Effect of astaxanthin on hepatocellular injury following ischemia/reperfusion

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Contents lists available at ScienceDirect

Toxicology

journa l homepage: www.e lsev ier .com/ locate / tox ico l

ffect of astaxanthin on hepatocellular injury following ischemia/reperfusion

ulten D. Cureka, Aysegul Corta, Gultekin Yucela, Necdet Demirb, Saffet Ozturkb, Gulsum O. Elpekc,erna Savasd, Mutay Aslana,∗

Department of Biochemistry, Akdeniz University Medical School, Antalya 07070, TurkeyDepartment of Histology, Akdeniz University Medical School, Antalya 07070, TurkeyDepartment of Pathology, Akdeniz University Medical School, Antalya 07070, TurkeyDepartment of Pathology, Ankara University Medical School, Ankara 06100, Turkey

r t i c l e i n f o

rticle history:eceived 1 September 2009eceived in revised form 3 November 2009ccepted 3 November 2009vailable online xxx

eywords:staxanthiniverschemia–reperfusionxidative stress

a b s t r a c t

This study investigated the effect of astaxanthin (ASX; 3,3-dihydroxybeta, beta-carotene-4,4-dione),a water-dispersible synthetic carotenoid, on liver ischemia–reperfusion (IR) injury. Astaxanthin(5 mg/kg/day) or olive oil was administered to rats via intragastric intubation for 14 consecutive daysbefore the induction of hepatic IR. On the 15th day, blood vessels supplying the median and left lateralhepatic lobes were occluded with an arterial clamp for 60 min, followed by 60 min reperfusion. At theend of the experimental period, blood samples were obtained from the right ventricule to determineplasma alanine aminotransferase (ALT) and xanthine oxidase (XO) activities and animals were sacrificedto obtain samples of nonischemic and postischemic liver tissue. The effects of ASX on IR injury were eval-uated by assessing hepatic ultrastructure via transmission electron microscopy and by histopathologicalscoring. Hepatic conversion of xanthine dehygrogenase (XDH) to XO, total GSH and protein carbonyl levelswere also measured as markers of oxidative stress. Expression of NOS2 was determined by immunohis-tochemistry and Western blot analysis while nitrate/nitrite levels were measured via spectral analysis.

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Total histopathological scoring of cellular damage was significantly decreased in hepatic IR injury follow-ing ASX treatment. Electron microscopy of postischemic tissue demonstrated parenchymal cell damage,swelling of mitochondria, disarrangement of rough endoplasmatic reticulum which was also partiallyreduced by ASX treatment. Astaxanthine treatment significantly decreased hepatic conversion of XDHto XO and tissue protein carbonyl levels following IR injury. The current results suggest that the mecha-nisms of action by which ASX reduces IR damage may include antioxidant protection against oxidative

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. Introduction

Ischemia–reperfusion injury of the liver is an important clinicalroblem in many clinical conditions such as liver transplantation,epatic surgery for tumor excision, trauma and hepatic failure afteremorrhagic shock (Lemasters and Thurman, 1997; Olthoff, 2001).artial or, mostly, total interruption of hepatic flow is often neces-ary when liver surgery is performed. This interruption of bloodow is termed as “warm ischemia” and upon revascularization,hen molecular oxygen is reintroduced, the organ undergoes arocess called “reperfusion injury” that causes deterioration of

Please cite this article in press as: Curek, G.D., et al., Effect of astaxanthin(2009), doi:10.1016/j.tox.2009.11.003

Urgan function (Hasselgren, 1987). Although the mechanisms byhich organ damage occurs in I/R injury are incompletely under-

tood, it has been suggested that reperfusion of the liver followingschemia, triggers the activation of kupffer cells causing oxygen

∗ Corresponding author. Tel.: +90 242 2496891; fax: +90 242 2274482.E-mail address: [email protected] (M. Aslan).

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300-483X/$ – see front matter © 2009 Published by Elsevier Ireland Ltd.oi:10.1016/j.tox.2009.11.003

© 2009 Published by Elsevier Ireland Ltd.

free radical formation, production of tumor necrosis factor-� (TNF-�) and interleukin-1 (IL-1) (McCord, 1985; Adkison et al., 1986;Carden and Granger, 2000). Elevated levels of the pro-inflammatorycytokines TNF-� and IL-1 promote PMN recruitment and activationwhich also generates reactive oxygen species (ROS) and leads tothe release of proteases (Colletti et al., 1996; Jaeschke et al., 1990).Although increased expression of NOS2 and elevated tissue nitrite(NO2

−) and nitrate (NO3−) occurs in animal models of I/R (Isobe et

al., 2000) formation of ROS can scavange NO produced by the sinu-soidal endothelial cells and lead to vasoconstriction and narrowingof the sinusoidal lumen (Aslan and Freeman, 2002).

Reports showing that antioxidant deficiency exacerbate I/Rinjury and the beneficial outcome of pharmacological interventionssuch as superoxide dismutase, catalase, vitamin E, or desferriox-

on hepatocellular injury following ischemia/reperfusion. Toxicology

amine, support ROS as a major pathophysiological component of 49

I/R (Atalla et al., 1985; Marubayashi et al., 1986; Drugas et al., 50

1991). 51

Astaxanthin is a naturally occurring carotenoid pigment and is 52

a powerful biological antioxidant (Palozza and Krinsky, 1992). It 53

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Samples were prepared for transmission electron microscopy (TEM) as previ- 173

ously described (Yücel et al., 2006). Tissues were prefixed by immersion in 2.2% 174

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Fig. 1. Structure of astaxanthin (3,3′-dihydroxybeta, beta-carotene-4,4-dione).

xhibits free radical scavenging activity and protects against lipideroxidation and oxidative damage to cell membranes, cells, andissues (Lim et al., 1992). Astaxanthine has a molecular structureimilar to that of �-carotene. However, it has 13 conjugated doubleonds, in contrast to 11 in �-carotene, which gives it significantlyreater antioxidant capacity (Shibata et al., 2001). Moreover, ASXas hydroxyl groups in the 3 and 3′ positions, making the moleculeighly polar (Fig. 1) and dramatically enhancing its membrane

unction to protect against degenerative conditions (Shibata et al.,001). Because of its polar end groups, ASX spans the cell mem-rane bilayer allowing it to sit near the lipid/water interface, whereree radical attack first occurs and contributes to cell membrane

echanical strength (Palozza and Krinsky, 1992). Astaxanthin sta-ilizes free radicals by adding them to its long double-bond chainather than donating an atom or electron to the radical. Conse-uently, it can resist chain reactions that occur when a fatty acid

s oxidized, thus allowing it to scavenge or quench longer thanntioxidants that cannot stop this chain reaction (Kurashige et al.,990).

The liver has effective mechanisms for inactivating and thenxcreting foreign substances through biotransformation. Theseunctions can lead to significant release of free radicals and oxida-ion byproducts and therefore it is important to have mechanismshat protect liver cells against oxidative damage. It has been shownhat astaxanthin is much more effective than vitamin E in pro-ecting mitochondria from rat liver cells against lipid peroxidationGuerin et al., 2003; Kurashige et al., 1990).

Previous studies on animal models of IR-induced myocardialnjury have shown a linear correlation between the plasma con-entrations of astaxanthin and the extent of infarct size reductionGross and Lockwood, 2004, 2005) Based on this information, theurrent experimental protocol was designed to determine whetherhe tissue protective effect of ASX could be observed in a rat modelf liver IR injury. We aimed to determine whether ASX, a potentntioxidant, had any effect on plasma levels of liver enzymes, tis-ue markers of oxidative stress and histopathologic alterations inn experimental rat model of hepatic I/R.

. Materials and methods

.1. Animals

All experimental protocols conducted on rats were performed in accordanceith the standards established by the Institutional Animal Care and Use Commit-

ee at Akdeniz University Medical School. Male Wistar rats weighing 350–450 gere housed in stainless steel cages and given food and water ad libitum. Animalsere maintained at 12 h light–dark cycles and a constant temperature of 23 ± 1 ◦C

t all times. Rats were randomly divided into two groups of 12 animals each whicheceived olive oil or 5 mg/kg/day ASX (3,3-dihydroxybeta, beta-carotene-4,4-dione;igma–Aldrich Chemie, Steinheim, Germany) dissolved in olive oil via oral gavageor 14 consecutive days before the induction of hepatic I/R.

.2. Rat model of hepatic ischemia–reperfusion injury

Animals were fasted 12 h before surgery, but allowed to drink tap water adibitum. Rats were anesthetized intraperitoneally with a mixture of ketamine


25 mg/kg, Richter Pharma AG, Wels, Austria) and xylazine hydrochloride (8 mg/kg,lfasan International B.V., Woerden, Holland). A model of lobar (70%) hepatic warm

schemia was performed according to a method previously described (Jaeschke etl., 1990; Karaman et al., 2006). After shaving and disinfecting the abdomen withetadine, a complete midline incision was made. The portal vein was exposed andessels supplying the median and left lateral hepatic lobes were clamped for 60 min.

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Reperfusion followed for 60 min via removal of the microvascular clip. The caudaland right lobes retained an intact portal and arterial blood flow, in addition to venousoutflow. These lobes served as control and also prevented intestinal congestion. Theabdomen was kept closed throughout the experimental period and body temper-ature was maintained by placing rats under warming lamps. Blood samples wereobtained before and after the experiment, from the tail vein and the right ventricle,respectively. At the end of the experimental period, liver was perfused with 0.9%NaCl injected from the left ventricle en route for inferior vena cava. Tissue samplesobtained from the left and median lobes of the liver accounted for I/R while dis-sected right lateral and caudate lobes served as nonischemic group. Obtained livertissues were either flash frozen in liquid nitrogen and stored at −70 ◦C; or fixed forhistological evaluation.

2.3. XO/XDH and ALT enzyme-activity measurements

All tissues were weighed and homogenized in ice-cold 50 mmol/L sodium phos-phate buffer (pH 7.4). Homogenates were centrifuged (40,000 × g for 30 min at 4 ◦C),and supernatants were stored at −80 ◦C. XDH and XO activity were determined witha fluorometric assay described previously (Beckman et al., 1989; Aliciguzel et al.,2003). This assay is based on the conversion of pterin to the fluorescent productisoxanthopterin and is performed with and without methylene blue to determineXO/XDH activity and XO activity, respectively. Enzyme activities are calculated onthe basis of the linear increase in fluorescence. Plasma alanine aminotransferaseactivity was measured on an automated analyzer (Roche/Hitachi Diagnostic Sys-tems).

2.4. Histopathological evaluation of liver sections

Paraffin sections stained with hematoxylin and eosin were evaluated by twopathologist blinded to the experimental condition. 20 high-power fields (HPF, 200×)were evaluated in all sections for congestion, intracellular edema and necrosisas previously described (Yilmaz et al., 2004). Congestion and intracellular edemawas scored as follows: 0 = none, 1 = present in zone III, 2 = present in zones II–III,3 = present in zones I–II–III. Necrosis was scored as follows: 0 = none, 1 = singleor focal necrosis, 2 = submassive necrosis, 3 = massive necrosis + infarction. Totalhistopathological score was obtained by summation of all scores given to eachparameter.

2.5. Immunohistochemical staining

Liver tissues were fixed in 10% buffered formalin solution, washed in phos-phate buffered saline (pH 7.4), embedded in paraffin and cut into 4-�m sections.Peroxidase staining was performed as previously described (Aslan et al., 2007).Briefly, sections were deparaffined, rehydrated and washed with Tris bufferedsaline. Endogenous peroxidase activity was blocked by incubating tissue sectionswith 3% hydrogen peroxide for 5 min prior to application of the primary antibody.Primary antibody incubations were at 25 ◦C for 60 min using rabbit polyclonal anti-NOS2 (1:100 dilution, Santa Cruz Biotechnology, Santa Cruz, CA). After sectionswere washed they were immunostained with an avidin–biotin complex kit (Dako,Carpinteria, CA) followed by hematoxylin counterstaining. Negative controls wereperformed by replacing the primary antibody with nonimmune serum followed byimmunoperoxidase staining. Presence of a red-brown colored end-product in thecytoplasm was indicative of positive staining. Counterstaining with hematoxylinresulted in a pale to dark blue coloration of cell nuclei. All stained tissue sectionswere visualized via light microscopy (Olympus IX81, Tokyo, Japan).

To obtain a quantitative standard for NOS2 immunostaining within the differentexperimental groups morphometric analysis was performed as previously described(Yücel et al., 2005). The immunostaining scores, obtained according to both the per-centage and intensity of positive stained cells, were statistically analyzed by SigmaStat (version 2.03) software for windows. The percentage of the positive stained cellswas scored as follows: 0 = less than 5% of the cells/high powered field (HPF, 40×)are stained, 1 = 5% to less than 30% of cells/HPF are stained. 2 = 30% to less than 50%of cells/HPF are stained. 3 = more than 50% of cells/HPF are stained. The intensity ofstaining within each counted cell was also scored as follows: 0 = no staining, 1 = weakstaining (pale red-brown), 2 = moderate staining (red-brown), 3 = strong staining(dark red-brown). A final immunostaining score was obtained for all sections byadding the two scores.

2.6. Transmission electron microscopy


glutaraldehyde in 0.1 mol/L (pH 7.4) Sorensen’s phosphate buffer for 4 h and post- 175

fixed in 1% osmium tetroxide in 0.1 mol/L (pH 7.2) sodium phosphate buffer. The 176

samples were dehydrated in graded ethyl alcohol series and embedded in Araldite 177

CY212. Ultrathin sections (70 nm) were prepared (Leica Ultracut UCT) and con- 178

trasted with uranyl acetate and lead citrate for examination by transmission electron 179

microscope (LEO 906E, Oberkochen, Germany).

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Table 1Plasma and liver activity of alanine aminotransferase, xanthine oxidase and xanthine dehygrogenase. ALT, alanine aminotransferase; XO, xanthine oxidase; XDH, xanthinedehygrogenase. All values are mean ± SEM. Statistical analysis was performed by Kruskal–Wallis one-way analysis of variance on ranks and all pairwise multiple comparisonswere via Dunn’s method.

Control IR ASX ASX + IR

Liver XO (mU/g protein) 16.8 ± 2.2 (n = 8) 22.2 ± 0.86a,b (n = 10) 6.4 ± 0.73c (n = 10) 10.3 ± 0.64c (n = 10)Liver XDH (mU/g protein) 24.2 ± 0.8d (n = 8) 12.3 ± 0.5 (n = 10) 21.8 ± 0.5d (n = 10) 12.2 ± 0.4 (n = 10)Liver XDH:XO (mU/g protein) 1.6 ± 0.2e (n = 8) 0.5 ± 0.03 (n = 10) 3.8 ± 0.5f (n = 10) 1.2 ± 0.1e (n = 10)Plasma XO (mU/mL) 1.98 ± 0.3 (n = 8) 5.4 ± 0.2g (n = 10) 1.47 ± 0.09 (n = 10) 3.3 ± 0.4h (n = 10)Plasma ALT (mU/mL) 43.4 ± 2.8 (n = 8) 4607.6 ± 502g (n = 8) 49.9 ± 2.9 (n = 8) 4263.5 ± 727g (n = 8)

a p < 0.001 vs. ASX and ASX + IR.b p = 0.014 vs. control.c p < 0.01 vs. control.d p < 0.001 vs. IR and ASX + IR.

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e p < 0.05 vs. IR.f p < 0.05 vs. IR and ASX + IR.g p < 0.05 vs. ASX and control.h p < 0.05 vs. ASX.

.7. SDS-PAGE and Western blot analysis

Liver tissue was homogenized in 2 mL ice-cold homogenizing buffer (50 mM2HPO4, 80 �M leupeptin Sigma–Aldrich, Steinheim, Germany), 2.1 mM Pefa-loc SC (SERVA, Heidelberg, Germany), 1 mM phenylmethylsulfonyl fluorideSigma–Aldrich), 1 �g/mL aprotinin (SERVA; pH 7.4). Homogenates were cen-rifuged (40,000 × g, 30 min, 4 ◦C) and supernatants were stored at −80 ◦C untilnalyzed. Western blot analysis was performed as previously described (Aslan andanatan, 2008). Briefly, tissue proteins were separated by SDS-PAGE and transferredo nitrocellulose membranes. A rabbit polyclonal antibody against NOS2 (1:800 dilu-ion; BD Transduction Laboratories, San Jose, CA) was used for immunoblot analysis.


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orseradish peroxidase-conjugated goat anti-rabbit IgG (1:10,000 dilution; Zymedaboratories, San Francisco, CA) was used as a secondary antibody, and immunore-ctive proteins were visualized by chemiluminescence via ECL reagent. Proteinseparated by SDS-PAGE were also visualized by Electro-Blue Staining solution pro-eins were washed, separated by SDS-PAGE, and visualized by GelCode Coomassielue stain reagent (Pierce Chemical Company, Rockford, IL).

ig. 2. Hematoxylin and eosin staining of liver sections. Hepatic photomicrographs ofschemia–reperfusion; CV, central vein. Bar 200 �m.

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2.8. Measurement of tissue protein carbonyl content

Protein-bound carbonyls were measured via a protein carbonyl assay kit (Cat.#1005020 Cayman Chemical, Ann Arbor, MI). The utilized method was based on thecovalent reaction of the carbonylated protein side chain with 2,4-dinitrophenyl-hydrazine (DNPH) and detection of the produced protein-hydrazone at anabsorbance of 370 nm. The results were calculated using the extinction coefficientof 22 mM−1 cm−1 for aliphatic hydrazones and were expressed as nmol/mg protein.

2.9. Measurement of tissue GSH content


EDTotal GSH levels were measured by a commercially available GSH assay kit 204

(Cat. #703002. Cayman Chemical, Ann Arbor, MI). Retina harvested from enucleated 205

globes was homogenized in ice-cold phosphate buffer (50 mM K2HPO4, contain- 206

ing 1 mM EDTA, pH 7). Homogenates were centrifuged (10,000 × g, 15 min, 4 ◦C) 207

and supernatants were deproteinated in 10% metaphosphoric acid (Sigma–Aldrich, 208

Steinheim, Switzerland). The GSSG was reduced to GSH by GSH reductase in the 209

representative rat are shown from each of the four groups. ASX, astaxanthin; IR,

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Table 2Histopathological scores of liver sections. Values are mean ± SD. Statistical analysis was performed by Kruskal–Wallis one-way analysis of variance on ranks and all pairwisemultiple comparison procedures were by Dunn’s method.

Group Congestion (n = 6) Intracellular edema (n = 6) Necrosis (n = 6) Total score (n = 6)

Control 0.17 ± 0.41 0.17 ± 0.41 0 0.33 ± 0.52IR 2.5 ± 0.5* 2.0 ± 0.6* 1.67 ± 0.5* 6.16 ± 0.75*

ASX 0.17 ± 0.41 0 0.17 ± 0.41 0.33 ± 0.52ASX + IR 1.33 ± 1.5 1.7 ± 0.8* 0.8 ± 0.4 3.8 ± 1.7*,**

* p < 0.05; compared to ASX and control.** p < 0.05; compared to IR alone.

Table 3Liver protein carbonyl, GSH and nitrite/nitrate levels. All values are mean ± SEM. Statistical analysis was by one-way analysis of variance with all pairwise multiple comparisonprocedures (Tukey test).

Protein carbonyl GSH Nitrite and nitrate

n = 6 (nmol/mg protein) n = 10 (nmol/mg protein) n = 7–11 (�M)

Control 5.2 ± 1.3 4.8 ± 0.4 2.3 ± 0.3IR 49.9 ± 4.4* 2.7 ± 0.2# 11.7 ± 1.1#

ASX 5.7 ± 0.8 4.8 ± 0.4 2.3 ± 0.5ASX + IR 16.5 ± 1.8** 3.5 ± 0.3** 5.8 ± 0.2##

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* p < 0.001 compared to control, ASX and ASX + IR.** p < 0.05 compared to control and ASX.# p < 0.001 vs. control and ASX.

## p ≤ 0.01 vs. ASX and control.

ssay cocktail of the kit containing DTNB (5,5′-dithiobis-2-nitrobenzoic acid, Ell-an’s reagent), glucose-6-phosphate dehydrogenase, GSH reductase, NADP+ and

lucose-6-phosphate. The sulfhydryl group of GSH reacts with DTNB to give a yel-ow colored 5-thio-2-nitrobenzoic acid (TNB) which is measured at an absorbancef 405 nm. The values of total GSH for each sample were calculated from theirespective slopes using a GSH standard curve.

.10. Nitrite and nitrate assay


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006). Briefly, samples were transferred to an ultrafiltration unit and centrifugedhrough a 10-kDa molecular mass cut-off filter (Amicon, Millipore Corporation, Bed-ord, MA) for 1 h to remove protein. Analyses were performed in duplicate via thereiss reaction using a colorometric assay kit (Calbiochem, Darmstadt, Germany).

ig. 3. Transmission electron micrographs of liver from all experimental groups. ASX, astaeticulum. Bar 2 �m.

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RProtein concentrations were measured at 595 nm by a modified Bradford assay usingCoomassie Plus reagent with bovine serum albumin as a standard (Pierce ChemicalCompany, Rockford, IL).

3. Results


E3.1. ALT, XO and XDH enzyme activity 226

Plasma ALT and XO levels were significantly increased in all IR 227

groups. Treatment of ASX significantly decreased liver XO levels 228

under basal conditions and increased XDH/XO ratio when com- 229

xanthin; IR, ischemia–reperfusion; M, mitochondria; N, nucleus; ER, endoplasmatic

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Cig. 4. (A) Immunostaining of NOS2 in the liver. Hepatic photomicrographs oschemia–reperfusion; CV, central vein. Bar 200 �m. (B) Quantitation of NOS2 inhe different groups were analyzed via Kruskal–Wallis one-way analysis of varianp < 0.05 compared to control and ASX.

ared to both control and IR groups. XDH/XO ratio was greater in/R livers treated with ASX as compared to the non-treated IR groupTable 1).

.2. Histological analysis


Congestion, intracellular edema and necrosis were significantlyreater (*p < 0.05) in IR and ASX + IR groups as compared to ASX andontrol (Fig. 1, Table 2). Although congestion, intracellular edemand necrosis showed no significant difference among IR groupsreated or non-treated with ASX, the total histopathological score

esentative rat are shown from each of the four groups. ASX, astaxanthin; IR,iver. Values are mean ± SD. The differences in the immunostaining score amongranks and all pairwise multiple comparisons were performed by Dunn’s method.

was significantly decreased (**p < 0.05) in ASX treated IR vs. IR alone(Fig. 1, Table 2).

Electron microscopy of postischemic tissue also demonstratedcell damage, swelling of mitochondria, disarrangement of roughendoplasmatic reticulum which was also partially reduced byastaxanthine treatment (Fig. 2).


3.3. Protein carbonyl, GSH and nitrite/nitrate levels 245

Ischemia/reperfusion caused a significant increase in liver pro- 246

tein carbonyl levels in both ASX treated and non-treated groups. 247

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Fig. 5. (A) Coomassie blue staining of liver proteins separated by PAGE. (B) NOS2Woos

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ECestern blot analysis of liver homogenates. 50 ng of NOS2 standard and 45 �g

f total tissue protein was loaded. Western blot analysis revealed an immun-detectable 130 kDa protein band present in IR groups. STD, molecular weighttandards; C, control; A, astaxanthin; IR, ischemia–reperfusion.

SX treatment significantly reduced levels of protein carbonyl for-ation in IR (Table 3). Ischemia/reperfusion significantly decreasedSH levels and caused a significant increase in liver nitrate/nitrite

evels in both ASX treated and non-treated groups (Table 3). ASXreatment had no significant effect on GSH and nitrite/nitrate levelsn IR.

.4. Nitric oxide synthase-2 expression

Fig. 3A shows hepatic photomicrographs representative fromach of the four groups. Increased NOS2 staining was localized tohe pericentral hepatocytes in both IR groups and not evident inhe control and ASX treated groups. The immunostaining scores,btained according to both the percentage and intensity of positivetained cells were statistically increased in IR group rats comparedo control and ASX groups (Fig. 3B). Western blot analysis of hep-tic extracts using an anti-NOS2 antibody confirmed the observedmmunostaining and revealed an immunodetectable 130 kDa pro-ein band present only in IR and ASX + IR groups (Fig. 4A). Fig. 4Bs Coomassie based staining of tissue proteins that were separatedor Western blot analysis and shows equal protein loading in eachane.

. Discussion

This study examined the effect of ASX treatment on liver injuryesulting from I/R. Astaxanthin was dissolved in olive oil anddministered at a dose 5 mg/kg/day via oral gavage for 14 consec-tive days before the induction of hepatic I/R. Plasma appearancend tissue accumulation of ASX was studied previously in rodents


fter single- and multiple-dose regimens (Showalter et al., 2004).ne time dosing at 500 mg/kg resulted in significant appearance ofSX in plasma (0.2 mg/L) and liver (0.9 mg/L) (Fig. 5).

ASX treatment showed no significant effect on plasma XO andLT activity following liver I/R injury, however it did decrease basal

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levels of hepatic XO activity and increased liver XDH/XO ratio in I/R(Table 1). Considering that XOR-specific activity in the liver is muchgreater than in plasma (Kooij et al., 1992), a small amount of enzymereleased from splanchnic tissues can cause a significant increase incirculating plasma levels of the enzyme (Aslan et al., 2001).

Xanthine dehydrogenase (XDH) is a molybdopterin-containingflavoprotein that oxidizes hypoxanthine to xanthine, and xanthineto uric acid. It has two identical subunits containing FAD, molyb-denum, and Fe-S clusters that facilitate electron transfer fromsubstrate to the electron acceptor NAD+ (Enroth et al., 2000). Xan-thine dehydrogenase can be converted to XO by either proteolyticcleavage of the amino terminus or more rapidly by thiol oxidationleading to intramolecular disulfide formation (Enroth et al., 2000;Amaya et al., 1990; McKelvey et al., 1988). Only the dehydroge-nase form of the enzyme can reduce NAD+ and form NADH and XOremains a major source for superoxide (O2

•−) and hydrogen per-oxide (H2O2) production. Treatment of ASX under basal conditionssignificantly decreased liver XO levels and increased XDH/XO ratiowhen compared to control (Table 1). Likewise, treatment of ASXprior to IR injury increased XDH/XO ratio when compared to non-treated IR livers. This effect of ASX is likely due to the preventionof thiol oxidation which will lead to a decrease in intramoleculardisulfide formation. Prevention of intramolecular disulfide forma-tion within XDH will decrease conversion of the enzyme to XO andincrease the XDH/XO ratio as seen in this study.

ASX treatment leads to a slight protection in I/R livers asreflected by significantly lower total histopathological score (Fig. 1,Table 2). As stated previously, the total histopathological scorewas obtained by summation of all scores given to intracellularedema, congestion and necrosis. Although histopathological evalu-ation revealed that intracellular edema, congestion and necrosis inIR livers treated with ASX was greater than control, these findingswere much more severe in non-treated liver tissue that underwentIR. This result is reflected in the total score which is significantlylower in ASX treated livers that underwent IR as compared to non-treated IR livers.

Treatment with ASX significantly decreased protein carbonylformation following I/R injury (Table 3). Protein carbonyl forma-tion is a widely utilized marker for protein oxidation (Stadtman andOliver, 1991). Carbonyls, formed following reactive oxygen species-mediated oxidation of sugar and membrane lipids, are able to formadducts commonly known as CO-proteins (proteins bearing car-bonyl groups) with structural proteins, causing alterations in theirbiological activity (Shacter, 2000). Reactive carbonyl groups on pro-teins can also be formed by direct oxidation of protein side-chains(Reznick and Packer, 1994). Astaxhantin is a potent scavenger ofoxygen radicals and inhibits the oxidation of proteins (Naguib,2000; Palozza and Krinsky, 1992).

As stated in the introduction, neutrophil-sinusoidal endothe-lium adherence observed during liver IR injury is, in part, a responseto reactive oxygen radicals, as well as being a response to cytokineswith chemotactic properties. Activated neutrophils can secreteseveral enzymes, such as MPO and elastase, which are indirectlyinvolved in tissue injury (Weiss, 1989). Treatment of ASX sig-nificantly decreased liver XO levels under basal conditions andincreased XDH/XO ratio in both control and IR groups. ASX treat-ment also significantly reduced hepatic levels of protein carbonylformation following IR injury. The effect of ASX treatment ondecreased XO activity will diminish a major source of hepatic O2

•−

and H2O2 production during I/R and alleviate tissue destructionin response to reactive oxygen radical formation as observed via


histopathological evaluation herein. 340

Increased expression of NOS2 and elevated tissue nitrite (NO2−) 341

and nitrate (NO3−) levels observed herein (Figs. 3 and 4 and Table 3), 342

are in agreement with previous animal models of liver I/R injury 343

(Isobe et al., 2000). In many cases, NOS2 inhibitors significantly 344

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mprove organ function in tissue I/R injury (Isobe et al., 1999). Sim-larly, I/R-induced tissue injury is attenuated in the liver of NOS2−/−

ice (Lee et al., 2001).Although ASX had no significant effect on NOS2 expression and

O2−/NO3

− levels, it still may provide some protection during I/Rnjury by scavenging ROS which can directly react with. NO lead-ng to the formation of secondary species capable of oxidation anditration reactions (Aslan et al., 2003).

Based on reported data it is concluded that ASX treatment canffer limited protection in liver I/R injury by reducing oxidant-nduced protein carbonyl formation and conversion of XDH to XO.he observed effect of ASX on liver enzymes, and oxidative stressas also reflected by a minor protection against histopathologic

lterations.

onflict of interest statement

The authors declare that there are no conflicts of interest.

cknowledgements

This study was supported by a grant (No: 2007.04.0103.002)rom Akdeniz University Research Foundation.

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Effect of astaxanthin on hepatocellular injury following ischemia/reperfusion

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