II

Carmen Solcan,* Mihaela Gogo,* Viorel Floristean,* Bogdan Oprisan,t and Gheorghe Solcan*2

*Faculty of Veterinary lvtedicine. [Iniuers'ity of Agricultural Sci,ences and Veterinary Med'ic'ine, Mihai'|,

Sad,oueaiu Atley 8, tasi, lOO4gg, Romanial and, fFaculty of Med,'ici,ne, Gr. T. Popa Uni,uers'ity of Medi'ci'neand Phnrm,ocy, tJn'iuers'ital'i'i Street 16, Iag'i, 700115, Roman'ia

ABSTRACT The leaves and berries of sea buckthorn(SB; Hi,ppophae rhamno'ides; family Elaeagnaceae) arcmedically claimed as having phytoantioxidant, antiin-flammatory, and anticancerous properties in humans'This study evaluated the hepatoprotective activity ofoil from SB berries against toxicity induced by afla-

toxin E}1 (AFB1) in broiler chickens. The toxicity of

AFB1 led to lower total serum proteins and specificallyreduced albumin (P < 0.001). Serum aspartate amino-transferase increased from 191.14 + 11.56 to 218.80 *13.63 (P < 0.001). When chickens were simultaneouslydosed with AFB1 and an extract of SB berries' sub-sequent histology of the liver showed a significant re-

Key wordsz Hippophae rhamno,id,es, aflatoxin 81, broiler chicken, hepatoprotection, immunohistochemistry

2013 Poultry Science 92:966-974http: //dx.doi.org/10.3382 / ps.2072-02572

INTRODUCTION

Sea buckthorn (SB) berries (Hi'ppophae rhamnoides)contain more than 200 bioactive components, many'

vitamins (including A, 81, B.2, C, E, F, K, and P)'

carotenoids, tocopherols, sterols, fl avonoids, phenolics,

Iipids, ascorbic acid, citric acid, and more than 15 mi-

croelements (including Fe, Mn, B, Al, K, F, Ti, and so

on). The oils are also rich in essential fatty acids, n-3,

n-6, n-7, and n-9 (Rosch et aI., 2003). Sea buckthorn re-

duces liver damage by antioxidant activity (Chauhan et

a1.,2007; Peng et al., 2010; Yang et al., 2011), making

it hepatoprotective (Geetha et al., 2008). In addition,

SB oil has a cytoprotective action on liver damage in-

duced by toxic chemicals such as carbon tetrachloride,

acetaminophen, and ethyl alcohol (Geetha et al', 2008)

The aflatoxin 81 (AFBI) is a secondary metabolite

of the Aspergillus flauus and Aspergi'llus paras'it'icus filn-

gi and is found in grains and other foods and feedstuffs

as a natural contaminant. It is a potent liver toxin and

duction of necrosis and fatty formation compared withchickens treated with AFB1 alone. Immunohistochemi-cal results indicated that COX2, Bcl-2, and p53 werehighly expressed in the liver of AFBl-treated chickensand their expression was significantly reduced by SBoil supplementation. The levels of AFB1 residues in

chickens livers were significantly reduced by SB oil from460.92 * 6.2 nglmL in the AFB1 group to 15.59 + 6.1ng/ml in the AFB1 and SB oil group. These findingssuggest that SB oil has a potent hepatoprotective activ-ity, reducing the concentration of aflatoxins in liver anddiminishing their adverse effectq

an extremely potent mutagen having teratogenic effectsand causing hepatocellular hyperplasia, hepatic necro-

sis, cirrhosis, biliary hyperplasia, and acute liver dam-age in animals (Murphy et al., 2006)' Aflatoxins affect

many species including humans, dogs, pigs, dairy cat-

tle, and chickens (Bruke et al., 2005). Currently, there

are no readily available, scientifically proven therapeu-

tic options, and this study was designed to determine if

SB oil has efficacy as a hepatoprotective action against

aflatoxin-induced liver damage in chickens.

MATERIALS AN D I'IETHODS

Plant Material

The sea buckthorn (Hippophae rhamno'ides) berries

were collected from Iaqi County, Romania, and extract-

ed according to the method described by Kumar et al'

(2011).

Experimental Birds

Ross 308 broiler chickens, 6 d old, 79.8 * 0'75 g'

of either sex, bred in the animal house of our faculty

were used in the study. The animals were maintained at

standa,rd conditions of temperature' humidity, and light

966

@2013 Poultry Science Association Inc.Received .Iune 26, 2012.Accepted December 23' 20L2.lThe authors decla,re that no conflict of interest exists'2Corresponding author: gsolcan@uaiasi.ro

HEPATOPROTECTIVE EFFECT OF SEA BUCKTHORN 967

on a standard pellet diet nith s'ater ad libitum. This

study was approved b1' the local ethical committee.

Materials

AII solvents, chemicals. solution-.. and reagents usedin the study were of analltical grade (Sigma ChemicalsCo.) and AFB1 ELISA kit R from Biopharm (Da'rm-

stadt, Germany). Antibodies to Bcl-2 Oncoprotein, cy-

clooxygenase-2 (COX-2), and p53 protein (DO-7) wereprovided by Vector Laboratories. USA (Burlingame,

CA) and diluted with a Vectastain Elite ABC kit.

Acute Toxicity Model of AflatoxinPoiso n ing and Asses sm entof Hep ato protective Effects

One hundred Ross 308 broiler chickens were selected

when 6 d old and randomly divided in 4 groups (n :

25): 3 experimental (E1, E2, E3) and 1 control' Chick-

ens were housed and maintained in a I2L:L2D cycle

under a constant temperature of 24 t 1"C with free

access to food and drinking vrater' The chickens were

acclimatized to conditions for 1 wk before experiments,and then the experimental groups were treated for aperiod of 28 d as follows:

1) E1 received 54 pg of AFBl/kg of BW per d, bygavage, diluted in sterilized sunflower oil' Pure

crystalline AFB1 (99.5%) was dissolved in chlo-

roform (1 mg/10 mL) and then mixed with 90

mL of sterilized sunflower oil.2) E2 received 54 pg of AFBl/kg of BW per d and

0.6 mL of SB oil/kg of BW Per d;3) E3 received 0.6 mL of SB oil/kg of BW per d'

The BW of the chickens was recorded on d 7, 14,

21, and 28 of treatment, respectively, when 5 chickens

from each group were euthanized by an overdose of

thiopentone sodium. The biood was collected by cord

pnt "t.tt",

and serum was used for estimating biochemi-

cal pa,rameters: total proteins (TP), albumins, globu-

Iinsf aspartate amino transaminase (AST)' and alanine

amino transaminase (AtT). The liver was collected for

mactoscopic, histologic' and electron microscopic ex-

amination.Residues of a,flatoxin from liver samples were deter-

mined by ELISA after the method described by Fernd'n-

dez et al. (1994), at d 7 and 28 of poisoning from all

euthanized chicks.

H i sto p ath o I o gi c al Sfudies

Four samples from each chicken were randomly evalu-ated (at a magnification of 40x, 10 fields).

lmmunohistochemistry

Fixed liver samples were trimmed, embedded inparaffin, and sectioned at 5 ptri Th"se were dewaxed,

and the epitopes in the sections were revealed by be-

ing heated at 95'C in 10 mmol of citrate acid buffer

at pH 6, for 10 min in a microwave, and then were

left at room temperature for 20 min. The slides were

then washed twice in PBS (pH 7.5) for 5 min. Tissue

sections were incubated with primary anti-p53, anti-

COX-2, and anti-Bcl-2 antibodies and diluted 1:100 at

room temperature in a humid chamber for t h. After

being washed with PBS, slides were incubated with the

secondary antibody, horseradish peroxidase goat ant!

mouse IgG, for t h, in a humid chamber, at 4'C, then

washed with PBS and incubated with the 3'3'-diamino-benzidine substrate for 7 min and counter-stained with

Harris hematoxylin, clarified in xylene, and mounted'

The liver sections were examined microscopically and

classified as (-) no lesion, (+) mild, (+*) moderate:(+++) intense, and (**t*) severe.

Electro n Microsc op i c lnvdstigafions

Cell ultrastructure was assessed in fixed liver sam-

ples. Briefly, control and treated liver tissues were fixed

in 2% gluta.raldehyde in 0.1 M sodium cacodylate buf-

fer, pH 7.2, postfixed' in 2% OsO4' dehydrated in an

etha-nol series, and embedded in Epoxy 1510. Ultrathin

sections were prepared on a Reichert OmU3 (Vienna'

Austria) microtome, stained with 1% uranyl acetate

and lTo phosphotungstic acid, and viewed and photo-

graphed with a Phitlips CM 100 transmission electron

microscope. Three or more 300-mesh copper grids were

examined from each sample in a blinded fashion'

Statistical AnalYsis

Results of biochemical blood parameters and AFB1

residues in liver were expressed as mean t SD' The

values were subjected to 1-way ANOVA software' The

variance in a set of data was estimated by Dunnett's

t-test. The minimum level of significance was fixed at

0.05.This study was approved by the local ethical com-

mittee.

RESULTS

Weight Variation of Chickens

slices of liver fiom each chicken (in all groups) Table 1 shows that there was a significant reduction

;;;";";d in 10% buffered neutral fot*uil" (pri of the weight of chickens jn group E1 (AFB1-tt1t"d.)

7.4), paraffin embedded, cut in sections of 5 pm, tiien "oTPryg

*itn tft" control group and group E3 (SB oil

stained with hematoxylin-eosin and observed under a only). The weight of chickens {rom E2 group (treated

low-power microscope for any pathological changes. witir'AFgr and SB oil) was situated between E1 and

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SOLCAN ET AL.

E3 or the control group, being significantly (P < 0.05)increased in comparison with the E1 group.

Blood Biochemistry Analyses

Blood biochemistry analyses are presented in Table2. The AFB1 determined a significant reduction of TP,albumins, globulins and increased the AST activity (P< 0.001) in the El goup (AFB1 treated) in compari-son with the control Broup, E2, and E3 group, suggest-ing that hepatocellular necrosis occured.

The biochemical parameters in chickens from E2group (treated with AFB1 and SB oil) were situatedbetween El and E3, being significantly (P < 0.001)decreased in comparison with the E1 group, suggestingthe hepatoprotective effect of SB oil.

Liver Pathology

The histology of the livers from the control groupof chickens showed normal hepatocytes with well-pre-served cytoplasm, prominent nuclei, nucleoli, and cen-tral veins. In these livers, hepatocyte expression of p53was undetectable by immunohistochemistry; Bcl-2 andCOX-2 were rarely detected.

In chickens from group E1, treated with AFB1 for 2wk, there was intense liver pathology including diffusehydropic degeneration, dilatation of Disse spaces, andvessel congestion. In some sections, there was initiationof new bile duct formation at hepatic triads. Prolif-eration of epithelial cells of the bile duct was observedand hepatocytes showed moderate necrotic changes insome fields. Intense degenerative changes, consistingof severe microvesicular steatosis of hepatocytes, wereobserved by d 28. Other lesions of the liver includedcongestion of sinusoids, focal to multifocal a,reas of he-patocyte granula^r swelling, degenerative and necroticareas, pseudoacina,r reaxrangement with mononuclearcell infiltration, and intense staining for p53, Bcl-2, andCOX-2 expression by hepatic cells (Figwe 1).

In more severe cases, there was foci of proto-onco'genic cells, atrophy of hepatocytes with scattered smalldroplet fat vacuoles, enlargement of Kupffer cells, focalcholestasis, and progressive fibrosis in the periphery ofthe acinus (rather than concentric around the centralvenules). AIso, in Kupffer cells, p53 was variably ex-pressed and Bcl-2 and COX-2 were intensely/widelydetected (Figure 2).

In group E2 (treated with AFB1 * SB oil) on d14, moderately dilated liver sinusoidal spaces were ob-served. There was a mild congestion and cellular aggre'gation around blood vessels. Cytoplasm of hepatocytesshowed mild vacuolation. On d 28, a focal infiltrationwith inflammatory cells and moderate hydropic degen-eration was observed. Congestion of blood vessels athepatic triads was moderate. On d 21 a^nd 28, therewas evidence of lymphoid aggregation in the liver. Resi-dent hepatic stem/progenitor cells were also identifiedin large numbers in livers in this group (Figure 3). Ex-

HEPATOPROTECTIVE EFFECT OF SEA BUCKTHORN 969

Table 2. Effects of diets containing sea buckthorn (SB) oil, aflatoxin 81 (AFBI), or both, on total proteins (TP), albumins (ALB),globulins (GLB), alanine anino transaminase (ALT), and aspa.rtate amino transaminase (AST)

Item TP (g/dl-) ALe (g/dl,) GLB (g/dl) ALT (ru/L) AST (IUIL)

Controld 7d 1 4d21d 2 8

E1 (AFB1)O J

d 1 4d 2 1d 2 8

E2 (AFBI + SB)d 7d 1 4d27d 2 8

E3 (sB)d 7d 1 4d21d 2 8

3.32 + 1.013.29 + 0.893.27 + 1.093.25 + 1.03

2.51 + 0.8r2.42 + 0.98"2.12 + 1.09a1.86 + l .Ma

3.2r + L.2P'b3.01 + 0.98a,b2.91 + 1.118'b2.71 + 1.614'D

3.34 + 2.10b3.41 + 1.81b3.44 + 2.02b'c3.39 + 2.43b,c

1.85 * 0.27r.75 + 0.241.82 + 0.36r.73 * 0.29

1.55 + 0.2541.38 + 0.3541.28 + 0.$a1.08 + 0.244

1.83 * 0.34b1.63 + 0.23a'b1.52 + 0.354'b7.42 + 0.23a,b

1.87 + 0.13b1.76 + 0.34b'c1.84 + 0.38b'c1.73 * 0.29b'c

1.51 + 0.291.40 + 0.221.41 * 0.311.34 + 0.35

0;91 * 0,2940,82 + 0.3841.09 + 0.3941.00 :t 0.244

1.51 + 0.39b1.54 + 0.31b1.09 + 0,35&'b1.28 * 0.25b

1.51 t 0.29b1.60 * 0.40b1.54 + 0.31b'c1.57 + 0.43b'c

62.80 * 2.2459.39 t r.2757.30 + 2.7056.51 + 1.36

48.76 + 1.13a47.40 + 2.65d40.43 + 3.68434.5t + 2.48e

57.36 + 232a,b55.28 * 2.24a'b50.01 + 3.01e'b43.69 + 2.158'b

63.41 + 2.60c59.28 r 3.V2a'c57.47 + 2.80c56.61 + 2.26c

t43.t4 * 5.77149.12 + 10.86145.40 + 10.56176.62 + 9.57

i91.14 + 11.566197.81" + 8.79d209.34 + 11.41a218.80 + 13.684

166.24 * 12.114,b,t74.24 + 9.56s'b17?.10 + 8.580'b152.03 + 12.03b

147.16 4 9;32a,b,c149.24 + 10.61b'c145.20 + 11.98b'c176.10 + t4.44?'b;c

aVery significant differences between control and experimental (E1, E2' E3) groups (P < 0'001)'bVery significant differences between E1 and E2 or E3 groups (P < 0.001).cVery signilicant differences between E2 and E3 groups (P < 0.001).

pression of Bcl-2 was more apparent in other cell types'including inflammatory cells and sinusoidal lympho-cytes. Increa,sed COX-2 expression was associated withincreased amounts of the anti-apoptotic Bcl-2 protein(Figure 3). In this group of chickens, liver expressionof p53, Bcl-2, and COX-2 were all moderate in hepa-tocy'tes and in Kupffer cells. Score lesions were mod-erate. Notably, those changes were less severe in theAFBI+SB oil-treated chickens (group E2) than in theAFBl-treated chickens (group E1; Tables 3 and 4).

In group E3 (SB oil treated), the histology of theIiver was not changed, being similar with that of thecontrol group (Table 4).

Electron Microscopy

The surface of hepatic cells from the control groupwas smooth, with large spherical nucleus and nucleolishowing fibrillo-granular network structure. The cy-toplasm showed a granular appea,rance. There was aprofuse amount of rough endoplasmic reticulum espe-cially around the nuclea,r envelope and between the mi-tochondria (Figure 4).

In chickens treated with SB (group E3), the nuclei ofhepatic cells contained a large amount of electron-lu-cent euchromatin, scattered areas of heterochromatin,scattered and distinct nucleolus. The cytoplasm of thehepatic cells contained a fairly large amount of roughand smooth endoplasmic reticulum with many roundedmitochondria (Figure 4).

On d 21 and 28 of AFB1 exposure (group E1)' theultrastructure of hepatocytes displayed shrinkage of thecytoplasm, condensation of nucleus and large nucleoli'and swollen or vacuolized mitochondria with decreasedor no christae. The rough endoplasmic reticulum (RER)

was enla,rged to surround the mitochondria. The lumenof the bile canaliculi in the E1 group wa,s expanded incompa,rison with the control group due to shortening ofmicrovilli. It was almost impossible to distinguish theother organelles in this group (Figure 5).

When broilers treated with AFB1 were supplementedwith SB oil (E2 group), remarkable changes were ob-served, with differentiation of the hepatic cells ratherthan multiplication. The nuclei of all the hepatic cellscontained small masses of condensed heterochroma-tin distributed throughout the nucleus, and the cellsshowed a large number of mitoclondria. The cytoplas-mic vacuoles were sparse and the fat droplets were notobserved (Figure 6).

The AFB1 residues in liver significantly and progres-sively decreased in the SB oil-treated group (Table 4).

DISCUSSION

The significantly increased BW in E2 group in com-paxison with the E1 group is a sensitive indicator ofthe positive effect of SB oil against toxicity of AFB1.The weight of the chickens that received AFB1 in theirfeed was lowered from 61.04% on d 7 to 54.347o on d28 of the experiment, in comparison with control birds,which is consistent with ea,rlier reports (Ortatatli andOguz, 2001). These changes were largely reversed bythe simultaneous administration of SB oil. These find-ings suggest a protective effect of SB oil against thedeleterious liver effects of AFB1.

Significantly (P < 0.001) reduced serum TP levelscompaxed with controls and compared with E2 groupwas observed in the E1 group. A mechanism of actionof the AFB1 is related to the inhibition of protein syn-thesis, which was previously reported (Kubena et al.,

SOLCAN ET AL,

Figure 1. Liver of chickens in experimerrtal (E)1 group on d 28

of e*fosrr.e to aflatoxin 81. a) Epithelial cells of the bile duct (BC)

protiflration. Bcl-2-positive ceil (arrows; Bcl-2 x 900); b) presence of

necrotic areas lasteiisk; cyclooxygenase-2 x 60]; c) p5&positive cells

(arrows; p53 x'900). Color version available in the online PDF'

1991). Additionally, a decrease in the concentration of

whole plasma proteins and in albumin, globulins have

been proposed as indicatom of the alteration in protein

synthesis observed in aflatoxicosis. This damage mightbe used as arr indicator in early dia,grrosis of aflatoxicosis

Figure 2. Liver of experimental (E)1 group on d 28 of exposure to

aflatoxin 81. a) Foci of prooncogenic cdlls (arrows; cyclooxygenase-2x 60); b) atrophy of hepatocl'tes, enlargement of Kupffer and steliate

cells bcl-z-positive (arrows; Bcl-2 x 900); c) p5&positive cells (a'rrows;

p53 x 900). Color version available in the online PDF.

and thus preventive meaaures could be adopted (Jindai

et al., 1994).The AFB1 induced a divergent effect on the plasma

activity of ALT and AST enzymes ALT decreased in

the AFBl-treated birds and in AFB1 + SB oil-treated

HEPATOPROTECTIVE EFFECT OF SEA BUCKTHORN

Table 3. Report of residues of allatoxin 81 (AFB1) distribution in liver

97r

Day of experiment

2 lL4T01

ControlEl (AFBI)E2 (AFBI + SB3)E3 (SB)

\D2\D\D

ND94.95 + 6.529.82 + 7.r*

ND

ND460.38 + 5.974.52 * 6.7,

ND

ND402.84 + 6.955.59 + 6.1*

ND

ND406.92 * 6.215.59 + 6.1*

ND

1T0 : time zero: day before the beginning of experiment2ND = not detected.3SB : sea buckthorn.xP < 0.05.

groups (E1 and E2). In turn, AFB1 increased the activ-ity of AST in E1 and E2 in comparison with the controlgroup. Manning and Wyatt (1990) aiso reported thatAST was elevated in aflatoxin-intoxicated birds.

The histological lesions of AFBl-treated group arein agreement with previous reports (Mollenhauer et

al., 1989; Ledoux et al., 1999). During liver injury byAFB1, Kupffer cells undergo activation and proliferateunder the influence of activating mediators and anti-apoptotic factors (Moreira, 2007).

Exposure to AFB1 increased synthesis rate of p53that became detectable by immunohistochemistry, butwas reduced by SB oil supplementation. Accumulationof p53 in response to inflammatory stress as well asits contribution to inflammation-induced injury is alsonoted in published data (Schd,fer et al., 2003; Staib etal., 2005). The extranuclear activities of p53 have beenreported to involve the physica,l or functional interac-tions (or both) with certain members of the Bcl-2 pro-

tein family (Chipuk et al., 2004; Jiang et al., 2006).The product of the Bcl-2 proto-oncogene is thoughtto be a negative regulator (suppressor) of apoptosis inmammalian cells. In the liver, in patients with cirrhosis,Bcl-2 has been detected in areas of cholangial prolif-eration and focally in hepatocytes. In normal liver tis-sue, Bcl-2 was rarely detected (Charlotte et al., 1994).Increased COX-2 expression has been associated withincreased amounts of Bcl-2 expression. In vitro studiesdemonstrated that COX-2 may stimulate angiogenesis(Tsujii et al., 1998), a critical step in the progression ofneoplasms to more aggressive grades and in the devel-opment of metastases. Also, COX-2 may regulate theinvasive potential of tumor cells (Fosslien, 2001)' The

COX-2 is characteristically expressed at low levels or

not at all in normal tissues. It is increased in responseto a range of stimuli such as mitogens, hormones, andproinflammatory mediators (Smith and Langenbach,2001).

Our results also showed that the level of AFB1 accu-

mulation in the livers of chickens of the E1 group pro-

gressively increased. In group 82, supplementation of

chickens by SB oil significantly decreased the residues

of aflatoxin by the end of experiment.The potential hepatoprotective role of SB oil may

be associated with its antioxidant constituents such as

selenium, canotene, tocopherol, phenolic compounds,and vitamin E and C working individually or in syn-

ergy. The SB oil has been shown to be effective againstfree radical-induced cellular transformation, and it can

inhibit cytochrome P450-mediated reactions involvedin the formation of reactive metabolites of the hepato-

Figure 3. Liver of experimental (E)2 group on d 28 of exposure to

aflatoxin Bl and sea buckthorn oil. Oval cells (arrow) are thought to

be the stem cells of the liver. They appear only to become active under

certain circumstances [e.g., when hepatocytes cannot repair damage(cyclooxygenase-2 x 90b)]. Color version available in the online PDF'

972 SOLCAN ET AL.

Table 4. Classification of histological and immunohistochemical lesions of the iiver duringthe experi-

mentl

Control E1 (AFBI) E2 (SB + AFBI) E3 (sB)

Hepatocyte cell degenerationHepatocytes necrosisDilatation of Disse sPacesVessel congestionExpression p53Expression Bcl-2Expression COX-2

+

++++

+++++

+++++++++

+++++++

T T

T

T T

++++++TT

+

+++

tBt, pZ -ta E3 are experimental groups; AFB1 = aflatoxin 81; SB : sea buckthorn'

toxins. Liver regeneration is a heterogeneous phenom-

enon involving the proliferation of different cell types

in response to injury. Adult stem cells may give rise

to a progeny of oval cells iocated within the intrahe-

patic biliary tree. They may infiltrate the parenchyma

tetween the hepatic cords' giving rise to both mature

hepatocytes uttd biliuty epithelia (Azuma et al', 2003)'

At tn" ultrastructural level, the cytopiasm of the he-

patocytes in AFBl-treated chickens showed different

sizes of vacuoles. The cytoplasmic vacuolisation implies

increased permeability of cell membranes. Also, the cy-

toplasm displayed an increased number of mitochon-

dria and bailooning of the mitochondrial cristae' Such

ultrastructural lesions were also described by Ergiin

et al. (2006). The pathological effects of AFB1 in the

Figure 5. Electron nicrograph of liver in chigkens treated vdth

aflatJ*in I}1 and sea bucktholn oil. a) Apoptotic hepatocytes (AH)'

iarge bilia"ry canaliculi (bc), electrolucent cltosol (electron nucroscopy

>< Epooit dl hepatocytes showing the normal^appeararce of the nuclei

iNl, ""i,i# ""creof

(n), a large number of -mitocho.ndria (M) wiih

iiloon-lite cristae, rough endoplasmic reticulum (R-E)' and vacuoles

(Vl electron microscoPY x 10,000).

Figure 4. Electron micrograph of hepatocytes'.a).Control group;

b) sei buckthorn oil group. N : nucleus, n : nucleolus, M : mitc'

cilo"ariu, RE : endoilasmic reticulum, V : vacuole (electron micros-

copy x 16,000).

HEPATOPROTECTIVE EFFECT OF SEA BUCKTHORN 973

Figure 6. Electron micrograph of the liver aftcr 28.d of exposure

to anito"i" B1. a) Hepatocytesf mitochondria (M) oval in shape and

different in size and cisterns dilatation of the smooth endopiasmic r-e-

ticulum (RE; electron microscopy x 6,000) b) biliary canaliculi (BC)

"nt*g"a'1"t""t.on microscopy x 10,000); N.: nucleus; n : nucleoius;

M :"mitocfrondria; RE : ieliculum endoplasmic; V : vacuole (elec-

tron microscopy x 10,000)'

liver are mainly due to the degradation products, either

the carbonium ion or the diazoalkane' These 2 reac-

tive metabolites may react with some vital compounds

of the liver, such as DNA or proteins, by alkylation'

The regenerative potential of SB oil was supported by

the finling of a large number of mitochondria and in-

Geased RER around the nucleus. Extensive RER re-

flect the activity of the cell producing large amounts

of proteins needed for normal differentiation' This out-

come could be attributed to the antimutagenic effect of

SB oil, minimizing DNA damage carued by AFBI' An-

other plausible explanation is that SB oil might prevent

transflrmation of wild-type p53 into mutant p53' The

wild-type p53 is a tumor suppressor gene involved in

several-difierent mechanisms including gene transcrip-

tion, DNA synthesis and repair' and programmed cell

death (Helton and Chen, 2007).In conciusions' otu results suggest that SB oil intake

decrease the severity of AFB1 toxic effects in the liver'

This protective action was evident on body growth, mi-

croscopic changes in the liver, serum protein' and albu-

min concentration, AST activity and p53, COX-2, and

Bcl-2 expression and decrease of micotoxin residues in

the liver. These data suggest that SB oil might be used

to prevent the effects of AFBf ingestion. To our knowl-

ed[e, this represents the first report of this approach

in chickens and supports the potential therapeutic use

of SB oil.

ACKNOWLEDGMENTS

The research was financed by a Romanian Gov-

ernment grant, CNCSIS (UEFISCDI) PN II Idei

1118/2003. The authors also thgnk Stuart Carter from

University of Liverpool for correction of the mamrscript

and valuable suggestions in analysis of data'

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