histological scoringSections from the lower lobe of the left lung were fixedwith a buffered formalin solution, cut at 3–5-mm sec-tions and stained with hematoxylin–eosin. The biopsieswere blindly evaluated, according to a previouslydescribed scoring system, appropriately modified toinclude all main features seen,13 which included alveolarcollapse, alveolar and interstitial edema, hemorrhageand infiltration by inflammatory cells. Each lung sectionwas given a score of 0–4 for each parameter.6 Total lunginjury score of each section was calculated by adding the

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separate scores. Additionally, separate specimens fromthe same region were processed at 60°C for 96 h forwet-to-dry determination.

StatisticsData are expressed as mean 1 standard deviation.Analysis of hemodynamic and respiratory parameters(Table 1) during time (inside groups) was made withone-way repeated measures ANOVA and the Bonferronicorrection for multiple comparisons. Mean valuesbetween the two groups at sequential time-points werecompared with multivariate one-way ANCOVA, usingthe baseline values as covariance. Differences betweengroups for parameters measured only at 24 h were ana-lyzed with ANOVA. In all tests, values of P < 0.05 wereconsidered statistically significant.

RESULTS

Hemodynamic and respiratory parameters

MEAN ARTERIAL PRESSURE decreased in group Cwithout differing from group DF, while heart rate

was lower at 12 h in group DF. PaO2 decreased earlierand to lower levels in group C, being significantly lowercompared to the DF group both at 0 and 24 h (Table 1).PaCO2 steadily increased but did not differ betweengroups. Mean pulmonary artery pressure and pulmo-

nary vascular resistance increased in both groups, butpulmonary vascular resistance was significantly lower at0 h and 24 h in group DF. Pulmonary capillary wedgepressure was not altered in either group.

Systemic biochemical indicesSerum IL-6 increased in group DF at 12 and 24 h afterreperfusion, being significantly higher at 12 h comparedto controls (Fig. 1). Total bilirubin and lactate dehydro-genase increased in controls but not in the DF group,being significantly lower compared to group C. Levelsof g-glutamyl-transferase were also lower in group DFat 6, 12 and 24 h (Table 2).

Total non-heme ironGroup DF exhibited significantly lower total non-hemeiron levels in lung and liver tissues compared to controls(Fig. 2).

Oxidative stress markersPlasma MDA levels at 24 h post-reperfusion were sig-nificantly lower in group DF compared to group C(Fig. 3).

In liver tissue, both MDA content and protein carbo-nyls were significantly lower in group DF compared togroup C (Fig. 4).

Table 1 Respiratory parameters in control (C) and desferrioxamine (D) groups

Parameter Group Baseline (I) 0 h (II) 6 h (III) 12 h (IV) 24 h (V)

PF ratio (kPa) C 69 1 15 49 1 15 47 1 16 41 1 15i 27 1 11I,ii,iii

DF 72 1 8 68 1 8a 60 1 10i,II 58 1 13ii 47 1 14I,ii,a

PaCO2 (kPa) C 5.0 1 0.3 4.8 1 1.2 5.2 1 1.2 4.7 1 0.5 6.4 1 0.5I,ii,IV

DF 4.4 1 0.5 4.8 1 1.3 5.3 1 1.2 4.9 1 0.4 6.5 1 1.5pH C 7.43 1 0.05 7.49 1 0.08 7.46 1 0.09 7.47 1 0.07 7.39 1 0.08iv

DF 7.48 1 0.07 7.42 1 0.06 7.44 1 0.02 7.39 1 0.04 7.29 1 0.19MAP (kPa) C 13.8 1 2.1 11.5 1 2.9 10.9 1 1.7 9.3 1 1.3 8.8 1 1.6i,ii

DF 14 1 3.2 11.2 1 1.7 9.9 1 2.5 10.5 1 1.6 9.6 1 3.2HR (b.p.m.) C 90 1 7 110 1 19 105 1 17 121 1 21 133 1 21

DF 107 1 24 112 1 41 111 1 17 104 1 37a 120 1 18MPAP (kPa) C 2.1 1 0.3 2.4 1 0.3 2.7 1 0.5 2.8 1 1.1 3.5 1 0.3I,II,III

DF 2.4 1 0.1 2.4 1 0.3 2.4 1 0.4 2.9 1 0.3I,ii,iii 3.3 1 0.8PCWP (kPa) C 1.2 1 0.3 1.2 1 0.1 1.2 1 0.1 1.2 1 3 1.3 1 0.3

DF 1.3 1 0.3 1.2 1 0.3 1.5 1 3 1.5 1 0.3 1.3 1 0.3PVR (dynes s-1 cm-5) C 129 1 68 163 1 29 277 1 192 128 1 67 434 1 100I,II,IV

DF 148 1 43 90 1 47a 122 1 58 175 1 50ii 227 1 165a

PF ratio, PaO2/FiO2 ratio; MPAP, mean pulmonary artery pressure; PCWP, pulmonary wedge pressure; PVR, pulmonary vascularresistance; DAPP, dynamic airway plateau pressure. Data are expressed as mean 1 standard deviation. aP < 0.05 between groups at thegiven time-point; iP < 0.05 compared to time-point I (baseline) inside the same group; iiP < 0.05 compared to time-point II (0 h) insidethe same group; IIP < 0.01 compared to time-point II (0 h) inside the same group; iiiP < 0.05 compared to time-point III (6 h) inside thesame group; IVP < 0.01 compared to time-point IV (12 h) inside the same group.

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Lung tissue analysis revealed lower nitrotyrosinecontent and protein carbonyls in group DF compared tocontrols (Fig. 5).

Pulmonary enzymesSuperoxide dismutase activity in lung tissue was signifi-cantly lower in group DF compared to group C (Fig. 6).This applied to both Mn-SOD and Cu/Zn-SOD, and forthe total SOD activity. PAF-AcH in lung tissue was alsolower in group DF (Fig. 7).

Lung histologyThe most prominent changes in lung histology werealveolar collapse and infiltration by neutrophils andlymphocytes. Hemorrhage and edema were alsocommon in histology. Animals that received DFrevealed significantly improved alveolar collapse and

Table 2 Liver tests

Parameter Group 0 h (I) 6 h (II) 12 h (III) 24 h (IV)

TB (mg/dL) C 0.24 1 0.08 0.69 1 0.29i 0.81 1 0.40 1.28 1 0.51i,ii

DF 0.30 1 0.10 0.38 1 0.30b 0.54 1 0.30a 0.26 1 0.16b

LDH (u/L) C 622 1 238 1331 1 621 1338 1 440i 1119 1 369i

DF 742 1 172 766 1 296b 916 1 600a 877 1 318a

AST (u/L) C 374 1 211 697 1 395 976 1 280 921 1 304i

DF 202 1 114 683 1 385 561 1 231b 724 1 473

AST, aspartate transaminase; LDH, lactate dehydrogenase; TB, total bilirubin. aP < 0.05 between groups (C, controls; DF,desferrioxamine) at the given time-point; bP < 0.01 between groups at the given time-point; iP < 0.05 compared to time-point I (0 h)inside the same group; iiP < 0.05 compared to time-point II (6 h) inside the same group.

0 h0

500

1000

1500

2000

2500

6 h

Time

#

**

Seru

m IL-6

(pg/m

L)

12 h 24 h

C

DF

Figure 1 Serum interleukin-6 (IL-6) levels after reperfusion(0 h) of the remnant liver in control (C) and desferrioxamine(DF) groups. Error bars represent standard deviations.*P < 0.05 at 12 h in DF group compared to 0 and 6 h;#P < 0.01 at 24 h in DF group compared to 12 h; †P < 0.05between the two groups at 12 h.

C DF

0

5

10

15

20

25

30

*

Tota

l non-h

em

e iro

n levels

in the liv

er

(μg/g

r of w

et tissue)

C DF

0

10

5

15

20

30

25

35

40

**Tota

l non-h

em

e iro

n levels

in the lung (

μg/g

r of w

et tissue)

Figure 2 Total non-heme iron content of liver and lung tissues at 24 h of liver reperfusion in controls (C) and desferrioxamine-treated animals (DF). *P < 0.05; **P < 0.01. Error bars represent standard deviations.

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C DF

0

0.4

0.8

1.2

1.6

2

*

Pla

sm

a M

DA

(μM

)

Figure 3 Plasma malondialdehyde (MDA) concentration at24 h of liver reperfusion in controls (C) and desferrioxamine-treated animals (DF); *P < 0.05. Error bars represent standarddeviations.

Figure 4 Liver tissue concentrations ofmalondialdehyde (MDA) and proteincarbonyls 24 h after liver reperfusion incontrols (C) and desferrioxamine group(DF); *P < 0.05; **P < 0.01. Error barsrepresent standard deviations.C DF

0

1

2

3

4

5

6

7

8

**

Liv

er

tissue M

DA

(p

mol/μg

pro

tein

)

C DF0

0.51

1.52

3

4

2.5

3.5

4.55

5.5

*

Pro

tein

carb

onyls

in liv

er

tissue

(nm

ol/m

g p

rote

in)

C DF

0

1

2

3

4

5

6

7

8

9

10

11

*

C DF0

20

40

60

80

100

120

140

160

180

*

Pro

tein

carb

onyls

in lung t

issue

(nm

ol/m

g p

rote

in)

Nitro

tyro

sin

e c

onte

nt of lu

ng

(nM

/mg p

rote

in)

Figure 5 Lung tissue concentrations of protein carbonyls and nitrotyrosine 24 h after liver reperfusion in controls (C) anddesferrioxamine group (DF); *P < 0.05. Error bars represent standard deviations.

Total SOD Mn-SOD

C

DF

Cu, Zn-SOD0

5

10

15

20

25

30

35

40

* *

*

SO

D a

ctivity

(units/m

g p

rote

in)

Figure 6 Lung tissue concentrations of total superoxidedismutase (SOD), manganese-dismutase (Mn-SOD), andcopper/zinc-superoxide dismutase (Cu/Zn-SOD) 24 h afterliver reperfusion in controls (C) and desferrioxaminegroup (DF); *P < 0.05. Error bars represent standarddeviations.

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lesser infiltration by inflammatory cells (Table 3, Fig. 8).Lung wet-to-dry ratio did not differ between groups(3.85 1 0.6 in group C, 3.27 1 0.9 in group DF;P > 0.05).

DISCUSSION

THE MAIN POINT of our study is that iron chelationeffectively alleviates lung injury following major

hepatectomy. DF administration significantly decreasednon-heme iron levels in the liver and the lung andresulted in reduced lipid and protein oxidation in theliver, the systemic circulation and the lung. Liver testsrevealed an improvement of liver function when DF wasused, while serum IL-6 levels increased significantly. Inparallel, DF promoted significant decreases in alveolarcollapse and inflammatory infiltration of the lungs,improving gas exchange and pulmonary circulatory

response to reperfusion. The reduced oxidative stressand inflammation in the lungs resulted in a significantdecrease in the reactive oxidant- and inflammatory-induced pulmonary enzymes.

Liver resection can lead to lung injury through variousmechanisms. Hepatic reperfusion causes an oxidativeburst in the injured liver, releasing oxidative moleculesfrom the Kupffer cells.1,2,14 We chose a 150-min durationof liver ischemia because shorter duration in the swine isnot considered to result in injury meaningful for thera-peutic interventions.15 In a previous work, we identifiedthat oxidative stress is an important component of lunginjury during surgical acute liver failure, even when noreperfusion takes place.6 In the present setting, DF sig-nificantly reduced lipid and protein oxidation in theliver, leading to decreased circulating MDA levels. Inaddition, cytokines released during hepatic reperfusionand/or liver failure, spread into systemic circulation,causing a systemic inflammatory response.7,8,16 This isfurther enhanced by bacterial translocation, which hasbeen depicted as an important cause of acute lung injuryafter hepatic reperfusion.17

Both systemic and pulmonary oxidative damage occurfollowing liver IR.14,18 Available iron increases duringinflammatory insults and may contribute to the devel-opment of lung injury.19,20 DF acts by chelating ironand inhibiting the generation of hydroxyl radicalsthrough the Fenton–Haberweiss reaction (Fe+2 + H2O2

→ Fe+3 + OH- + OH).20 In addition, DF directly scav-enges peroxyl radicals and inhibits activity of peroxyni-trite.21 These reactive oxygen and nitrogen species areresponsible for oxidative damage to proteins, lipids andDNA.22 Inhibition of the above iron-mediated oxidativereaction has been shown to protect the lung duringvarious inflammatory insults.6,8,14 In our experiment, DFeffectively reduced total non-heme iron levels in theliver and the lung. Although the evaluation of redox-active iron would be more accurate, sensitive methodsto detect the free-iron pool in tissues are lacking.23 Nev-ertheless, the clearly reduced iron levels in liver and lungtissue, along with the decreased nitrotyrosine andprotein carbonyl content of the lung in the presentexperiment, reveal a protective effect on protein nitra-tion and oxidation. This could be important for pre-servation of proteins, epithelial cells and surfactantfunctions, which are severely compromised duringhepatic IR and liver failure.6,24

A consequence of the oxidative burst in the lung is theupregulation of defensive enzymes. Superoxide dismu-tases, consisting of the Mn-SOD and the Cu/Zn-SOD,are responsible for reducing O-

2 in the mitochondria

C DF

0

700

600

500

400

300

200

100

*

PA

F-A

cH

activity in lung tis

sue

(pm

ol/m

in/m

g p

rote

in)

Figure 7 Platelet-activating factor acetylhydrolase (PAF-AcH)in pulmonary tissue 24 h after liver reperfusion in control (C)and desferrioxamine group (DF); *P < 0.05. Error bars repre-sent standard deviations.

Table 3 Lung histological evaluation (hematoxylin–eosin)

Parameter C DF

Alveolar collapse 3.0 1 1.1 1.5 1 0.8*Edema 1.7 1 0.5 1.3 1 0.5Hemorrhage 1.5 1 1.2 1.8 1 1.0Inflammatory cells 3.2 1 0.8 1.8 1 0.8*Total lung injury score 9.5 1 3.0 6.5 1 2.0

*P < 0.05.

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and the cytoplasm, respectively. Several causes of lipidand protein oxidation, such as bleomycin and ozone,increase pulmonary SOD activity, in a reactive effort tocounteract the increased oxidative burden in the lung.25

Herein, the decreased activities of Mn-SOD and Cu/Zn-SOD after liver reperfusion treated with DF are in accor-dance with the reduced levels of oxidation, showing areduced need for native antioxidant protection.

Interleukin-6 is an important component of inflam-mation after IR and resection of the liver. Increaseddoses of IL-6 during liver IR were shown to protect theliver from damage.26 Suggested mechanisms are notonly the anti-inflammatory effects of IL-6, but alsoTNF-a reduction and hepatocyte proliferation.3 Theimproved liver function tests combined with the

increased IL-6 levels are indicative of the beneficial effectof DF on the liver. More importantly, the increasedlevels of IL-6 may indicate an improved regenerationprocess, because IL-6 is essential for hepatocyte prolif-eration and promotes liver repair.27–29 Therefore, IL-6, byimproving liver function, may be an important media-tor of lung injury prevention by DF in this setting.

Moreover, IL-6 may have protected the lung also byacting on the lung itself. It has been shown that IL-6attenuates lung injury after intratracheal instillation ofendotoxin or hyperoxic injury and that it also reducessystemic inflammation during septic shock.30–32 In addi-tion, downregulation of TNF-a is considered a signifi-cant pathway of IL-6 protection during liver IR.3 Becausereduction of TNF-a was shown to mediate pulmonary

a b

c d

Figure 8 Histological sections of lung tissue from control (a–c) and desferrioxamine (d) showing: (a) extensive atelectatic changes(hematoxylin–eosin [HE], original magnification ¥120); (b) a field of lymphocytic infiltration organized in a lymphoid nodule(HE, original magnification ¥250); (c) exfoliated pneumonocytes (HE, original magnification ¥250); and (d) no remarkablehistological findings.

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protective effects of DF during liver IR, IL-6 may havealso acted through TNF-a to improve pulmonaryparameters in our experiment.8 These anti-inflammatoryeffects of IL-6 could be considered as an additional pro-tective mechanism by which DF protects the lung afterextended hepatectomy.

The main histological findings in this experimentwere the reduced degree of lung infiltration by inflam-matory cells and the improved alveolar collapsewhen antioxidative treatment was applied. The reducedinflammation in the lung could be either a direct effectof DF through an effect on inflammatory cell activity orindirect through an increase of IL-6 levels.30,31 In addi-tion, IL-6 was recently shown to alter lung inflammationso that oxidative stress was decreased.33 Therefore, IL-6may offer a positive feedback mechanism for antioxida-tive protection during lung injury. On the other hand,the suppression of pulmonary PAF-AcH in the context ofreduced oxidative stress and inflammatory cells in thelungs seems to be the consequence rather than a media-tor of the anti-inflammatory effects of iron chelation.Other studies have shown that both lipid oxidation andinflammation induce PAF-AcH release which furtheraggravates oxidative damage.34 Thus, although PAF-AcHdoes not seem to be implicated in the protective actionof DF, its downregulation may itself entail the advantageof decreased lysophosphatidylcholine production, anissue that should be further studied.34 The reducedinflammation and oxidative damage have possibly con-tributed to alveolar cells and surfactant preservation,resulting in decreased atelectasis and improved gasexchange. The improved oxygenation in the DF groupmay have lead to maintenance of pulmonary arterypressure, although the latter could have also resultedfrom reduced formation of inflammatory mediators.35

Although the mild hypercapnia was not improved ingroup DF, one should consider that the animals wereventilated conventionally for over 24 h, so that the clini-cal indices of lung injury were not obscured. Therefore,to some extent, ventilator-induced injury may accountfor the increased wet/dry ratios, hindering ventilationand efficient CO2 elimination, which DF would notbe expected to improve. In addition, increased CO2

production as has been documented following liverreperfusion may have also contributed to the mildhypercapnia.36

In conclusion, iron chelation with DF after hepatec-tomy resulted in improved systemic oxygenation andwas accompanied by reduced inflammation and oxida-tive stress in the liver, the systemic circulation and thelung. We showed that the decreased lung protein nitra-

tion and oxidation and the anti-inflammatory effect ofIL-6 on the lung could be important mediating mecha-nisms. The defensive pulmonary enzymes, SOD andPAF-AcH do not seem to be directly implicated in theprotection offered by DF, at least in the present settingof liver IR, but their downregulation confers further evi-dence that the DF dose used suffices as an intra- andpostoperative antioxidant treatment to attenuate oxida-tive stress in the lung. However, appropriate dose–response experiments should elucidate the optimaldosing scheme of DF. The improved liver functions alsocontributed to less pulmonary damage, because the lungwas exposed to milder liver failure. Although treatmentwith DF provided improved circulatory and respiratoryparameters, our opinion is that the deteriorating state ofthe animals would not allow many animals to survivethe extended ischemia and hepatectomy for longbeyond the 24 h. However, prolonged observationunder intensive care in future experiments could give aninsight into a possible survival benefit from DF in thisexperimental model. In our opinion, the efficacy andsimplicity of the treatment support further research onthe role of DF during hepatic resection surgery and post-operative care.

ACKNOWLEDGMENTS

THIS WORK WAS co-funded by the EuropeanSocial Fund and National Resources (EPEAEK II)

PYTHAGORAS.

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